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2017-11-27T10:20:40.959Z | 2017-11-01T00:00:00.000 | 23642704 | {
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} | pes2o/s2orc | 1H-NMR-Based Metabonomics of the Protective Effect of Coptis chinensis and Berberine on Cinnabar-Induced Hepatotoxicity and Nephrotoxicity in Rats
Coptis chinensis Franch has been used in Traditional Chinese Medicine (TCM) for treating infectious and inflammatory diseases for over two thousand years. Berberine (BN), an isoquinoline alkaloid, is the main component of Coptis chinensis. The pharmacological basis for its therapeutic effects, which include hepatoprotective effects on liver injuries, has been studied intensively, yet the therapy of liver injuries and underlying mechanism remain unclear. We investigated the detoxification mechanism of Coptis chinensis and berberine using metabolomics of urine and serum in the present study. After the treatment with Coptis chinensis and berberine, compared with the cinnabar group, Coptis chinensis and berberine can regulate the concentration of the endogenous metabolites. PLS-DA score plots demonstrated that the urine and serum metabolic profiles in rats of the Coptis chinensis and berberine groups were similar those of the control group, yet remarkably apart from the cinnabar group. The mechanism may be related to the endogenous metabolites including energy metabolism, amino acid metabolism and metabolism of intestinal flora in rats. Meanwhile, liver and kidney histopathology examinations and serum clinical chemistry analysis verified the experimental results of metabonomics.
Introduction
Metals (minerals) have been used in Traditional Chinese Medicine for a long time. According to the Pharmacopoeia of China (2015), ten kinds of minerals are listed, including cinnabar (HgS) and realgar (As 4 S 4 ). Cinnabar (96% as HgS) has long been used for its sedative and hypnotic effects in TCM prescriptions [1,2], such as Zhusha Anshen Wan (ZSASW) [3], An-Gong-Niu-Huang Wan [4], etc. Although cinnabar has many special medical effects, the safety of cinnabar is becoming of more and more concern to the public due to the toxic effects caused by the high mercury content [5]. The actual Hg content in ZSASW is about 11-13%, i.e., 110 mg Hg/g, which is 110,000 times higher than the European Drug and Food Standards (1 µg Hg/g) [6]. Consumption of food and drugs can cause cinnabar and other minerals to enter the body, and in some cases can cause liver and kidney injuries [3]. Therefore, it is of great significance to find a drug or effective component which has a protective effect on liver and kidney toxicity induced by cinnabar.
Coptidis chinensis (CR, Huanglian in Chinese) is a Chinese herbal medicine used in Traditional Chinese Medicine (TCM) for thousands years to treat syndromes for its detoxifying and heat-clearing effects [7,8]. For instance, Huanglian Jiedu decoction has been used for hepatitis and liver dysfunction therapy [9]. It was reported that Coptis chinensis had hepatoprotective effects on carbon tetrachloride-induced liver injury through regulating the abnormal metabolism of transaminase [10], yet the mechanism of its liver protective effects remains unexplored. Many studies have shown that many biological activities of Coptis chinensis are due to its main chemical component berberine [11][12][13]. Berberine (molecular formula C 20 H 19 NO 5 ), an isoquinoline alkaloid, is isolated from Coptidis rhizome [14]. Berberine has many pharmacological and biological activities and previous studies have shown that it has beneficial effects such as hypoglycemic, antioxidant and anti-inflammatory activity [15]. Recent studies have found that berberine has a good protective effect on liver and kidney injury caused by mercury by increasing the expression of Bcl-2 protein in liver and kidney [16]. However, no study of the protective effect of Coptis chinensis and berberine on the metabolic pathways of liver and kidney has been reported.
Metabonomics is the science of comprehensively profiling small molecule metabolites in cells, tissues, or whole organisms, the application of which has led to an understanding of the pathophysiologic mechanisms of cardiometabolic diseases, defining predictive biomarkers for those diseases, and also assessing the efficacious effects of administered drugs. As an important part of systems biology, metabonomics refers to a qualitative and quantitative analysis of all low molecular-weight metabolites produced by cells, tissues or organisms within a specific period [17]. It has been well known that a disease can cause pathological and physiological changes in the body, which may cause subsequent changes in these low-molecular-weight metabolites [18]. Metabonomics these are not interchangeabledo not mix has become more and more useful for the characterization of the metabolic changes and the biomarkers involved in toxicological mechanisms [19,20]. NMR spectroscopy, which requires minimal sample preparation [21], provides a rapid, non-destructive and high-throughput method, and is been one of the most powerful techniques used in metabonomics studies.
To elucidate the underlying mechanisms of the protective effects of Coptis chinensis and berberine on liver and kidney injury caused by cinnabar, in the present study a 1 H-NMR-based metabolomic method was applied to evaluate the systemic metabolic consequences.
Biochemical Characteristics and Histopathology
The clinical biochemical results showed the parameters in the serum samples from the rats treated with Coptis chinensis + cinnabar and berberine + cinnabar were different from those of the cinnabar group on day 10 of continuous administration (Table 1). The elevated level of alanine aminotransferase (ALT) and aminotransferase (AST) in the cinnabar-treated groups indicated that the liver was damaged, releasing ALT and AST into the blood [22]. Furthermore, the creatinine (CREA) concentration was significantly increased, which reflected a renal function impairment. Compared to the cinnabar group, the Coptis chinensis + cinnabar and berberine + cinnabar groups showed milder and diminished toxic symptoms. The distributions of ALT values in the Coptis chinensis and berberine groups were less diffuse and closer to the mean value of the control group. The biochemical characteristics demonstrated that Coptis chinensis and berberine could thus effectively reduce the cinnabar-caused injury in rats.
Histopathological examination revealed that the main lesions showed diffuse hepatocyte degeneration, necrosis, apoptosis and significant swelling (ballooning degeneration) in the cinnabar group ( Figure 1). By contrast, no liver and renal damage was observed in the Coptis chinensis and berberine groups. reflected a renal function impairment. Compared to the cinnabar group, the Coptis chinensis + cinnabar and berberine + cinnabar groups showed milder and diminished toxic symptoms. The distributions of ALT values in the Coptis chinensis and berberine groups were less diffuse and closer to the mean value of the control group. The biochemical characteristics demonstrated that Coptis chinensis and berberine could thus effectively reduce the cinnabar-caused injury in rats. Histopathological examination revealed that the main lesions showed diffuse hepatocyte degeneration, necrosis, apoptosis and significant swelling (ballooning degeneration) in the cinnabar group ( Figure 1). By contrast, no liver and renal damage was observed in the Coptis chinensis and berberine groups. Figure 2 shows 1 H-NMR spectra of urine from rats in each group. Eighteen endogenous metabolites, i.e., lactate, valine, alanine, acetate, pyruvate, succinate, α-ketoglutarate, citrate, creatinine, choline, trimethylamine oxide (TMAO), betaine, taurine, glycine, fumarate, phenylalanine, hippurate and formate were identified in the samples.
Analysis of Urine Sample 1 H-NMR Spectroscopic
PLS-DA analysis of the urine spectrometry results show that compared with the control and the cinnabar groups, both the Coptis chinensis and berberine ones appeared close to the control group, but significantly separated from the cinnabar group ( Figure 3A,D). The PLS-DA scores plot also revealed that the Coptis chinensis and berberine groups could easily be distinguished from the cinnabar group along t1 ( Figure 3B,E). The corresponding loadings plot ( Figure 3C,F) combined with the VIP values from the pattern recognition model, screened out potential biomarkers for the differentiation of Coptis chinensis, berberine and cinnabar groups, The 1 H-NMR-detected relative integral levels of metabolites indicating differences between the cinnabar group and Coptis chinensis and berberine groups are listed in Table 2. Figure 2 shows 1 H-NMR spectra of urine from rats in each group. Eighteen endogenous metabolites, i.e., lactate, valine, alanine, acetate, pyruvate, succinate, α-ketoglutarate, citrate, creatinine, choline, trimethylamine oxide (TMAO), betaine, taurine, glycine, fumarate, phenylalanine, hippurate and formate were identified in the samples.
Analysis of Urine Sample 1 H-NMR Spectroscopic
PLS-DA analysis of the urine spectrometry results show that compared with the control and the cinnabar groups, both the Coptis chinensis and berberine ones appeared close to the control group, but significantly separated from the cinnabar group ( Figure 3A,D). The PLS-DA scores plot also revealed that the Coptis chinensis and berberine groups could easily be distinguished from the cinnabar group along t1 ( Figure 3B,E). The corresponding loadings plot ( Figure 3C,F) combined with the VIP values from the pattern recognition model, screened out potential biomarkers for the differentiation of Coptis chinensis, berberine and cinnabar groups, The 1 H-NMR-detected relative integral levels of metabolites indicating differences between the cinnabar group and Coptis chinensis and berberine groups are listed in Table 2.
Compared with cinnabar group, the level of α-oxoglutarate, citrate, TMAO and hippurate were significantly increased, and lactate, alanine, acetate, pyruvate, creatine, choline and taurine were significantly reduced in the Coptis chinensis and berberine groups.
1 H-NMR Spectroscopic Analysis of Serum Samples
Representative 600 MHz 1 H-NMR spectra of serum obtained from controls and treated groups are shown in Figure 4. Endogenous metabolite assignments were based on chemical shifts reported in the literature [23]. Coptis chinensis (C) derived from 1 H-NMR spectra of urine. PLS-DA scores plot from control group, group cinnabar, group berberine (D); group cinnabar and group berberine (E); and corresponding loadings plot group cinnabar and group berberine (F) derived from 1 H-NMR spectra. Key: control group ( ); group Cinnabar ( ); group Coptis chinensis ( ); group berberine ( ). score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group. score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group. score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group. Compared with cinnabar group, the level of α-oxoglutarate, citrate, TMAO and hippurate were significantly increased, and lactate, alanine, acetate, pyruvate, creatine, choline and taurine were significantly reduced in the Coptis chinensis and berberine groups.
1 H-NMR Spectroscopic Analysis of Serum Samples
Representative 600 MHz 1 H-NMR spectra of serum obtained from controls and treated groups are shown in Figure 4. Endogenous metabolite assignments were based on chemical shifts reported in the literature [23]. The PLS-DA scores plots ( Figure 5A,B,D,E) reveal an obvious separation of the control, Coptis chinensis and berberine groups from the cinnabar group. At the same time, we observed that there is no difference between the Coptis chinensis, berberine and control groups. The corresponding loadings plots, which show which metabolites contributed most to the separation of samples in the The PLS-DA scores plots ( Figure 5A,B,D,E) reveal an obvious separation of the control, Coptis chinensis and berberine groups from the cinnabar group. At the same time, we observed that there is no difference between the Coptis chinensis, berberine and control groups. The corresponding loadings plots, which show which metabolites contributed most to the separation of samples in the score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group. score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group.
Pathway Analysis
In addition, to understand which pathways were the most relevant to the Coptis chinensis and berberine protective effect on acute hepatic and kidney injury, metabolic network was mapped by Metaboanalyst 3.0 (http://www.metaboanalyst.ca). A total of sixteen biomarkers were subjected to pathway analysis [23], and the associated metabolic pathways of each substance with their FDR values are summarized in Figure 6 and score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group.
Pathway Analysis
In addition, to understand which pathways were the most relevant to the Coptis chinensis and berberine protective effect on acute hepatic and kidney injury, metabolic network was mapped by Metaboanalyst 3.0 (http://www.metaboanalyst.ca). A total of sixteen biomarkers were subjected to pathway analysis [23], and the associated metabolic pathways of each substance with their FDR values are summarized in Figure 6 and Table S1 (Supplementary Materials). score plots, are presented in Figure 5C,F. The major biochemical changes identified in serum from the corresponding loadings plot and relative integral levels of metabolites (Table 3) showed the level of α-ketoglutaric acid, alanine, and TMAO were increased, and the levels of pyruvate, lactate, creatine, leucine, isoleucine and choline were decreased in the Coptis chinensis and berberine groups compared with the cinnabar group. These endogenous metabolites showed no significant differences in the Coptis chinensis and berberine groups compared with the control group.
Pathway Analysis
In addition, to understand which pathways were the most relevant to the Coptis chinensis and berberine protective effect on acute hepatic and kidney injury, metabolic network was mapped by Metaboanalyst 3.0 (http://www.metaboanalyst.ca). A total of sixteen biomarkers were subjected to pathway analysis [23], and the associated metabolic pathways of each substance with their FDR values are summarized in Figure 6 and Table S1 (Supplementary Materials).
). Table 3. 1 H-NMR-relative integral levels of metabolites in serum samples of groups control, cinnabar, Coptis chinensis and berberine.
Pathway Analysis
In addition, to understand which pathways were the most relevant to the Coptis chinensis and berberine protective effect on acute hepatic and kidney injury, metabolic network was mapped by Metaboanalyst 3.0 (http://www.metaboanalyst.ca). A total of sixteen biomarkers were subjected to pathway analysis [23], and the associated metabolic pathways of each substance with their FDR values are summarized in Figure 6 and Table S1 (Supplementary Materials). The pathways with the impact value > 0.1 were considered as the most relevant pathways for the liver injury. In this study, taurine and hypotaurine metabolism; glyoxylate and dicarboxylate metabolism; valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; pyruvate metabolism; citrate cycle (TCA cycle) and glycolysis or gluconeogenesis pathways were important metabolic pathways with impact factors of 0.42, 0.40 0.33, 0.29, 0.24, 0.17, 0.012, respectively. The pathway analysis indicated that the alleviation effect of Coptis chinensis and berberine was connected to alterations in energy metabolism (TCA cycle), amino acid metabolism (valine, leucine and isoleucine biosynthesis).
Discussion
A large number of studies have reported that substances containing heavy metals such as HgS, cadmium (Cd), etc., can cause liver and kidney toxicity [24]. Heavy metals may enter the body through food, drugs, and decoration materials, etc. [1], and human intake of heavy metals for a long time will cause liver and kidney damage, and further lead to various types of liver diseases. In this work NMR metabonomics technologies were applied to elucidate the detoxification and mechanism of Coptis chinensis and its main component berberine on liver and kidney toxicity caused by cinnabar. The metabolites pathway analysis based on the potential biomarkers indicates that TCA cycle, lipid metabolism and amino acid metabolism were involved in the metabolic changes of urine and serum.
Lactate is a substance produced by the metabolism and movement of the human body, but its concentration generally does not rise. Only when the lactic acid production process increases, but cannot be excreted in time, will the concentration increase [25]. It is formed by the combination of pyruvate and hydrogen. If the energy metabolism of the rat body is normal, it does not produce any accumulation, breaks lactate down into water and carbon dioxide, and generates heat. The increase in the corresponding integral area shows that the metabolism of lactate in the cinnabar group has abnormally accelerated. In addition, the levels of acetate and creatine anhydride in serum of the cinnabar group were significantly increased. Increases in the amount of acetate in the serum indicate a fat metabolism disorder in the liver mitochondria [26]. Compared with the cinnabar group, the content of lactate, acetate and creatine in the urine of rats decreased to a level close to the control when the Coptis chinensis and berberine were administered. All the above results suggest that Coptis chinensis and berberine can improve the metabolism of hepatic mitochondria.
Trimethylamine oxide is the oxidation product of trimethylamine. It has the function of maintaining the concentration balance between the cell body fluid and the external body fluid environment. Studies have shown that the metabolism of trimethylamine oxide is related to the disorder of intestinal flora [27]. When the content of TMAO in the urine of rats was increased, it The pathways with the impact value > 0.1 were considered as the most relevant pathways for the liver injury. In this study, taurine and hypotaurine metabolism; glyoxylate and dicarboxylate metabolism; valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; pyruvate metabolism; citrate cycle (TCA cycle) and glycolysis or gluconeogenesis pathways were important metabolic pathways with impact factors of 0.42, 0.40 0.33, 0.29, 0.24, 0.17, 0.012, respectively. The pathway analysis indicated that the alleviation effect of Coptis chinensis and berberine was connected to alterations in energy metabolism (TCA cycle), amino acid metabolism (valine, leucine and isoleucine biosynthesis).
Discussion
A large number of studies have reported that substances containing heavy metals such as HgS, cadmium (Cd), etc., can cause liver and kidney toxicity [24]. Heavy metals may enter the body through food, drugs, and decoration materials, etc. [1], and human intake of heavy metals for a long time will cause liver and kidney damage, and further lead to various types of liver diseases. In this work NMR metabonomics technologies were applied to elucidate the detoxification and mechanism of Coptis chinensis and its main component berberine on liver and kidney toxicity caused by cinnabar. The metabolites pathway analysis based on the potential biomarkers indicates that TCA cycle, lipid metabolism and amino acid metabolism were involved in the metabolic changes of urine and serum.
Lactate is a substance produced by the metabolism and movement of the human body, but its concentration generally does not rise. Only when the lactic acid production process increases, but cannot be excreted in time, will the concentration increase [25]. It is formed by the combination of pyruvate and hydrogen. If the energy metabolism of the rat body is normal, it does not produce any accumulation, breaks lactate down into water and carbon dioxide, and generates heat. The increase in the corresponding integral area shows that the metabolism of lactate in the cinnabar group has abnormally accelerated. In addition, the levels of acetate and creatine anhydride in serum of the cinnabar group were significantly increased. Increases in the amount of acetate in the serum indicate a fat metabolism disorder in the liver mitochondria [26]. Compared with the cinnabar group, the content of lactate, acetate and creatine in the urine of rats decreased to a level close to the control when the Coptis chinensis and berberine were administered. All the above results suggest that Coptis chinensis and berberine can improve the metabolism of hepatic mitochondria. Trimethylamine oxide is the oxidation product of trimethylamine. It has the function of maintaining the concentration balance between the cell body fluid and the external body fluid environment. Studies have shown that the metabolism of trimethylamine oxide is related to the disorder of intestinal flora [27]. When the content of TMAO in the urine of rats was increased, it showed that the intestinal flora of rats was destroyed and the metabolism was disordered [28]. TMAO has been suggested as a link between gut microbiota and disease [29]. Gut microbiota play a critical role in several metabolic processes in the human body [30]. Coptis chinensis and berberine can significantly regulate the abnormal metabolism of trimethylamine oxide caused by cinnabar. The results suggest that Coptis chinensis and berberine ensure a good regulatory balance in the intestinal flora of the organism.
The main role of taurine is to participate in the regulation of environmental homeostasis in animals [31]. One of the standards for judging liver injury is the content of taurine in animal urine. Bile acid synthesis mainly contains taurine, and cholestasis in the liver can cause the bile acid concentration to increase, which can lead to an increase in the renal excretion of taurocholate acid. Taurine is likely to be a possible urinary marker of liver damage [32]. The level of taurine was restrained in the Coptis chinensis and berberine groups compared with the cinnabar group. The levels of taurine in the Coptis chinensis and berberine groups were close to those of the control group. This suggested that Coptis chinensis and berberine have protective effect on liver by regulating the taurine metabolism.
Creatine is synthesized in the liver and stored in the muscles. When the body's energy is insufficient, creatine is phosphorylated and releases energy in skeletal muscle, and a series of changes are produced to produce creatinine. Creatinine is excreted through the glomerulus through the urine and is almost reabsorbed by the renal tubules. When the animal's liver is damaged, it can lead to impaired energy metabolism and promote the production of creatinine. In addition, the increase of creatinine content is related to renal dysfunction [33]. A significant increase of creatine in serum was also observed after cinnabar adminstration. By contrast, after the administration of Coptis chinensis and berberine, the metabolism of creatine returned to normal, which implied that Coptis chinensis and berberine could prevent the liver and kidney damage induced by cinnabar.
Succinate, citrate and 2-OG are vital intermediates in the TCA cycle, which is the core metabolic pathway for energy, which promotes the oxidative decarboxylation of acetyl-CoA and produces reduced equivalents, FADH 2 and NADH [34]. The level of the three endogenous metabolites were decreased in the cinnabar groups. However, succinate, citrate and 2-OG showed a reversion tendency in the Coptis chinensis and berberine groups, which indicated that the Coptis chinensis and berberine could restore the energy metabolism in rats.
Animals and Drug Administration
Adult male Wistar rats (SPF, 180 ± 20 g, animal license No. SCXK-(Military) 2013-004) were purchased from the Experimental Animal Center of Shenyang Pharmaceutical University. All the animal experiments were approved by the national legislations of China and local guidelines. After acclimatization to the environment for ten days, the rats were maintained under standard laboratory conditions (23 ± 1 • C, 45 ± 15% relative humidity, and 12 h/12 h light/dark cycle) in individual metabolic cages with freely available food and water. All the rats were randomly divided into four groups (n = 6): control group (injected water), cinnabar group (treated with cinnabar), Coptis chinensis group (treated with cinnabar and Coptis chinensis French), and berberine group (treated with cinnabar and berberine). All animals were treated by intragastric administration for consecutive eight days. The dosage of cinnabar was 1.8 g/kg according to the literature [3]. The dosage of Coptis chinensis was 2.7 g/kg [3], and the dosage of berberine waas 100 mg/kg according to the literature [16].
Sample Collection and Pretreatment
Urine samples were collected over dry ice in tubes overnight (from PM 7:00 to AM 7:00). And all the urine samples were immediately stored at −20 • C until NMR spectroscopic analysis was conducted. Blood samples from sacrificed rats were centrifuged at 14,000 rpm for 10 min at 4 • C. All the serums samples were stored at −80 • C for the NMR spectroscopic analysis and serum biochemistry assays.
Serum samples (500 µL) were mixed with D 2 O (60 µL) and TSP (40 µL), and transferred to 5 mm NMR tubes. TSP acts as internal standard reference (δ 0.00 ppm) and D 2 O was used for locking the signal.
Data Reduction Analysis of 1 H-NMR Spectra
Urine measurement conditions: 1 H-NMR spectral measurements were acquired on an AV600 spectrometer (Bruker Biospin, Ettlingen, Germany) at 298 K. Typically, 32 free induction decays (FIDs) were collected into 64 k data points over a spectral width of 12,019.23 Hz with a relaxation delay of 3 s and an acquisition time of 2.73 s. Number of scans is 64.
Serum measurement conditions: 1 H-NMR spectra of these samples were also recorded on the Bruker-AV600 spectrometer at 298 K. Pulse program is a 1D experiment with-T 2 filter using the Carr-Purcell-Meiboom-Gill sequence. Thirty-two FIDs were collected into 64 k data points over a spectral width of 12,019.23 Hz with a relaxation delay of 3 s and an acquisition time of 2.73 s. Power level for presaturation is 50 dB, and number of scans is also 64.
All the serum and urine spectra were baseline corrected and manual phase adjustments of the spectrua were performed. Data sets were zero-filled to 64 k data points. Each urine or serum 1 H-NMR spectrum was segmented into integrated regions at 0.04 ppm, corresponding to the chemical shifts δ 0.2-10.0 using MestReNova 11.0.2 (Mestrelab Research SL, Norwich, CT, USA). The δ 4.2-6.0 region was excluded to eliminate the effect of the water resonance and urea signals. The integral data were normalized to a constant unit area to reduce the effects of variation in concentration differences. Finally, the exported data was input into the SIMCA-P 13.0 software package (Umetrics AB, Umea, Sweden) for PLS-DA analysis. The goodness of fit for a model was evaluated according to the three quantitative parameters: R 2 X was the explained variation in X, R 2 Y was the explained variation in Y, and Q 2 Y was the predicted variation in Y. The range of the parameters was between 0 and 1, and the values approaching 1 indicate good fit for the model [35].
Statistical Analysis
All the data are expressed as mean ± standard deviation (SD). The significance testing was determined using one-way ANOVA followed by Dunnett's test by SPSS 19.0 (IBM SPSS Inc, Chicago, IL, USA). p < 0.05 was considered as statistically significant.
Conclusions
The results clearly showed that the metabolic profiles of the Coptis chinensis and berberine groups were remarkably similar to those of the control group. In this work, the metabolite responses indicating the main liver and kidney protective effects of Coptis chinensis and berberine against liver and kidney damage induced by cinnabar were investigated by 1 H-NMR-based metabonomics for the first time. 1 H-NMR spectroscopy in conjunction with histopathology and clinical biochemical assays provided a special insight into the relationship between the metabolite changes and the toxicity to tissues. The corresponding biochemical pathways of energy metabolism, amino acid metabolism and gut microbiota disorder provided new suggestions for a systematic and holistic evaluation of the protective mechanism of Chinese herbal medicines on hepatic and renal injury caused by minerals.
Supplementary Materials: The following are available online. Table S1: The pathway analysis potential target metabolites. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2011-12-01T00:00:00.000 | 15751885 | {
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} | pes2o/s2orc | Radical-Scavenging Activity of Dietary Phytophenols in Combination with co-Antioxidants Using the Induction Period Method
The radical-scavenging activity of dietary phytophenols has been investigated by many researches due to their antioxidant, anti-inflammatory and anticancer property but the radical-scavenging effect of 2-phytophenol and the phytophenol:co-antioxidants, vitamin C and thiol combination under nearly anaerobic conditions still remains unknown. The radical-scavenging activity for seventeen phytophenols and for six synthetic phenols (positive controls) was investigated using the induction period method in the polymerization of methyl methacrylates (MMA) initiated by thermal decomposition of benzoyl peroxide (BPO) by monitoring differential scanning calorimetery (DSC). The kinh for the phytophenols was likely with the range 0.5 × 103 M−1s−1−2.2 × 103 M−1s−1, whereas that for synthetic phenols, hydroquinone and galvinoxyl, was with the range 7 × 103 M−1s−1−8 × 103 M−1s−1. Also, the additive scavenging effect of the (−)-epigallocatechin (EGC):(−)-epicatechin (EC) and the (+)-catechin:epicatechin (EC) combination was observed at 1:1 molar ratio, whereas that of the EC:quercetin combination showed the cancel (prooxidative) effect. Furthermore, the EGC:ASDB (L-ascorbyl 2,6-dibutylate) or 2-ME (2-mercaptoethanol) combination showed the prooxidative effect. Such enhancement of prooxidation in the combination may increase their toxic effects due to their cooxidation. Also, the synergic, additive or cancel effects of the flavonoid:vitamins E combination on the induction period in the BPO (a PhCOO* radical) and 2,2′-azobisisobutyronitrile (AIBN, an R* radical) systems are discussed.
Introduction
Dietary phytophenols are approximately divisible into single-ring phenolic acids such as ferulic acid, p-coumalic acid and caffeic acid, double-ring biphenols such as curcumin and tricyclic phenols (flavonoids) such as (+)-catechin, (−)-epicathechin (EC), (−)-epigallocatechin (EGC) and (−)-epigallocathechingallate (EGCG). They are well known to have antioxidant anti-inflammatory and anticancer properties [1]. Their probable mechanism is free radical scavenging and selective interference with various factors of abnormal proliferation of mammalian cells [1]. The phytophenols in every cup of tea or cafe may act as antioxidants to help neutralize the harmful effects of free radicals in biosystems.
Benzoyl peroxide (BPO) as an initiator is used widely for denture based resins. In combination with BPO and tertiary amines, self-curing denture-repair resins are widely used in dentistry. Currently, initiators such as camphoroquinone and benzyl as a photopolymerization system are widely used in dental materials [2]. BPO and related peroxides as an active molecule were previously reported to induce the lipid peroxidation of cellular membranes and consequently cell damage [3]. Due to the fact that dentures and restorative materials remain in the oral cavity for a very long time, BPO and related initiators may be involved in allergy and inflammatory activity [4][5][6]. Harmful free radicals derived from dental materials may be scavenged by dietary phytophenols found in food [7]. We investigated previously the radical scavenging activity of various dietary phytophenols such as polyphenols [8][9][10], single-ring phenolic acids [11], biphenols and related-phenols [12] using induction period method in polymerization of MMA initiated by BPO and/or AIBN by monitoring DSC. However, the comparative antioxidant activity for mono-ring phenols, biphenols and tricyclic phenols and for their combination was not sufficiently investigated. In the present study, we investigated the radicalscavenging activity for 13 phytophenols and butylated hydroxytoluene (BHT), a typical synthetic antioxidant, and 2-flavonoid combination and the flavonoid:co-antioxidant, ascorbate or thiol combination using the induction period method in polymerization of MMA initiated by thermal decomposition of BPO.
Characterization of the Radical-Scavenging Activity
Polymerization curves were derived from the DSC thermogram using the integrated heat evoked by the polymerization of MMA. Typical time-exotherm and time-conversion curves for capsaicin are shown in Figures 1 and 2, respectively. As shown in Experimental section, using Equations (3), (6) and (7), the stoichiometric factor (n) (i.e., the number of radicals trapped by each inhibitor molecule), k inh and kinetic chain length (KCL) for indicated phytophenols were determined. Results are shown in Table 1. We also examined the induction period (IP) and inhibition rate of propagation (Rp inh ) when BPO varied with 0.1-1.0 mol% without an inhibitor. Note that a certain amount of oxygen in air was contained in the DSC sample pan and the estimated oxygen was approximately 8.128 × 10 −8 mol/L. The relation between the induction period (A) or the initial rate of polymerization (propagation rate, Rp) (B) is shown in Figures 3 and 4 respectively.
As shown in Figure 3, induction period (IP) and initial rate of polymerization (Rp) were linearly dependent on the square root of the initiator concentration. Also, the Rp was proportional to the concentration of the monomer (data not shown). Thus, the kinetic studies cab be applicable using the induction period. The kinetic chain length (KCL) was calculated by equation 7. KCL of control and phenolic inhibitors at 1 mol% BPO is shown in Table 1 As an example, the induction period and initial rate of polymerization for capsaicin were calculated on the base of data of Figure 2. The relationship between the induction period or the initial rate of polymerization and the concentration of capsaicin is shown in Figure 4, respectively. The induction period was linearly increased as the concentration was increased. Whereas, the initial rate of polymerization was linearly decreased as the concentration was increased.
Radical-Scavenging Activity
The n and k inh value for dietary phytophenols and synthetic phenols were determined and results are summalized in Table 1 . trans-Cinnamic acid did not inhibit the polymerization because there is no phenolic OH group in the ring. Hesperetin and p-coumalic acid constituted the group of the largest k inh value, whereas curcumin, catechin and EGCG was a group of the smallest value. The k inh for hesperetin was approximately four-fold greater than that for EGCG. EGCG, EGC and catechin, flavan derivatives were a group for the smallest KCL value of 421-501 (Table 1). Comparing their KCL to that of control, the KCL for EGCG was reduced to about 30%, whereas that for EGC and catechin was reduced to about 20%. This finding demonstrated that these compounds are the efficient chain-breaking antioxidant. Although hesperatin, n-propyl gallate, quercetin and resveratrol are a polyphenol group, their KCL value was 548-610, indicating that these compounds were a group of the weak chain breaking compounds and also that their activity was similar to that for monophenols, ferulic acid, eugenol, isoeugenol, chlorogenic acid and caffeic acid. On the other hand, the k inh for synthetic phenols declined in the order galvinoxyl (8.2) > HQ (7.0) > DPPH (3.1) >> p-cresol (1.0) > bisphenol A (0.8) > BHT (0.8). Although galvinoxyl and DPPH are stable radicals, they were also a potent inhibitor with the large k inh value. As a polymerization inhibitor, HQ preferably prevented polymerization of MMA, resulting from the large k inh and small KCL value.
Polyphenol Combinations
The result of radical-scavenging effect of the combination of EC:catechin, EC:EGC and EC:quercetin at 1:1 molar ratio is shown in Table 2. The B/A value at the molar ratio of 1:1 for the EC:quercetin combination was approximately 0.8, showing that when the experimental IP for the mixture was compared with the total sum of the IP of EC + quercetin, the IP decreased by approximately 20%. The radical-scavenging activity for the EC; quercetin combination balanced out. Similarly, the B/A ratio of the EC:EGC combination was approximately 0.9, showing approximately the decrease in 10%. The radical-scavenging activity for the EC:EGC combination balanced out but the activity was relatively smaller than that for the EC:quercetin combination. In contrast, that for the EC:catechin combination was 1, showing that the radical-scavenging effect for this mixture was additive.
Polyphenol: Co-Antioxidant Combination
Next, the radical-scavenging activity for EGC or epicatechin in combination with ASDB or 2-ME was investigated. ASDB and 2-ME were used as a biological model for vitamin C and glutathione (GSH), respectively, because water-soluble vitamin C and GSH were unable to dissolve in MMA. The result is shown in Table 3. The EGC:ASDB and the EGC:2-ME combination balanced out, i.e., a cancelling effect was noted.
Discussion
Previously quantitative in vitro studies of the radical-scavenging activity of Burton and Ingold reported previously the kinetics of radical-scavenging activity of phenolic compounds in the chlorobenzene-styrene system initiated by azobisisobutyronitrile (AIBN) at 30 °C using induction period method [13]. In this method, autooxidations were carried out under 760 torr of O 2 in an automatic recording gas pressure transducer apparatus. Styrene has no abstractable hydrogens and forms a polymeric peroxyl radical. The end of inhibition period can be calculated by inhibited oxygen -time curves on the base of the interception of a base and initial lines. By contrast, we investigated the kinetics of radical-scavenging activity of phenolic compounds in polymerization of MMA initiated by thermal decomposition of AIBN and/or BPO using differential scanning calorimetry (DSC) [8][9][10]. The model using induction period method was able to explain the mechanism of radical-scavenging activity and to predict the chain-breaking activity of phenolic compounds, because measurement by DSC is highly sensitive. Also, the DSC system in the present study was performed by the induction period method under aerobic conditions. The oxygen tension under a 15 torr oxygen atmosphere is similar to that in many tissues [14,15], suggesting that oxygen is scarce in living cells and that the radical-scavenging activity of phytophenols in vivo may differ considerably from that observed under aerobic conditions. The n value for EGCG was the greatest among the compounds tested, followed by EGC and catechin ( Table 1). As shown in Table 2, the radical-scavenging activity for EGC, catechin, EC and quercetin was investigated and it was found that the n value for EGC, EC, catechin and quercetin was 3.15, 3.83, 3.69 and 2.46, respectively. EC and catechin showed a similar value to that shown in Table 1. The antioxidant effect of (+)-epimer, catechin and (−)-epimer, EC was similar. The inhibitory effect of catechins on peroxidation of soybean phosphatidylcholine liposomes was investigated by 2,2′-azobis(2-aminopropane)hydrochloride (AAPH)-induced oxidation and it was found that the n value for EC, EGC and EGCG was 3.2, 1.1 and 1.7, respectively [16]. The n value for EGC and EGCG, particularly the latter, was less than that in the present study. This may be depend on the experimental conditions. In general, the n value of monophenols is 2. The n value for caffeic acid, ferulic acid and BHT is near 2. That for p-coumaric acid, hesperetin, eugenol and n-propyl gallate it is near 1, suggesting the formation of dimer derived from the monomer-monomer coupling reaction due to radical oxidation [8,11]. Also, HQ with the n value of 1 may undergo dimerization. Tricyclic phenols (flavonoids) such as catechin, EGC and EGCG showed the large n value due to their large number of phenolic OH groups.
Javanovic et al. previously investigated oxygen radical-scavenging activity for flavonoids and it was found that their inhibition rate of super oxygen radical (k inh ) at 20 °C was with the range 3 × 10 2 M −1 s −1 -5 ×10 4 M −1 s −1 [17]. Zhou et al. previously reported that the green tea polyphenols could reduce α-tocopheroxyl radical to regenerate α-tocopherol with rate constants of 0.45, 1.11, 1.31, 1.91, and 0.43 ×10 2 M −1 s −1 for EC, EGC, ECG, EGCG, and gallic acid (GA), respectively, in sodium dodecyl sulfate micelles [18]. In the present study, the k inh (M −1 s −1 ) for green tea polyphenols, EGCG, ECG and catechin was 0.5 × 10 3 , 0.85 × 10 3 and 0.66 × 10 3 , respectively. That for p-coumalic acid and hesperatin was approximately two-fold greater than that for EGCG or EGC. Although under different experiment conditions, the k inh value for catechin polyphenols was different, the difference in the relative k inh between polyphenols fell with a range of several-fold. EGCG, EGC, catechin with the small k inh showed their small KCL value. From the present finding of the k inh , EGCG scavenged much free radicals but released them at relatively short time. EGCG was found to be potent chain-breaking antioxidant, resulting from the small KCL.
There are many radical-scavenging methods for polyphenols [19]. Their scavenging effect on O 2 − declined in the order EGCG > ECG > EC ≈ EGC [19]. Tea catechins are effective 1,1′-diphenyl-2picrylhydrazyl (DPPH) antagonists. Anti-DPPH activity was EC < (+)-catechin < EGC < EGCG [20]. The n value for tea catechins and related compounds was investigated by the DPPH method and indicated that for catechin, EC, EGC, ECG and EGCG they was estimated to be 2, 2, 5, 7 and 10, respectively [21]. The n for EGC and EGCG was calculated from the data in Table 1, and it was found that their value was similar to that for above described report [21]. Anti-DPPH activity for the gallated catechins, EGCG and n-propyl gallate was greater than that for nongallated catechins, EGC. The antioxidant activity for tea polyphenols is also radical-dependent and medium dependent. In general, EGCG has been recognized as a potent radical scavenger. The synergic scavenging effect of two-catechins combinations on free radicals such as O 2 − was investigated, indicating that the effect of the EGCG:ECG combination was the strongest, followed by the ECG:EC and the EC:EGC combination. [19]. In the present study, however, no synergic scavenging effect of the EGC:EC and the EC: (+)-catechin combination, was found. By contrast, a prooxidative (cancelling) scavenging effect of the EGC:ASDB or 2-ME combination was observed. Also, the prooxidative effect of the EC:quercetin combination was observed. The synergistic antioxidant mechanism of α-tocopherol with green tee polyphenols, EC, EGC, ECG, EGCG and GA was previously reported and it was found that these polyphenols could reduce the α-tocopheroxy radical to regenerate α-tocopherol [18,22,23]. Also, the flavonoid:vitamin C combination caused a synergic antioxidant effect in an in vitro lipoprotein oxidation model [24]. We previously investigated the free radical interaction between α-tocopherol or ASDB and methyl gallate (MG), EC, EGC, ECG or EGCG. This showed a synergic radical-scavenging effect of the α-tocopherol:MG, EC, EGC or ECG combination on the induction period in the polymerization of MMA initiated by thermal decomposition of AIBN [9]. Whereas, there was no synergic scavenging effect of the ASDB:MG, EC, EGC or ECG combination and conversely, the prooxidative scavenging effect was observed for their combination [9]. On the other hand, using the induction period method initiated by BPO, a synergic radicalscavenging effect of the δ-tocopherol:EC or EGCG combination alone was observed, whereas a prooxidative radical-scavenging effect of the α-, β-or γ-tocopherol:EC or EGCG combination was observed. Also, the ASDB:EC or EGCG combination showed a prooxidative radical-scavenging effect [10]. The radical-scavenging effect of the mixture of flavonoid and vitamin E showed clearly the great contradictory difference between the AIBN and BPO systems. These findings may be involved in the induced decomposition of BPO. Frank et al. reported that dietary flavonoids, quercetin, catechin and epicatechin increase α-tocopherol in rat and protect the vitamin from oxidation in vitro [23]. Also, Wiegand et al. reported that dietary flavonoids, quercetin and catechin do not affect vitamin E status in growing rats [24]. These contradictory findings suggested that the vitamin E status in the presence of flavonoids may be dependent on radical species.
In the previous our studies, a synergic radical scavenging effect of the vitamin E:flavonoid combination was found under R* radical conditions derived from the AIBN system [9]. This suggested that the antioxidant activity in vivo due to bioactive compound such as vitamins and GSH ocurrs under nearly anaerobic conditions because biological systems tend to have low oxygen tension [14,15]. Interestingly, the radical-scavenging effect of the flavonoid:vitamin C combination oxidized by both radicals R* and PhCOO* was the prooxidative one [9,10]. That of the EGC:2-ME combination was also prooxidative ( Table 3). The radical-scavenging effect of the EC:2-ME combination oxidized by both radicals R* and PhCOO* was the prooxidative (data not shown). Green tea polyphenols are wellknown to possess antioxidant and anticancer activity. Cancer cells are anaerobic in the metabolism and have very poor mechanisms for absorbing adequate amounts of antioxidants [25,26]. Dietary phytophenols not only interact directly with free radicals, but also can react with other redox-based antioxidant substances. These redox cycles of vitamin E, vitamin C or GSH form a well-known antioxidant network. Such antioxidant network could prevent chronic inflammation and cancer due to free radical reactions in biological systems. Further studies may be necessary to clarify the radicalscavenging effect of phytophenols in the presence of vitamin C and vitamin E and GSH. On the other hand, the inhibition of polymerization for phenolic inhibitors is very important in the chemical industry. In the present study, HQ in addition to galvinoxyl showed one-order grater k inh value than that for EGC and EGCG and its KCL was the smallest (454). We have seen HQ as a potent inhibitor, in the fresh light.
In vivo experiments are too complex to amenable to simple interpretation and, hence, we employed physical-chemical studies using the induction period method in the radical polymerization of MMA under nearly anaerobic conditions. We expect that induction period, n, kinh, KCL values for dietary phytophenols determined in the present study will be relevant for the development of compounds that mimic their biological activity.
Materials
The following chemicals were obtained from the indicated companies: (
Methods
The experimental resin consisted of MMA and BPO with or without phenolic antioxidants. BPO were added at 0.1, 0.2, 0.5 and 1.0 mol% and additives were used at 0.001, 0.01, 0.02, 0.05 and 0.1 mol% when BPO was 1.0 mol%. The combination study was carried out for 0.01 mol% for each phenol with 1.0 mol%. Approximately 10 µL of the experimental resin (MMA: 9.12-9.39 mg) was loaded into an aluminum sample container and sealed by applying pressure. The container was placed into a different scanning calorimeter (model DSC 3100: Mac Science Co., Tokyo, Japan) maintained at 70 °C, and the thermal changes induced by polymerization were recorded for appropriate time periods. Exothermic curves for the polymerization of MMA with BPO in the presence of capsaicin, a typical compound is shown in Figure 1. The heat produced due to polymerization of MMA was 13.0 kcal/mol in these experiments.
Polymerization curves break when an inhibitor is consumed (Figure 2). These breaks are sharp and provided a reliable measure of the induction period of the antioxidant inhibitor. The presence of oxygen retards polymerization because oxygen reacts with MMA radicals (MMA*) activated by the initiator, and the produces a non-radical product. Thus, polymerization of the control was slightly inhibited, even though the reaction was carried out in a sealed DSC pan, because the pan contained a small amount of oxygen since it had been sealed in air. At an early stage in each run, tangents were drawn to the polymerization curves. The induction period for each test compound was determined from the length of time between the zero point on the abscissa and the point of intersection of the tangent with the polymerization curve. The induction period (IP) was calculated at the difference between the induction period of the specimen and that for the control. The initial rates of polymerization in the absence (Rp con ) and presence (Rp inh ) of the phytophenols were calculated from the slope of the first straight line on each plot of the conversion rate during MMA polymerization [8]. The Rp inh value represents the rate of inhibition of initial polymerization by the antioxidant.
Measurement of Rate of Initiation
Initiation of BPO, an initiator: The induction period method was also used to determine the rate of initiation of polymerization (R i ) due to thermal decomposition of BPO using in Equation (1): in which [IH] 0 is concentration of the initiator at time zero and the IP is the induction period. DTBMP was used to determine R i , since the stoichiometric factor, n, is known to be 2.00. In the case of MMA = 9.4 M and BPO = 0.1 M at 70 °C gave an R i value of 2.28 × 10 −6 M s −1 .
Measurement of Stoichiometric Factor (n)
The relative n value in Equation (3) can be calculated from the induction periods in the presence of inhibitors.
n = R i [IP] / [IH]
( where [IP] is the induction period in the presence of an inhibitor. The number of moles of peroxy radicals trapped by the relavant phenol is calculated with respect to 1 mole of inhibitor moiety unit.
Measurement of the Inhibition Rate
When R i is constant, i.e., when new chain are started rata constant rate, a steady-state treatment can be applied and the initial rate of polymerization of MMA is given by Equation (4) where MMA represents methyl methacrylates and k p and k t are the rate constants for chain propagation and termination, respectively. The k p /(2k t ) 1/2 rate of polymerization of MMA (9.4 M) by BPO (0.1M) at 70 °C was 9.86 × 10 −2 M −1/2 s −1/2 [8]. The k t was estimated to be approximately 3.8 × 10 7 and therefore, the k p was approximately 930.
Rp inh = (k p [MMA]R i ) / (n k inh [IH]) (5) in which is the initial rate ofpolymerization with an inhibitor.
[MMA], n, [IH] and kp are defined above and kinh is the rate constant for scavenging (inhibiting) of MMA radicals by a phytophenol antioxidant.
Conclusions
The radical-scavenging activity in the polymerization of MMA initiated by thermal decomposition of BPO under nearly anaerobic conditions was investigated by the induction period method for thirteen dietary phytophenols and one artificial phenol (BHT). The stoichiometric factor (n), k inh , KCL for these compounds was determined. Their anti-DPPH activity was also determined. The radicalscavenging effect of the EC:catechin, EGC or quercetin combination and the EGC:ASDB or 2-ME combination at molar ratio 1:1 were investigated. The EGC:quercetin, ASDB or 2-ME combinations showed prooxidative effects. The synergistic, additive and cancelling (prooxidative) radicalscavenging effects of the combination were discussed. | v3-fos-license |
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} | pes2o/s2orc | Cytotoxic Effect of Methanol Extracts and Partitions of Two Mexican Desert Plants against the Murine Lymphoma L 5178 YR
Pachycereus marginatus (DC.) Britton & Rose and Ibervillea sonorae (S. Watson) Greene have been used in the Mexican traditional medicine for the treatment of various diseases, including cancer. The present study aims to investigate the cytotoxic activity of these plants against a murine lymphoma. Soxhlet extraction of dried and powdered plant material was performed with methanol. Also, a further partitioning of these methanolic extracts with hexane and ethyl acetate was achieved. The in vitro cytotoxic activity against the murine lymphoma L5178Y-R cell line was assessed via the colorimetric MTT assay. The methanol extract from P. marginatus exhibited high cytotoxic activity (up to 94%) at concentrations ranging from 3.9 to 500 μg/mL; however, hexane and ethyl acetate partitions from this methanolic extract showed lower but significant (p < 0.05) concentration-dependent cytotoxicity (hexane partition up to 94% at 500 μg/mL; ethyl acetate partition up to 94% at 65.5 μg/mL). The methanolic extract and partitions derived from I. sonorae also showed significant (p < 0.05) and concentration-dependent cytotoxicity against L5178Y-R cells at concentrations ranging from 7.81 to 500 μg/mL (methanolic extract up to 63% at 500 μg/mL; hexane partition up to 76% at 250 μg/mL; ethyl acetate partition up to 73% at 500 μg/mL). These results demonstrate that the methanol extracts and partitions from P. marginatus and I. sonorae possess significant cytotoxic activity against the murine lymphoma L5178Y-R and validate the ethnobotanical use of these plants for the treatment of diseases consistent with cancer symptomatology. Previous scientific reports describe the isolation of isoquinoline alkaloids of P. margiCorresponding author. R. Quintanilla-Licea et al. 1522 natus as well as cucurbitacins from I. sonorae, phytochemicals that could be responsible for their observed cytotoxic activity in this research. The direct extraction with methanol of medicinal plants allows extracting of both high and low-polarity compounds, contrary to the simple extraction with water that only allows obtaining compounds of high polarity. The subsequent partition of the methanol extract with a solvent of low polarity (hexane) and another of medium polarity (ethyl acetate) allows making a preliminary fractionation of the bioactive molecules present in the plant that will facilitate the bioguided chromatographic isolation of the pure compounds responsible for the biological activity of the plant.
Introduction
Cancer is a worldwide disease that is one of the leading causes of death [1].In 2012 there was reported an estimate of 14.1 million adults diagnosed with cancer around the world and 8.2 million of deaths from this condition in the same year [2].Its ascending frequency and global distribution, ignoring gender or age, encourage researchers to seek new alternatives that contribute to actual anticancer therapies [3].
As in other developing countries, cancer incidence and mortality have been on the rise in Mexico [4], where a large section of the population uses herbal medicines for the treatment of several diseases [5].Three hundred plant species belonging to 90 botanical families used for cancer treatment have been recorded in Mexico, of which only 181 have been experimentally analyzed.The remaining 119 plant species are being used in the empirical treatment of diseases consistent with cancer symptomatology [6].
Some studies have reported the use of aqueous extracts of Cactaceae and Cucurbitaceae, recollected in desert areas of North Mexico, for the treatment of malignant and benign tumors.The patients received phytotherapy as only causal treatment.This therapeutic approach has demonstrated no adverse reactions or clinical and laboratory events, improving the quality of life and survival [7].
The aim of this work is to evaluate the possible cytotoxic activity of Pachycereus marginatus (DC.)Britton & Rose (cactacea) and Ibervillea sonorae (S.Watson) Greene (cucurbitacea) against the L5178Y-R murine lymphoma.These plants are commonly found in popular markets in Mexico, and their ethnobotanical use includes the treatment of cancer [6], but until now there are no sufficient scientific studies available related to their use against said disease.
Pachycereus marginatus
Pachycereus marginatus (Figure 1(a)) is a columnar cactus known in Mexico as "Órgano, Chilayo or Orégano de zopilote" [8] [9], and it is used popularly as living fence.Flowers and fruits are edible.From the stems a dye is prepared for hair coloring, giving an intense black color [10].The bark is used for the treatment of kidney and bladder problems in the form of poultices placed at the height of the affected region.The sap of the plant is rubbed on the skin in case of loss of hair, skin infections and dryness problems.It is also used as a disinfectant and for the healing of wounds [11].P. marginatus is often confused with Lophocereus schottii (Engelm.)Britton & Rose, species used by the Mayos tribe of the Mexican State of Sonora for the treatment of cancer [12] [13].
Ibervillea sonorae
Ibervillea sonorae (Figure 1(b)), popularly called in Mexico "Guareque, Wareque, Wareke, Guereque or Wereke", is an interesting Cucurbitaceae because of the rapid change in its ethnobotanical use [14] [15].The root of this plant was considered an effective therapeutic resource in dermatological care in the indigenous area mayoyoreme in the Mexican State of Sinaloa and oral ingestion was not advisable, as being a plant with an extremely bitter taste and cathartic activity [16].As of the nineties of the last century the use of the Wereke for the treatment of diabetes is being reported, initially in the natural distribution area of the species and by the time in other Mexican regions [17].
Preparation of Plant Extracts
Vegetal material of the selected plants was cleaned, dried at room temperature and powdered.The extraction was carried out with methanol as unique solvent.188 g of P. marginatus were extracted with methanol, using a Soxhlet equipment for 40 h (4 portions of ca.47 g, each charge with 600 mL solvent).After filtration, the solvent was removed under reduced pressure.With the same procedure, 52 g of I. sonorae were extracted with 600 mL methanol.These methanolic extracts were submitted to a partition with n-hexane and ethyl acetate as described by Pérez-Castorena et al. [18].Each extract was dissolved in 200 mL methanol in a 500 mL separating funnel.Then, 200 mL hexane was added and stirred vigorously.After the formation of the two immiscible phases, the hexane phase (upper phase) was separated from the methanolic solution.This process was performed in triplicate.Finally, the hexane partition was concentrated under reduced pressure.The recovered methanolic phase was concentrated up to a volume of approximately 50 mL and was dissolved in 150 mL of distilled water.This aqueous methanol solution was transferred to a 500 mL separating funnel, added 200 mL of ethyl acetate and stirred vigorously.After the formation of the two immiscible phases, the ethyl acetate phase (lower phase) was separated.This process was performed in triplicate.The recovered ethyl acetate partition was concentrated in vacuum.Yields are shown in Table 1. 10 milligrams of each extract was dissolved in 10% aqueous ethanol to give a solution stock of 1 mg/mL.These solutions were sterilized by filtration using sterile 0.22 µm pore size filters (Merck Millipore, Co.) and submitted to cytotoxic assays.
Reagents, Culture Media and Cell Lines
Penicillin-streptomycin solution, L-glutamine, and RPMI 1640 were obtained from Life Technologies (Grand
Effect of Extracts on Murine Tumor Cell Growth
L5178Y-R is a cell line derived from a lymphoma induced in female DBA/2 mice treated with 3-methylcholanthrene and has been maintained by passes in vivo in its syngeneic host.The cells are resistant to X-rays but sensitive to UV radiation, also are sensitive to antineoplastic drugs, so they are ideal for cytotoxic activity assessments [19].
To determine the direct in vitro cytotoxic effect of the extracts, exponentially growing lymphoma L5178Y-R cells [20] were plated at 5 × 10 4 cells/mL in flat-bottomed 96-well plates (Becton Dickinson, Lincoln Park, NJ) in 100 µL of complete RPMI 1640 medium.These tumor cell cultures were then incubated for 44 h at 37˚C in 5% CO 2 in the presence of different concentrations, ranging from 3.9 to 500 µg/mL of the extracts in a volume of 100 µL.The extracts were dissolved in 10% ethanol as explained previously, and vehicles were tested using the same concentration of the solvent but without the extract.Vincristine was used as positive control.After incubation, 15 µL of MTT (0.5 mg/mL final concentration) were added to all wells, and cultures were additionally incubated for 4 h.Next, the plates were decanted and added 80 µL of DMSO to each well.Optical densities were then read in a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) at 570 nm.The percentage of cytotoxicity was calculated as follows:
Statistics
Results are expressed as mean ± SEM of the response of 3 replicate determinations per treatment.Level significance was assessed by Dunnet's t-test.P < 0.05 was considered significant.
Results
The in vitro cytotoxic effect of methanolic extracts and partitions of the Mexican plants Pachycereus marginatus (DC.)Britton & Rose and Ibervillea sonorae (S.Watson) Greene against lymphoma L5178Y-R cells was evaluated.
As shown in Figure 2, the crude methanol extract derived from P. marginatus revealed a remarkable cytotoxic activity against the L5178Y-R cell line with an inhibition up to 92% at the lowest concentration tested (3.9 µg/mL), and it kept this high activity up to the largest concentration of 500 µg/mL.The partitions derived from the methanolic extract of P. marginatus showed lower cytotoxicity at the lowest concentration tested (hexane up to 16%; ethyl acetate up to 7%).On the other side these partitions showed significant (p < 0.05) and concentration-dependent cytotoxicity against L5178Y-R cells at concentrations ranging from 7.81 to 500 µg/mL (hexane partition up to 94% at 500 µg/mL; ethyl acetate partition up to 94% at 65.5 µg/mL).The partitions from P. marginatus showed lower cytotoxic activity than the original methanolic extract at the same concentrations.
The methanolic extract of I. sonorae (Figure 3) demonstrated a little activity, with an inhibition of 6% at 3.9 µg/mL, but on the other side showed significant (p < 0.05) and concentration-dependent cytotoxicity against L5178Y-R cells at concentrations ranging from 7.81 to 500 µg/mL (up to 63% at 500 µg/mL).Hexane partition derived from the methanolic extract of I. sonorae did not show cytotoxic activity against the L5178Y-R cell line at the lowest concentration tested (3.9 µg/mL), whereas the ethyl acetate partition showed 31% inhibition at this concentration.Both partitions showed significant (p < 0.05) and concentration-dependent cytotoxicity against L5178Y-R cells at concentrations ranging from 7.81 to 500 µg/mL (hexane partition up to 76% at 250 µg/mL; ethyl acetate partition up to 73% at 500 µg/mL).The partitions from I. sonorae showed higher cytotoxic activity than the original methanol extract at the same concentrations.
Discussion
Medicinal plants have played a significant role in the discovery and development of new therapeutic drugs [21].In the last three decades the interest in phytochemicals and their possible applications in the pharmaceutical industry has been reborn [22] [23].Nowadays, approximately 30% of drugs used in industrialized countries come
Methanol
Hexane partition EtOAc partition from plants or are derived from plant secondary metabolites [24].Some plants have proven to be an important source of anticancer compounds [25].A successful case of natural anticancerous agent is Paclitaxel (Taxol), a diterpenoid isolated from the bark of Taxus brevifolia [26].
Pachycereus marginatus
According to the results mentioned above, and taking into account that Vincristine control caused up to 81% cytotoxicity against L5178Y-R cells at a concentration of 31.25 µg/mL (data not shown), the crude methanol extract of Pachycereus marginatus (DC.)Britton & Rose revealed a substantial cytotoxic effect against the murine lymphoma L5178Y-R (94% cytotoxicity at 31.25 µg/mL) (Table 2).As shown in Figure 2, in general, the partitions derived from the methanol extract of P. marginatus showed lower cytotoxic activity than the original extract at the same concentrations.At low concentrations (3.9 to 15.62 µg/mL) the hexane partitions showed higher cytotoxicity (up to 37%) than the ethyl acetate partitions, but starting from 31.5 µg/mL this situation was reversed and the partitions of ethyl acetate significantly increased their cytotoxic against the murine lymphoma L5178Y-R (up to 94%) with respect to the hexane partitions.At 31.25 µg/mL, no partition of P. marginatus surpassed the cytotoxic activity of Vincristine control (hexanepartition up to 35%; ethyl acetate partition up to 53%; Vincristine up to 81%).This relationship between cytotoxicity and concentration of methanol extract and partitions of P. marginatus suggests that the plant possesses active compounds of a wide range of polarities and that they work synergistically to confer its high activity to the crude methanolic extract, even at the lowest concentrations tested (3.9 to 31.25 µg/mL).The Cactaceae family comprises more than 1500 species, but until recently only a few of them have been tested for their chemopreventive and anticancer attributes [27].Mexico has the greatest richness of these plants, with 913 taxa, 80% of which are endemic to the country [28].It has been reported the biological activity of aqueous extracts of Lophocereus schottii, a cactus phylogenetic related to P. marginatus [29], against bacteria of medical importance and the human cervical cancer HeLa cell line [30].Orozco-Barocio et al. [31] validated that the ethanolic extract of L. schottii had an effect on L5178Y murine cells lymphoma.There is to date no report of similar biological activity of P. marginatus.
As said before, phylogenetic studies have shown that P. marginatus is highly related to Lophocereus schottii, and their relationship became more evident due to similar results on chemotaxonomic experiments.Similar alkaloids have been isolated from both cacti [32] [33].Cactus alkaloids are of simple chemical constitution.They are either substituted β-phenylethylamines, tetrahydroisoquinolines or 1-methyltetrahydroisoquinolines [34].The alkaloids of L. schottii and P. marginatus, however, are unique among the isoquinoline alkaloids by having an isobutyl group at C-1 [35], e.g.lophocerine and pilocereine, the last one being trimeric and is presumed to arise from lophocerine [36].
Isoquinoline alkaloids isolated from different natural sources have shown important cytotoxic and anti-carcinogenic activities [37] [38].Lophocerine and pilocereine (compounds of high polarity), both found in L. schottii and P. marginatus, might be responsible for the cytotoxic activity of these cacti.
Ibervillea sonorae
Ibervillea sonorae (S.Watson) Greene biological results reveal a lower cytotoxicity in comparison to the cactus, but important as well (38% cytotoxicity at 31.25 µg/mL; Vincristine control up to 81% at the same concentration) (Table 3).As shown in Figure 3, in general, the partitions derived from the methanol extract of I. sonorae showed higher cytotoxic activity than the original extract at the same concentrations.At low concentrations (3.9 to 7.81 µg/mL) the ethyl acetate partitions showed higher cytotoxicity (up to 38%) than the hexane partitions, but starting from 15.62 µg/mL this situation was reversed and the partitions of hexane significantly increased their cytotoxic activity (up to 67%) with respect to the ethyl acetate partitions.At 31.25 µg/mL, no partition of I. sonorae surpassed the cytotoxic activity of Vincristine control (hexanepartition up to 56%; ethyl acetate partition up to 37%; Vincristine up to 81%).These results might indicate that the active compounds in I. sonorae are those of low and medium polarity.Cucurbitaceae, commonly known as cucurbits or gourds, are a family of 95 genera in 15 tribes comprising 940 to 980 species that are essentially distributed in the tropical and subtropical regions, with hotspots of diversity in Southeast Asia, West Africa, Madagascar, and Mexico [39].There are about 148 species of Cucurbitaceae in Mexico [40].Although the roots and the fruits of these Cucurbitaceae species are very bitter, they have been used as folk medicines in some countries because of their wide spectrum of pharmacological activities such as anti-inflammation and anticancer effects.
Ibervillea sonorae has been used mainly to treat diabetes due to its hypoglycemic activity [41]- [43].Ruiz-Bustos et al. [44] evaluated the antimicrobial activity of I. sonorae against fungi as well as Gram-positive and Gram-negative bacteria.The cytotoxicity of aqueous extracts of I. sonorae against human cervix cancer and human breast cancer has been investigated [45], and a recent study showed that the methanol extract and hexane and ethyl acetate fractions of I. sonorae exhibit potent anti-proliferative activity against HeLa, M12AK.C3F6, A549, and RAW 264.7 cancer cell lines [46].Nevertheless, its cytotoxicity against L5178Y-R has never been established up to now.
During the last decades, a large number of cucurbitacins have been isolated from various plant species belonging to the Cucurbitaceae.More than 50 cucurbitacins have been identified, and they exhibit a wide variety of biological activities that include, but are not limited to, cytotoxicity, antiproliferation, anti-inflammation, antioxidant, antihepatotoxicity, antibacterial and antiviral properties, as well as antimetastatic properties and improved anticancer activity when combined with current chemotherapies [47]- [49].In the last ten years, cucurbitacins have shown to inhibit proliferation and induced apoptosis utilizing an extended array of in vitro and in vivo cancer cell models [50] [51].
From I. sonorae many cucurbitanes and cucurbitane-type glycosides have been isolated, e.g.Kinoins A-C [46] [52]- [54] and these cucurbitacins (compounds of medium polarity) might be responsible for the cytotoxic activity of I. sonorae observed in this research.
Conclusion
The studies presented in this article unveil the extraordinary therapeutic potential of two Mexican desert plants.
The results demonstrate that the methanol extracts and partitions from P. marginatus and I. sonorae possess cytotoxic activity against the murine lymphoma L5178Y-R and may validate the ethnobotanical use of these plants for the treatment of diseases associated with cancer [7].Cancer prevention or chemotherapy based on extracts or pure compounds derived from desert plants with known cancer-inhibiting properties might lead to promising alternatives to current cancer therapy.The cytotoxic activity of these plants could arise due to the occurrence of isoquinoline alkaloids in P. marginatus as well as cucurbitacins in I. sonorae, as is described in the scientific literature.Whereas single targeted chemotherapeutic drugs commonly lack efficacy and invoke drug resistance and adverse effects in cancer patients, traditional herbal medicines are seen as bright prospects for treating complex diseases, such as lymphoblastic leukemia, in a systematic and holistic manner [55]- [57].Further studies on the mechanisms of biological effects by which the extract exerts their cytotoxic effects are necessary.Given the high increase in the main types of cancer, it also needs to intensify studies on the capabilities of desert plants for the combat of this disease.
Figure 2 .
Figure 2. Effect of Pachycereus marginatus (DC.)Britton & Rose methanolic extract and its partitions over lymphoma L5178Y-R growth.The results are expressed as % of cytotoxicity.Data are mean ± SEM of triplicate cultures.Vincristine was used as positive control (data not shown).* p < 0.05 was considered significant.
Figure 3 .
Figure 3.Effect of Ibervillea sonorae (S.Watson) Greene methanolic extract and its partitions over lymphoma L5178Y-R growth.The results are expressed as % of cytotoxicity.Data are mean ± SEM of triplicate cultures.Vincristine was used as positive control (data not shown).* p < 0.05 was considered significant.
/mL) Direct methanol extraction of I. sonorae
Table 2 .
Effect of P. marginatus methanolic extract and its partitions on L5178Y-R cells toxicity.
*Effect of Pachycereus marginatus (DC.)Britton & Rose methanolic extract and its partitions over lymphoma L5178Y-R growth.The results are expressed as% of cytotoxicity.Data are mean ±SEM of triplicate cultures.Vincristine was used as positive control (data not shown).* p < 0.05, ** p < 0.01.
Table 3 .
Effect of I. sonorae methanolic extract and its partitions on L5178Y-R cells toxicity. | v3-fos-license |
2017-12-13T19:04:07.951Z | 2017-12-13T00:00:00.000 | 39156233 | {
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} | pes2o/s2orc | Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells
Outer membrane protein 25 (OMP25), a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK) signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308) and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10) were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK), and Jun-N-terminal kinase (JNK) from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.
Outer membrane protein 25 (OMP25), a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK) signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308) and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10) were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK), and Jun-N-terminal kinase (JNK) from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.
There are three groups of major outer membrane proteins (Omps) in Brucella (7). Group 1 Omps consist of two major Omps: Omp10 and Omp19. Group 2 Omps consist of two major Omps: Omp2a and Omp2b. Group 3 Omps consist of two major Omps: outer membrane protein 25 (Omp25) and Omp31. Omp25 was a primary protein that was released by Brucella when it invaded host cells (8). Omp25 was involved in attachment or invasion to the host cells and intracellular survival or reproduction of Brucella, which plays an important role in Brucella virulence. Omp25 mutant was attenuated in animals (9,10). Therefore, Omp25 is an important virulence factor of Brucella.
Mitogen-activated protein kinase (MAPK) is one signal transduction pathway in organisms. It is associated with many profiles and processes of the cell, such as auxesis, development, proliferation, differentiation, and apoptosis (11). MAPK includes four subfamilies: p38, ERK1/2, Jun-N-terminal kinase (JNK), and ERK5 (12). MAPK is implicated in bacterial pathogenesis as demonstrated by the induction of inhibition of ERK1/2 and p38 branches during infection with Salmonella typhimurium (13), Yersinia (14,15), Listeria monocytogens (16,17), and Mycobacterium (18). In the inflammatory response, MAPK signal pathway can mediate secretion of IL-8, interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and other cytokines by epithelial cells (19). In this report, we used S2308 and 2308ΔOmp25 to infect HPT-8 cells and mice, and the expression of cytokines were detected. In addition, we analyzed the effects of Omp25 on the MAPK signal pathway, with the aim to understand the function of Omp25 in the pathogenesis of Brucella.
Mice
Six-week-old BALB/c female mice were obtained from the Experimental Animal Center of the Academy of Military Medical Science (Beijing, China). Animals were maintained in barrier housing with filtered inflow air in a restricted-access room in pathogen-limited conditions. All experimental procedures and animal care were performed in compliance with institutional animal care regulations. And all experimental procedures and animal care were performed in Biosafety Level 3 Laboratory.
Brucella cell infection assay
HPT-8 cells were infected with S2308 and 2308ΔOmp25, as previously described (20). The bacteria for infection studies were prepared before the experiment was executed. S2308 and 2308ΔOmp25 were cultured in TSB at 37°C with 5% CO2 (vol/vol) until logarithmic growth phase. Then, 2 × 10 6 cells/ well were cultured in 6-well plates for 24 h at 37°C under 5% CO2, and then infected with S2308 or 2308ΔOmp25 at a multiplicity of infection (MOI) of 100 bacteria per cell. Culture plates were centrifuged at 350 × g for 5 min at room temperature. At 45 min post-infection, the cells were washed thrice with medium without antibiotics and then incubated with 50 µg/mL of gentamicin (Invitrogen, Carlsbad, CA, USA) for 1 h to kill extracellular bacteria. Afterward, the medium was removed and replaced with fresh DMEM with 10% FBS containing 25 µg/mL gentamicin (defined as time 0). Uninfected cells were used as control.
Detection of cytokines
HPT-8 cells were infected with S2308 and 2308ΔOmp25 accor ding to the above description.
Determination of MaPK Branches associated with the secretion of TnF-α
To confirm the MAPK signal pathway associated with the secretion of TNF-α, HPT-8 cells were pre-treated with p38 inhibitor (10 µM), or JNK inhibitor (10 µM) at 37°C for 1 h and then infected with S2308 or 2308ΔOmp25 at a 100:1 MOI according to the above description. At 4, 8, 24, and 48 h post-infection, supernatant was collected and measured the levels of TNF-α according to the above description. All assays were performed three times.
Western Blotting analysis
HPT-8 cells were infected with S2308 and 2308ΔOmp25 according to the above description. The activation of p38, ERK1/2, and JNK was detected in infected cells, as previously described (21). Briefly, at 24 h post-infection, supernatant was discarded and cells were lysed in ice-cold Radio Immunoprecipitation Assay Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min, then centrifuged at 16,000 × g for 30 min at 4°C. The supernatant was collected and concentration was detected with BCA protein assay kit (Sangon Biotech, Shanghai, China). 500 µg protein samples separated by 12% SDS-PAGE and electro-transferred to a nitrocellulose membrane using a Mini Trans-Blot Cell (Bio-Rad, Hercules, CA, USA) at 200 mA for 1 h. Unbound sites on the membrane were blocked in 5% nonfat milk in Tris-buffered saline Tween-20 (TBST) buffer for 1 h at room temperature. Then, the membrane was washed three times with TBST buffer and incubated with rabbit anti-human anti-p38,
Brucella infection in Mice and cytokine Measurement
BALB/c mice were infected with Brucella as previously described (22). Briefly, 6-week-old BALB/c female mice (n = 5 per group) were randomly divided into three groups. Group Tissue specimens BALB/c mice (n = 25 per group) were inoculated with S2308 and 2308ΔOmp25 according to the above description. At 2, 4, 6, 8, and 10 weeks post-immunization, mice were euthanized and uteruses were removed aseptically. The uteruses were collected, formalin-fixed, and paraffin-embedded, as previously described (24).
immunohistochemistry
The immunohistochemistry assay was performed as previously described (25). Briefly, the embedded paraffin will be serial sectioned by slicer, with a thickness of 5 µm and mounted on slide glasses coated with poly-l-lysine (Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, the tissue sections were routine deparaffinized and dehydrated with xylene and ethanol. After three times washed with distilled water, the tissue sections were microwaved in 10 mM citrate buffer (pH 6.0; Sigma-Aldrich, MO, USA) at 95°C for 10 min for antigen retrieval and naturally cooled at room temperature. Then, the endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide buffer (Sigma-Aldrich, MO, USA) for 10 min at room temperature. The tissue sections were incubated overnight at 4°C with primary antibody (rabbit anti-mouse anti-p38 or anti-ERK pAb;
statistical analysis
Cytokine production was expressed as the mean cytokine concentration ± SD. Statistical analysis was performed with Student's unpaired t-test. The differences between groups were and TNF-α levels were assayed by ELISA. TNF-α production is expressed as the mean cytokine concentration ± SD for each group of mice. Significant differences between the S2308 and 2308ΔOmp25 are indicated by * (P < 0.05). OMP25, outer membrane protein 25; TNF-α, tumor necrosis factor-α; PBS, phosphate-buffered saline.
resUlTs 2308ΔOmp25 induces lower levels of cytokines
To detect the expression level of cytokines, we collected supernatant from HPT-8 cells infected with S2308 and 2308ΔOmp25 and then monitored expression levels of cytokine TNF-α, IL-1, and IL-10 by ELISA. Supernatant from HPT-8 cells infected with S2308 produced higher amounts of TNF-α (Figure 1A), IL-1 (Figure 1B), and IL-10 ( Figure 1C) than did supernatant from uninfected cells (P < 0.05) and this difference increased with time. Slightly higher cytokine production levels were observed in 2308ΔOmp25-infected cells than in control cells (Figure 1), but there was no significant difference between 2308ΔOmp25 group and control group (P > 0.05).
expression of TnF-α associated with p38 Branch
We next evaluated which branch associated with secretion of TNF-α. HPT-8 cells were pre-incubated for 1 h with 10 µM p38, 10 µM ERK, or 10 µM JNK inhibitors and then infected with S2308 or 2308ΔOmp25 for 4, 8, 24, and 12 h. The levels of TNF-α were assessed in the supernatants of the S2308 or 2308ΔOmp25-infected p38 inhibitor, ERK inhibitor or JNK inhibitor-treated cells. At 4,8,24, and 12 h, the S2308 or 2308ΔOmp25-infected p38 inhibitor-treated cells produced higher levels of TNF-α than S2308 or 2308ΔOmp25-infected cells (P < 0.05; Figure 2A). However, there was no significant difference between S2308 or 2308ΔOmp25-infected ERK inhibitor and JNK inhibitor-treated cells and S2308 or 2308ΔOmp25infected cells (P > 0.05; Figures 2B,C). These results showed that p38 branch could induce secretion of TNF-α in S2308 or 2308ΔOmp25-infected cells.
2308ΔOmp25 activates Weak MaPK Pathway
To assess activation of p38 and ERK1/2 kinases, HPT-8 cells were infected with the 2308ΔOmp25 mutant and the parental strain S2308 at a MOI of 100. We found that at 24 h post-infection, the activation process triggered by S2308 resulted in a phosphorylation of the p38, ERK1/2, and JNK kinases (Figure 3). Infections with 2308ΔOmp25 induced a markedly weaker stimulation of p38, ERK1/2, and JNK kinases, with S2308 demonstrating a slightly higher capacity of activation than 2308ΔOmp25 mutant (Figure 3). These results show that OMP25 involved in activating of MAPK pathway.
2308ΔOmp25 induces lower levels of TnF-α in Peripheral Blood of Mice
To detect the expression level of TNF-α in animal, we collected sera from mice inoculated with S2308, 2308ΔOmp25, or PBS and then measured expression levels of TNF-α by ELISA. Serum samples from mice inoculated with S2308 produced higher amounts of TNF-α (Figure 4) than did serum samples from mice inoculated with 2308ΔOmp25 or PBS (P < 0.05) and this difference increased with time. Slightly higher cytokine production levels were observed in 2308ΔOmp25 immunized mice than in PBS immunized mice (P > 0.05) (Figure 4). Total TNF-α levels increased with time.
immunohistochemical staining
The immune complexes of p38 and ERK phosphorylation proteins were located in the cytoplasm of the uterus tissues, and they were strongly stained as tan or brownish yellow. The phosphorylation proteins in p38 and ERK signal pathways had been detected and found in the uterus tissues of S2308 immunized mice (Figures 5A and 6A). But the immune complexes of phosphorylation proteins in p38 and ERK signal pathways were weakly stained in the uterus tissues of 2308ΔOmp25 immunized mice (Figures 5B and 6B). These results showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK.
DiscUssiOn
Brucella could infect many kinds of cells, but the main host cells are macrophages and trophoblasts (26). In animals, abortion is associated with a rapid proliferation of Brucella within the placenta. Trophoblasts are primary cellular targets for Brucella in the natural host. The presence of high bacterial loads within placental trophoblasts ultimately results in disruption of the placenta and infection of the fetus. Omps of Brucella play an important role in the process of pathogen (27). Omp25 an important Omp, it involved in growth, colonization, and proliferation of Brucella (28). The Omp25 mutant strain attenuated Brucella infection abilities and changed the response of host cells (10). TNF-α is one of important factors that involved in many of the body's immune and inflammatory responses (29). The expression of TNF-α was related with Omp25 and ERK pathway (27). Our results found that there was a significant difference in the expression of TNF-α between S2308 and 2308ΔOmp25. These results suggested that Omp25 may play an important role in the progress of expression of TNF-α when Brucella infected cells. Phosphorylation of MAPK p38 pathway acts as a "switch" role in regulating the production of cytokines. The whole process is through a typical pathway: MAPKKK → MAPKK → MAPK (12). SB208035 is the inhibitor of p38 signal pathway. It has been reported that p38 inhibitors could inhibit the production of IL-1, IL-10, and TNF-α in peripheral blood cells (30). TNF-α could activate p38 pathway (31). Our results showed that 2308ΔOmp25 was weaker to activate p38 pathway in MAPK signal pathway than S2308. The reason may be the low expression of TNF-α in 2308ΔOmp25. It suggested that the production of TNF-α may be related with the activation of p38 pathway. When we used p38 inhibitors to deal with HPT-8 cells, and detected the expression of TNF-α in the culture medium. We found that the expression of TNF-α has been inhibited. It suggested that when S2308 or 2308ΔOmp25-infected HPT-8 cells, the p38 pathway in the MAPK signal pathway was related with the expression of TNF-α.
Brucella can lead many organs happening pathological damages, particularly in chronic infection stage. In the experiment, we found that the expressions of TNF-α significantly increased when S2308 or 2308ΔOmp25-infected mice or HPT-8 cells. It showed that TNF-α was one of cytokines that happened significantly change when Brucella infected hosts (organisms and cells), and it may be related to the progress of inflammation. From the result of immunohistochemistry, we found that phosphorylation proteins of p38 and ERK signal pathway in uterine tissues of mice. In addition, we also found that the immune complexes of phosphorylation proteins in p38 and ERK signal pathways were weakly stained in the uterus tissues of 2308ΔOmp25 immunized mice. These results suggested that Omp25 participated in phosphorylation of p38 and ERK signal pathway proteins. Previous studies have reported that the MAPKs are a target for immune intervention by virulent smooth Brucella (32). Our results further suggest that Omp25 played an important role in activating MAPK signal pathway in smooth Brucella.
In conclusion, we found that Brucella can affect the expression of TNF-α by activating MAPK signal pathway, and the expression was higher in S2308 than 2308ΔOmp25. It suggested that Omp25 played an important role in activating MAPK signal pathway when Brucella infected hosts. These results established theoretical foundation for further studying pathogenic mechanisms and proinflammatory mechanisms of Brucella.
eThics sTaTeMenT
The study was approved by the Institutional Committee of Post-Graduate Studies and Research at Shihezi University, China (No. 2012-9). All efforts were made to minimize animal suffering. | v3-fos-license |
2020-06-26T14:48:36.258Z | 2020-06-25T00:00:00.000 | 220058956 | {
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} | pes2o/s2orc | Deficiency of Nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 reduces atherosclerosis
Atherosclerosis is crucially fueled by inflammatory pathways including pattern recognition receptor (PRR)-related signaling of the innate immune system. Currently, the impact of the cytoplasmic PRRs nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2 is incompletely characterized. We, therefore, generated Nod1/Nod2 double knockout mice on a low-density lipoprotein receptor (Ldlr)-deficient background (= Ldlr−/−Nod1/2−/−) which were subsequently analyzed regarding experimental atherosclerosis, lipid metabolism, insulin resistance and gut microbiota composition. Compared to Ldlr−/− mice, Ldlr−/−Nod1/2−/− mice showed reduced plasma lipids and increased hepatic expression of the scavenger receptor LDL receptor-related protein 1 after feeding a high-fat diet for 12 weeks. Furthermore, intestinal cholesterol and its bacterial degradation product coprostanol were elevated in Ldlr−/−Nod1/2−/− mice, correlating with the increased abundance of Eubacterium coprostanoligenes as assessed by 3rd generation sequencing of the gut microbiota. Atherosclerotic plaques of Ldlr−/−Nod1/2−/− mice exhibited less lipid deposition and macrophage accumulation. Moreover, macrophages from Ldlr−/−Nod1/2−/− mice showed higher expression of the cholesterol efflux transporters Abca1 and Abcg1 and accordingly reduced foam cell formation. Deficiency of Nod1 and Nod2 led to reduced plaque lipid deposition and inflammatory cell infiltration in atherosclerotic plaques. This might be explained by diminished plasma lipid levels and foam cell formation due to altered expression of key regulators of the hepatic cholesterol pathway as well as differential intestinal cholesterol metabolism and microbiota composition. Electronic supplementary material The online version of this article (10.1007/s00395-020-0806-2) contains supplementary material, which is available to authorized users.
Introduction
Elevated levels of blood cholesterol and vascular inflammation are considered as the primary triggers of cardiovascular disease due to atherosclerosis, a chronic disease of arteries [15]. Subsequent fatal events such as myocardial infarction are still responsible for the most illness-related fatalities worldwide [28]. There is an ongoing debate on the weighting of trigger mechanisms or whether they equally contribute to the initiation and progression of atherosclerosis. In this regard, cholesterol-lowering strategies, such as statins, are widely used as primary as well as secondary prevention in patients with imminent or apparent atherosclerotic cardiovascular disease since the 90s of the past century [1]. Various pleiotropic effects-including anti-inflammatory effects-of statins have been reported; 47 Page 2 of 12 however, cholesterol-lowering is regarded as their principle mechanism of action. Although the inflammatory nature of atherosclerosis is known for quite some time, the first successful anti-inflammatory therapy for atherosclerotic disease using a monoclonal antibody targeting interleukin (IL)-1β has been reported just in 2017 [27].
Nucleotide-binding oligomerization domain-containing protein (NOD) are pattern-recognition receptors (PRR), and NOD1 and NOD2, previously known as caspase activation and recruitment (CARD) domain family member CARD4 and CARD15, represent the founding members of the NODlike receptor family. Different to other membrane bound PRRs, they operate as soluble cytosolic receptors recognizing conserved bacterial peptidoglycan fragments (muropeptides) in order to initiate an adequate immune response [8,25]. Expression of Nod1 and Nod2 has not only been shown in immune cells such as macrophages but also in vascular cells such as endothelial cells and smooth muscle cells [19,30]. Both NOD1 and NOD2 interact with the adapter protein receptor-interacting serine/threonine-protein kinase 2 and subsequently share a common downstream signaling cascade involving nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathways [12]. Important roles for NOD1 and NOD2 in various experimental disease models have been identified in recent years including their involvement in the regulation of the intestinal barrier function and the gut microbiota composition [22].
The role of Nod1 and Nod2 in atherosclerosis and insulin resistance has already been investigated in several experimental studies; however, with partially conflicting results. Administration of a NOD1 agonist enhanced atherosclerosis in apolipoprotein E (Apoe)-deficient mice [17], whereas Nod1/Apoe-deficient mice showed reduced atherosclerosis [13,17]. Following antibiotic pretreatment, NOD2 stimulation in addition to an oral gavage with the periodontal pathogen Porphyromonas gingivalis decreased atherosclerosis in Apoe-deficient mice, whereas P. gingivalis alone increased atherosclerosis in Nod2/Apoe-deficient mice [32]. Contrarily, Johansson et al. showed that stimulation with a NOD2 ligand aggravated atherosclerosis in low-density lipoprotein receptor (Ldlr)-deficient mice [16]. In regard to insulin resistance, Schertzer et al. [29] reported that especially NOD1 ligands induce insulin resistance, whereas NOD1/2-deficient mice were protected from high-fat diet-induced insulin resistance. Likewise, high-fat diet (HFD)-induced translocation of intestinal bacteria into adipose tissue and blood as well as insulin resistance is prevented in NOD1-deficient mice [3]. Opposing results were seen by Cavallari et al. [9] NOD2 stimulation reduced insulin resistance, whereas NOD1 stimulation worsened insulin resistance in obese mice without altering the composition of the microbiome.
The partially contradicting outcomes of these studies can potentially be attributed to different experimental settings, e.g. for experimental atherosclerosis different knockout (KO) animal models (Apoe-KO, Ldlr-KO mice, systemic vs. bone marrow-specific KO), with or without antibiotic pretreatment and with or without different NOD ligand or bacterial pathogen administration regimes were used. Besides periodontal pathogens, the gut microbiota have been shown to contribute to atherosclerosis and type 2 diabetes [21]. However, an exogenously applied bacterial load or NOD ligands in experimental models principally mimic severe systemic infection rather than a low-grade bacterial load. The latter presents a chronically evolving cardiovascular risk and more likely resembles the situation in patients. Most studies with NOD-deficient mice used a NOD-single knock-out even though bacterial-derived peptidoglycan motifs mostly activate both NOD receptors. Although NOD1 and NOD2 may be activated by different ligands, compensatory effects are conceivable due to joint down-stream signaling. Hence, in the present study we investigated hypercholesterolemic mice deficient for Nod1 as well as for Nod2 in regard to experimental atherosclerosis, lipid metabolism, insulin resistance and gut microbiota composition.
Glucose tolerance and insulin resistance
Glucose tolerance and insulin resistance tests were performed after 6 and 12 weeks of HFD on two consecutive days. Mice were fasted for 4 h and baseline glucose measurement was taken from a drop of tail vein blood using an OneTouch Ultra2 glucometer (LifeScan Inc. Milpitas, CA). Afterwards, mice were injected i.p. with 1.25 g glucose or 0.75 IU of insulin (Sanofi, Frankfurt, Germany) per kg body weight in a total volume of 200 μL buffered saline (PBS). Following injection, blood glucose was measured over a period of 2 h as stated above.
Plasma lipid analysis
Plasma samples were collected after 4 h of fasting and plasma total cholesterol and triglyceride levels were measured enzymatically using commercially available reagents (Roche Diagnostics, Mannheim, Germany) as reported previously [23]. Plasma samples were subjected to fast protein liquid chromatography gel filtration on an Äkta purifier equipped with a Superose 6 column (GE Healthcare, Little Chalfont, UK). Chromatography was done at a flow rate of 0.5 mL/min and lipoprotein fractions of 500 µL each were collected and assayed for cholesterol concentrations. Individual lipoprotein subclasses were isolated by tabletop sequential ultracentrifugation from 30 µL of plasma using a TLA-120.2 fixed-angle rotor (Beckman Coulter, Woerden, The Netherlands) and a 1.34 g/mL potassium bromide stock solution to adjust densities (densities, VLDL/ IDL: 1.006 < d < 1.019, LDL: 1.019 < d < 1.063, HDL: 1.063 < d < 1.21; each run 3 h at 100,000 rpm). After extensive dialysis against PBS, cholesterol concentrations within individual fractions were determined using a commercial colorimetric assay (Roche Diagnostics).
Fecal sterol analysis
Fecal samples were weighed and aliquots separated into bile acid and neutral sterol fractions as reported previously [10]. Briefly, samples (50 mg each) were first heated for 2 h at 80 °C in 1 mL alkaline methanol with 5α-cholestane added as internal standard and then extracted three times with petroleum ether 60-80. The top layer (ether) was used for neutral sterol analysis and the bottom layer (water) to profile bile acid species. The ether phase was dried under a stream of nitrogen, then a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide, pyridine and trimethylchlorsilan (5:5:0.1) was added followed by drying under nitrogen again; finally, samples were taken up in heptane. The water phase was transferred to Sep-Pak C18 cartridges, flushed out with 75% methanol followed by drying the eluate under a stream of nitrogen, and derivatized as above. Sterol analyses were performed by gas-liquid chromatography (Agilent, Santa Clara, CA) with a Chrompack CPSil19 column, Helium as carrier gas and including calibration curves for all analytes determined (covering a range between 0 and 50 nmol) [10].
Tissue preparation
After 4 h of starvation, blood samples were collected and subsequently mice were killed and aorta was perfused with PBS after opening of the hepatic portal vein. Heart, including aortic root and aortic arch, was dissected after perfusion with PBS. The aortic arch was separated, snap-frozen in liquid nitrogen and the heart including aortic root was embedded in Tissue Tek OCT (Sakura Finetek, Staufen, Germany) for histochemistry and immunohistochemistry. Organs and tissues including liver, cecum, ileum and aortic arch were collected into cryotubes, flash frozen and stored at − 80 °C.
Histochemistry and immunohistochemistry
Atherosclerotic burden was quantified in the aortic root by Oil Red O (Sigma-Aldrich) staining. Within the aortic root, serial cryostat sections (8 μm, CM3050S, Leica Microsystems) at the level of all three cusps were prepared and atherosclerotic lesions were analyzed by Oil Red O staining (2 h at 60 °C) for the measurement of atherosclerotic plaque burden. The remaining sections were used for immunohistochemical analysis of the atherosclerotic plaque composition. Air-dried sections were fixed in ice-cold acetone and stained with antibodies against MOMA-2 (reacts with monocytes/ macrophages, Acris Antibodies GmbH, Herford, Germany) or α-smooth muscle actin (Abcam, Cambridge, UK), visualized by appropriate secondary antibodies (ImmPRESS™ detection reagent, Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin (solution Gill No. 2, Merck KGaA, Darmstadt, Germany). Sirius Red staining was used to visualize collagen content in atherosclerotic plaques. Briefly, tissue sections were incubated with 0.1% Sirius Red F3BA (Sigma-Aldrich) in saturated picric acid for 1 h and then rinsed with 0.01 N HCl for 1 min twice. The sections were then dehydrated with 70% ethanol for 30 s. Structural mature type I collagen appeared bright orange-red in polarized light. Morphometric data on three sections were obtained using a light microscope (DMI3000 B and DM4000 B microscope, Leica Microsystems Wetzlar, Germany) and ImageJ software (National Institutes of Health, Bethesda, MD). The atherosclerotic plaque size was determined by calculating the percentage of the Oil Red O positive area of the total aortic root cross sectional area. MOMA-2, α-smooth muscle cell actin and Sirius Red were expressed as percentage of total plaque area.
Real-time PCR
Total RNA from snap-frozen liver, ileum or bone marrowderived macrophages was isolated using RNA-Solv ® Reagent (Omega Bio-tek, Norcross, GA) following the manufacturer's instructions and reverse-transcribed with SuperScript reverse transcriptase, oligo(dT) primers (Thermo Fisher Scientific, Waltham, MA) and deoxynucleoside triphosphates (Promega, Mannheim, Germany). Real-time PCR was performed in duplicates in a total volume of 20 µL using Power SYBR green PCR master mixture (Thermo Fisher Scientific) or TaqMan Fast Advanced Master Mix (Applied Biosystems, Foster City, CA) on a Step One Plus Real-time PCR system (Applied Biosystems) in 96-well PCR plates (Applied Biosystems). Real-time PCR using SYBR green was performed with an initial denaturation step at 95 °C for 10 min, followed by 40 PCR cycles that consisted of 95 °C for 15 s, 60 °C for 1 min and 72 °C for 10 s. In terms of TaqMan Fast Advanced Master Mix, real-time PCR was performed with an initial holding and denaturation step at 50 °C for 2 min followed by 20 s at 95 °C. The 40 PCR cycles consisted of 95 °C for 1 s and 60 °C for 20 s. SYBR Green and Taq emissions were monitored after each cycle. For normalization, expression of GAPDH was determined in duplicates. Relative gene expression was calculated using the 2 −ΔΔC t method. Real-time PCR primers were obtained from Microsynth AG (Balgach, Switzerland) and sequences are available in the Online supplementary methods.
Cholesterol efflux was determined as published previously [4]. Experiments were performed in triplicates using BMDM. BMDM were plated in 48-well plates (Corning, Corning, NY) and, after reaching confluence, loaded with 3 H-cholesterol (1 μCi/mL, NEN Life Sciences Products, Boston, MA) as well as acetylated LDL (50 µg protein/ mL) for 24 h to generate foam cells. After washing with PBS, cells were equilibrated in RPMI with 0.2% BSA for 18 h. After another washing with PBS, HDL (50 µg protein/mL, isolated from pooled healthy donors by ultracentrifugation) was added as acceptor. After 5 h, radioactivity within the supernatant was determined by liquid scintillation counting (Beckman LS6500, Beckman Instruments, Palo Alto, CA). Next, 0.1 M NaOH was added to the wells, plates were incubated for 30 min at room temperature and the radioactivity remaining within the cells was assessed by liquid scintillation counting. Efflux is expressed as the percentage of counts recovered from the medium in relation to the total counts present on the plate (sum of medium and cells). Values for unspecific efflux determined as release of 3 H-cholesterol from macrophages in the absence of HDL were subtracted from the individual values.
Microbiome analysis
From each mouse, 200 mg cecum was used and DNA was isolated with Stool-lysing Buffer (ASL) (QIAGEN) extracted with Phenol/Chlorophorm/Isomamylalcohol (Roth) and cleaned up with AMPure XP beads (Beckman Coulter, Krefeld, Germany). After concentration measurement and quality control, 16S rRNA-PCR amplification with Nanopore Barcode-Primer was performed (LSK-RAB204) (Oxford Nanopore, Oxford, UK). Afterwards, amplification products were controlled using gel electrophoresis. For sequencing, the sequencing library with a total amount of 100 fmoles PCR ampliconsDNA was prepared according to the instructions of the manufacturer and loaded onto a MinION FlowCell (Oxford Nanopore). In total, 1 × 10 6 sequences per sequencing run were detected. For basecalling Oxford Nanopore Technology's official command-line Albacore enabling FAST5 to FASTQ conversion was used. Afterwards FASTQ files were compiled and converted into FASTA files which were used for comparison with the local blast 16S rRNA database. Analysis was performed using MEGAN6 microbiome analysis tool from Eberhard Karls Universität Tübingen.
Statistical analysis
All data are represented as means ± SD. After normal distribution validation, groups were compared using parametric 2-tailed Student t-test or nonparametric Mann Whitney test as appropriate (GraphPad Prism, version 6.05; GraphPad Software, La Jolla, CA). A value of P < 0.05 was considered statistically significant. Real-time PCR was performed in technical duplicates.
Nod1/2-deficiency lowers plasma cholesterol levels
NOD1-and NOD2-signaling share many similarities in the initiation of inflammatory pathways [12,24]. Accordingly, we observed a comparable induction of inflammatory genes in bone marrow-derived macrophages (BMDM) such as tumor necrosis factor-α (Tnf-α) and interleukin-1β (Il-1β) by the NOD1 ligand l-Ala-γ-d-Glu-meso-diaminopimelic acid (Tri-DAP) and the NOD2 ligand muramyl dipeptide (MDP, Fig. 1a). Previous studies with Nod1-or Nod2-single KO-mice had revealed partially conflicting results in regard to atherosclerosis and insulin resistance [3,9,13,16,17,29,32]. In addition, the use of single Nod-KO mice potentially harbors the risk of compensatory effects by the other NOD member. To address the impact of Nod1 and Nod2 on experimental atherosclerosis and lipid metabolism, we crossed Nod1-and Nod2-deficient mice on the background of Ldlr-deficient mice. These mice are abbreviated as Ldlr −/− Nod1/2 −/− in the following; Ldlr −/− mice served as control. To induce hypercholesterolemic conditions, mice were fed an HFD for 12 weeks. Body weight gain at the end of the feeding period was slightly more pronounced in Ldlr −/− Nod1/2 −/− mice, but did not reach statistical significance (Fig. 1b). Fasting plasma glucose (Fig. 1c) and insulin levels (Fig. 1d) were similar between the groups and we likewise did not observe any effects on glucose tolerance (Fig. 1e) or insulin resistance (Fig. 1f) as possible signs of prediabetes in Ldlr −/− Nod1/2 −/− mice. Next, we analyzed plasma lipid levels as a critical parameter in the development of atherosclerosis. Plasma total cholesterol was significantly decreased in Ldlr −/− Nod1/2 −/− mice. Subsequent sequential ultracentrifugation analysis revealed that Ldlr −/− Nod1/2 −/− mice had significantly lower proatherogenic very-low-density lipoprotein (VLDL) cholesterol levels, whereas low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride (TG) levels were unchanged (Fig. 2a, Online Table 1). To explore the potential underlying mechanism, we next analyzed the hepatic expression levels of key factors for cholesterol and triglyceride metabolism and detected significantly enhanced hepatic expression of scavenger receptor lowdensity lipoprotein receptor-related protein 1 (Lrp1), apolipoprotein A1 (Apoa1), apolipoprotein B (Apob) and metabolic nuclear receptor liver X receptor alpha (Lxrα) in Ldlr −/− Nod1/2 −/− mice (Fig. 2b). However, intrahepatic cholesterol level itself was not altered in Ldlr −/− Nod1/2 −/− mice (Fig. 2c). In accordance with the results from Nod1/2-deficient mice, we found reduced hepatic mRNA expression levels of Lrp1 and Lxrα after injection of the NOD ligands Tri-DAP and MDP into Nod1/2-competent mice, whereas Apoa1 and Apob were not changed (Online Fig. 1).
Nod1/2-deficiency elevates intestinal cholesterol levels and alters intestinal microbiota composition
To identify potential mechanisms of an altered cholesterol metabolism in Ldlr −/− Nod1/2 −/− mice, we additionally investigated feces samples from the cecum after 12 weeks of HFD. Ldlr −/− Nod1/2 −/− mice showed a profound increase in cholesterol and its bacterial degradation products dihydrocholesterol and coprostanol (Fig. 2d). Primary and secondary bile acids were analyzed as well, but did not show any significant differences (Online Table 2).
Nod1/2-deficient mice have reduced atherosclerotic plaque burden and plaque macrophage content
In the Ldlr-deficient mouse model, we expected increased vascular inflammation under HFD conditions compared to chow diet (CD), which we exemplified by increased Tnf-α expression levels in atherosclerotic aortic tissue. Expression levels of Nod1 and Nod2-likewise involved in inflammation-were similar between CD and HFD conditions (Online Fig. 2a). In the liver, expression levels only for Nod1 were similar, whereas Nod2 was significantly enhanced by HFD (Online Fig. 2b). Next we analyzed the atherosclerotic plaque burden after 12 weeks of HFD. Oil Red O staining revealed significantly attenuated lipid deposition in plaques of the aortic root in Ldlr −/− Nod1/2 −/− mice compared to Ldlr −/− mice (Fig. 4a). However, no differences in lipid deposition in the thoracoabdominal aorta were found (Online Fig. 3). Focusing on the aortic root, we investigated the plaque macrophage content, since infiltrating monocytes and their subsequent differentiation to macrophages represent a crucial step for atherogenesis [15]. Staining for the general monocyte/macrophage marker MOMA-2 revealed reduced macrophage accumulation in plaques of Ldlr −/− Nod1/2 −/− mice (Fig. 4b). However, we did not observe any differences in total blood leukocytes between Ldlr −/− and Ldlr −/− Nod1/2 −/− mice (42,700 ± 11,300 counts vs. 45,700 ± 15,600 counts) or total monocytes and their subsets in peripheral blood and bone marrow (Online Fig. 4). Smooth muscle cell content as an indicator of plaque stability [15] was not affected by Nod1/2deficiency (Fig. 4c). In addition, smooth muscle cells are a major source of stabilizing collagens within atherosclerotic plaques [15]. Therefore, we assessed Sirius Red staining by polarized light microscopy, which displays structural mature type I collagen in bright orange/ red and found increased plaque collagen deposition in Ldlr −/− Nod1/2 −/− mice (Fig. 4d). In addition, we investigated pro-inflammatory gene expression in aortic arch tissue. We detected similar mRNA levels for C-C motif chemokine ligand 2 (Ccl2) but a trend towards decreased levels of Tnf-α and Il-6 in Ldlr −/− Nod1/2 −/− mice (Fig. 4e).
L r p 1 A p o a 1 A p o b A p o c 3 A p o e M t t p A b c a 1 A b c g 1 A b c g 5 A b c g 8 L x r S r-b 1
So r t 1 L p l n .s . Fig. 5a). Moreover, we observed a trend towards increased cholesterol efflux in BMDM from Ldlr −/− Nod1/2 −/− mice (Fig. 5c). We likewise observed a trend towards elevated Abca1 and Abcg1 expression levels in the aortic arch, a tissue with a high proportion of plaque macrophages (Online Fig. 5b).
Discussion
High cholesterol levels and comorbidities such as arterial hypertension or type 2 diabetes are pharmacologically targeted for the treatment of atherosclerosis since a while [5,15,26], whereas the inflammatory nature of the disease was targeted for the first time in 2017 [27]. NOD1 and NOD2 are soluble cytosolic immune receptors responsible for the recognition of bacterial peptidoglycan fragments. They share similarities in regard to structure and induction of inflammatory pathways-such as NF-κB and MAPK-in order to initiate inflammatory cytokine expression for immune defense [8,12,24,25]. Based on these similarities, we generated Nod1/Nod2-double KO mice on a Ldlr-deficient background (= Ldlr −/− Nod1/2 −/− ) which showed less lipid deposition and inflammatory cell infiltration in atherosclerotic plaques.
Considering the crucial role of NOD receptors in the initiation of inflammatory pathways, we expected especially an influence of Nod1/2-deficiency on vascular inflammation. However, we did not detect significant changes in Ccl-2, Tnf-α und Il-6 expression in the aortic arch in both groups. Reduction in plaque burden and monocyte content may be mediated by a lipid phenotype in the current study since Ldlr −/− Nod1/2 −/− mice showed reduced cholesterol plasma levels and diminished macrophage foam cell formation. Increased expression of hepatic cholesterol uptake pathways and elevated intestinal cholesterol levels suggested an accelerated cholesterol clearance from the circulation in Ldlr −/− Nod1/2 −/− mice. It fits in the picture that the intestinal abundance of the cholesterol-metabolizing Eubacterium coprostanoligenes, probably due to more favorable growth conditions, and their metabolic product coprostanol were found to be likewise enhanced. Effects of gut microbiota composition have been already associated with cardiovascular disease including atherosclerosis; however, little is known about the exact mechanisms [2]. An impact of NOD1 and NOD2 on the intestinal microbiota composition [21] also under dietary intake [7] has been already established.
Interestingly, many PRRs initially identified for pathogen recognition and subsequent inflammatory immune response turned out to play crucial roles in pathophysiological conditions including atherosclerosis. First observations were made for Toll-like receptors (TLRs), genetic deletion of Tlr4 was shown to reduce experimental atherosclerosis in Apoe-deficient mice [20]. Later on, similar results have been shown for many other TLRs in related models [18] and likewise for NOD receptors [13,16,17,32] which we confirm in our study using for the first time a Nod1/2-double KO model. However, an essential impact of bacterial components itself on atherosclerosis can be ruled out, since none of the conducted large clinical trials with antibiotic treatment in patients with cardiovascular disease demonstrated any longterm benefit [6]. In addition, adverse effects of antibiotic treatment on microbiota composition are conceivable. Later identified endogenous ligands for PRRs, such as degradation products of extracellular matrix components released during tissue damage or apoptotic cell material, rather than live pathogens seem to be responsible for PRR-dependent effects in atherosclerosis. Different to NOD receptors, endogenous ligands for almost every member of the TLR family have been described [14]. Moreover, dietary unsaturated fatty acids such as lauric acid (dodecanoic acid) from coconut oil have been shown to activate TLR4 [31] as well as NOD1 and NOD2 [34], demonstrating a potential influence of PRRs on Ccl2 mRNA (n-fold) α α the development of chronic inflammatory diseases by sensing dietary ingredients.
In the current study, we detected an effect of Nod1/2-deficiency on experimental atherosclerosis and lipid metabolism, whereas we did not observe effects on insulin resistance. As already mentioned, the current literature on NOD1 and NOD2 in this field shows partially divergent results [3,9,13,16,17,29,32], which are most likely attributable to different experimental settings such as the animal model used (Apoe-KO, Ldlr-KO), diet ingredients, feeding period, antibiotic treatment or the use of a specific pathogen challenge.
In addition, some limitations of our study are also worth mentioning. First of all, every murine animal model of experimental atherosclerosis only insufficiently reflects the situation in patients with atherosclerosis. Genetic deletion of certain factors, in our case systemic lifetime deficiency for Nod1 and Nod2, does not represent the physiological situation. We have selected Ldlr-deficient mice for our experimental study since their plasma lipoprotein profile, contrary to Apoe-deficient mice, more closely resembles that of humans [33]. However, due to deletion of vascular cholesterol clearing pathways, atherosclerosis development is experimentally accelerated and condensed to some weeks, whereas the disease itself develops over decades in humans. Moreover, patients usually die due to rupture of end-stage coronary plaque, which are not existing in mice. Therefore, care must be taken to translate results from experimental mouse studies on atherosclerosis to patients. However, a genome-wide association study has already shown that a rare variant of NOD1 is associated with intima-media thickness in patients [11] suggesting a potential role for the NOD pathway in cardiovascular disease.
Reduced plaque burden in Ldlr −/− Nod1/2 −/− mice in our study was restricted to the aortic arch, whereas we did not detect differences in the whole thoracoabdominal aorta, potentially due to different hemodynamic conditions in these distinct vascular beds. This might be also attributed to the use of a HFD containing rather low cholesterol levels (research diets, D12079B), which led on the one hand to moderate plaque load but on the other hand allowed us to simultaneously study metabolic effects.
In summary, we identified NOD1 and NOD2 as proatherosclerotic factors, mainly due to their influence on plasma cholesterol levels. Combined deficiency of Nod1 and Nod2 reduced lipid-loaded plaque area and plaque macrophage content. In our view, this is conceivably explained by reduced levels of total cholesterol, especially VLDLcholesterol (the observed effects are depicted in a cartoon in Online Fig. 6). Combined, our results establish NOD1 and NOD2 as another example for evolutionary conserved immune receptors with crucial functions for cholesterol metabolism translating to vascular pathology.
Acknowledgements Open Access funding provided by Projekt DEAL. We thank Daniela Beppler and Silke Brauschke for excellent technical assistance. | v3-fos-license |
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} | pes2o/s2orc | Chemical composition, phytotoxicity and cytogenotoxicity of essential oil from leaves of Psidium guajava L. cultivars
Natural products with biological activity, such as essential oils, can be used in the search for and development of ecological herbicides as an alternative to reduce the damage caused by synthetic herbicides. This work to aimed to determine the chemical composition and phytotoxic properties of the essential oils, at concentrations of 3000, 1500, 750, 375 and 187.5 μg/mL, of four cultivars of Psidium guajava (guava) evaluated on germination and root growth of plant models Lactuca sativa and Sorghum bicolor, as well as in the L. sativa cell cycle. Exposure to essential oils reduced germination and root growth in bioassays, especially at the highest concentration (3000 μg/mL). The essential oils interfered in the normal dynamics of the cell cycle of L. sativa at most concentrations, causing a decrease in the mitotic index and increasing of chromosomal alterations, evidencing aneugenic and clastogenic action. The biological activity of the oils was associated with the presence of sesquiterpenes and monoterpenes found here, such as caryophyllene oxide, (E) -caryophyllene, and limonene. Thus, the essential oils of cultivars of guava demonstrated the promising potential for use as natural herbicides.
Introduction
The use of synthetic herbicides in crop fields has been questioned due to the environmental impact and toxicological implications caused to farmers and consumers by the application of these products (Jabran et al., 2015;Ribas & Matsumura, 2009). Additionally, weeds are becoming increasingly resistant due to the few modes of action present in herbicides, increasing the demand for more efficient, sustainable, and safe active ingredients (Dayan & Duke, 2014).
Allelopathy is a biochemical strategy in which plants negatively influence or benefit the growth and development of other plants by the action of allelochemicals (secondary metabolites) released into the environment (Inderjit et al., 2011;Rice, 2012). These compounds found in natural products, such plant extracts and essential oils, are biodegradable and have a wide structural diversity with a wide variety of modes of action (Jana & Biswas, 2011). In this context, natural substances that have allelopathic activity have already been recognized as an alternative for the control of agricultural pests and the development of new herbicides (Dayan & Duke, 2014).
Essential oils are a complex mixture of volatile and lipophilic substances produced by secondary metabolism of individual plants. They have associated the adaptations to the environment, like the attraction of pollinators and seed dispersers, interactions with microorganisms and other plants, protection against predators, and abiotic stresses (Boaro et al., 2019). The literature presents several works that report on the biological activity of essential oils as herbicides, pesticides and biocidal agents (Giunti et al., 2021;Gruľová et al., 2020;Hazrati et al., 2017;Navarro-Rocha et al., 2020;Rajkumar et al., 2020;Taban et al., 2020). These effects are related to the complex interaction between the compounds present in essential oil, being mostly monoterpenes and sesquiterpenes. These interactions allow each compound to modulate or alter the effects of other ones (Sharifi-Rad et al., 2017).
Myrtaceae is an important family of pantropical plants with some species rich in essential oils (Beech et al., 2017;Wilson, 2010) known for its allelopathic potential and the biological activity Durazzini et al., 2019;Habermann et al., 2017;Scalvenzi et al., 2017). The genus Psidium is one of the most representative of the family with about more than 150 species of plants (Hayes, 1953). However, little is known about the allelopathic activity of its species, especially with regard to essential oils. Recently, studies of essential oils from different species of the genus Psidium, Psidium cattleianum (araçá-vermelho), P. myrtoides (araçá-roxo), P. friedrichsthalianum (araçá-azedo), P. gaudichaudianum (araçá) and P. guajava (guava), reported the ability of these oils to inhibit the germination of seeds and the growth of other plants, as Lactuca sativa, Sorghum bicolor and Lycopersicum esculentum, being considered potential sources of natural herbicides (Almeida et al., 2019;Vasconcelos et al., 2019).
Psidium guajava L. (Myrtaceae), popularly known as guava, is one of the most studied species of the genus. With pleasant taste and high nutritional value rich in antioxidant compounds and also has antimicrobial properties, guava stands out in the food industry and in popular medicine (Shu et al., 2012). Studies with the essential oil of P. guajava leaves showed the antimicrobial, antiproliferative, antiparasitic, anti-inflammatory, antioxidant and cytotoxic effects of the species (Chaturvedi et al., 2019;Jerônimo et al., 2021;Lee et al., 2013;Weli et al., 2019).
Essential oils extracted from leaves of different P. guajava cultivars grown under the same conditions and in similar environments have shown differential larvicidal activity on the Aedes aegypti L. (Mendes et al., 2017). It has also been shown that the genetic and environmental factor are an important constituent of the composition of the essential oils of guava . These studies show that the use of these oils can be enhanced by choosing the appropriate genotype. Different genotypes can promote variation in the composition of essential oils, leading to different properties and, therefore, are of great interest in the management and control of weeds.
Contemplating the above, this work aimed to (a) investigate of the chemical constituents of the essential oil from the leaves from four guava cultivars (Cortibel Branca LG, Cortibel VII, Paluma and Século XXI), (b) evaluate the allelopathic effects on the germination and initial development of the model plants Lactuca sativa L. and Sorghum bicolor (L.) Moench, and (c) assess of the cytogenotoxicity of the respective oils on the cell cycle of L. sativa.
General experimental procedures
The chromatographs used in essential oil analysis were by gas chromatography with flame ionization detector (GC-FID) model Shimadzu GC-2010 Plus and gas chromatography coupled to mass spectrometry (GC-MS) model Shimadzu GCMS-QP2010 SE. Seeds of L. sativa (commercial cultivar Crespa Grand Rapids -TBR) obtained from ISLA Sementes and S. bicolor (commercial cultivar Al Precious) from BRSEEDS. The 2% (w.v) acetic orcein dye was purchased from Sigma-Aldrich.
Plant material
Leaves were collected from cultivars Cortibel Branca LG (C4), Paluma (PAL), Século XXI (SEC), and the superior genotype of guava Cortibel VII (C7) in the experimental area of the Federal University of Espírito Santo in Alegre, ES-Brazil, altitude 254 m, coordinates 20° 45' 50" S 41° 31' 58" W, subtropical climate by Köppen-Geiger climate classification: Cwa, in the morning at breast height (1.3 m) and around the canopy diameter. The experiment followed a randomized block design (RBD) with three blocks. Samples were collected around treetop using young and old leaves from all plants and blocks in order to randomize any differences between them. After, the leaves were dried in the shade and at room temperature for one week.
Chemical characterization of essential oils
The essential oils were extracted from 500 g of dry leaves of the C4, C7, PAL, and SEC guava cultivars by hydrodistillation technique in Clevenger apparatus. The samples were divided and placed in two round-bottom volumetric flasks of 2000 mL with distilled water and distilled for approximately 4 h. The extractions used about 250 g of leaves in approximately 1000 mL of water. The hydrolate obtained was centrifuged at 5000 RPM (rotations per minute) for 5 min to separate the aqueous and oily phases. The essential oils (supernatant) were removed with a Pasteur pipette and stored in amber glass bottles in a freezer at -20ºC.
The chemical composition of the essential oils was determined by gas chromatography coupled to mass spectrometry (GC-MS) and the quantification by gas chromatography with flame ionization detector (GC-FID). The analyses were performed using a fused silica capillary column (30 m x 0.25 mm) with the stationary phase RTX®-5MS (0.25 μm internal diameter). Helium was used as the carrier gas with flow and linear rate of 2.80 mL min-1 and 50.80 cm seg-1 (GC-FID); and 1.98 mL min-1 and 50.90 cm seg-1 (GC-MS), respectively. The temperature of the injector was 220°C and FID and MS detector temperature of 240 °C and 200 °C, respectively. The initial temperature of the furnace was 40°C, which remained for 3 min and then gradually increased by 3°C/min until reaching 180ºC. Chemical constituents were identified by mass spectra obtained compared with data of spectral library, retention index (RI), and literature data (Adams, 2007;NIST, 2011). The GC-MS and GC-FID analyses were performed as described by Mendes et al. (2017).
Phytotoxicity analyses
The phytotoxicity effects of the essential oils of the cultivars of P. guajava was evaluated against the selected plants, L. sativa (dicot) and S. bicolor (monocot), in laboratory bioassays. These model plants have been widely used to verify the effects of chemical compounds on germination and initial development, as they are highly sensitive to toxic substances, have fast germination and low cost (Alves et al., 2018;Vasconcelos et al., 2019). The solutions of essential oils of P. guajava were prepared by intense mixed and agitation with the solvent composed of distilled water, acetone (2% v.v), and Tween 80® (0.05% v.v). Five oil concentrations were tested: 3000, 1500, 750, 375 and 187.5 µg/mL. The solvent used in the preparation of the solutions was previously compared with water, and both presented the same statistical results. So, the water was applied as a negative control in the analysis, omitting the solvent. The herbicide glyphosate was used as positive control, at 0.01% (v.v), the same concentration recommended for commercial use. Petri dishes were lined with filter paper and treated with 2 mL of the solutions of essential oils and negative and positive controls. In each treatment, 25 seeds of each plant model were used, with five replications each. The plates were wrapped with plastic film and incubated in a germination chamber (BOD), under photoperiod with 16 h of light, at 24±2°C where they remained for 120 h.
The germination percentage (GP)number of germinated seeds after 48 h of exposure to the treatments, calculated by the ratio between the number of germinated seeds times 100 divided by the total number of exposed seeds per repetition; germination speed index (GSI)number of germinated seeds counted every 8 h during the first 48 h of exposure to the treatments, calculated by formula (N1 * 1) + (N2 -N1) * 1/2 + (N3 -N2) * 1/3 + ... (Ny -(Ny-1)) * 1/y , where: Ny refers to the number of seeds germinated within a given period; y: represents the total number of time intervals; and root growth (RG)measured (in mm) with the aid of a digital caliper after 48 h of exposure to the treatments of model plants exposed to the essential oils of P. guajava cultivars were evaluated.
Cytotoxicity analyses
To evaluate the cytotoxicity of the essential oils of the different guava cultivars, meristematic cell slides of L. sativa (lettuce) roots were prepared. Only lettuce roots were used because they are considered a suitable model for microscopic analysis (cytotoxic analysis) to test the toxic effect of chemical compounds (Silveira et al., 2017). In addition, lettuce is highlighted because it presents high proliferative activity, rapid growth, high number of seeds, large chromosomes, high sensitivity to mutagenic and genotoxic compounds, and easily manipulated roots (Andrade-Vieira et al., 2014;Aragão et al., 2017). Lettuce roots were collected at 48 h of exposure in the phytotoxicity assays and fixed in ethanol: acetic acid (3:1) and stored at -20ºC to perform the analysis. The fixer was changed once after 10 min and once after 24 h. Lettuce is one of the most common species used in the evaluation of the toxic effects of substances. Besides the advantages mentioned earlier in this work, it presents a small number of chromosomes (2n = 2x = 18) with easy visualization under the microscope, and roots easy to manipulate for slide preparation and microscopic analyses (Matoba et al., 2007;Silveira et al., 2017).
The previously fixed roots were washed with distilled water and hydrolyzed in 5N HCl for 18 min at room Research, Society and Development, v. 10, n. 9, e6110917710, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17710 5 temperature. We prepare the slides using the crushing technique, stained with 2% (w.v) acetic orcein, before being covered by a coverslip and observed under the light microscope. The mitotic index (MI) was obtained by observation of 5000 cells in mitosis per treatment and controls (1000 cells per lamina). The frequency of nuclear alterations (NA) and chromosomal alterations (CA) in the cells observed was also evaluated according to Pinheiro et al. (2015).
Statistical analyses
The experiments were conducted in a completely randomized design (CRD) with five replications for each treatment (3000, 1500, 750, 375, and 187.5 µg/mL) and control (distilled water and glyphosate). The results of the analyses on phytoand cytogenotoxicity were further inspected in an analysis of variance (ANOVA), and the resulting mean values in a Dunnett's test at 5% significance for compared the treatments with the controls. All statistical analyses were performed using the R computing environment (R Development Core Team, 2020).
Chemical characterization of essential oils
Twenty-nine compounds were identified in the essential oils from leaves of guava cultivars in GC-MS analyses.
Were only considered compounds with a relative area above 1% after normalization, which accounted for 96.8% to 98.4% of the total compounds present in the four cultivars. The compounds that had a relative area above 10% were considered major compound; these are presented in Table 1 adapted from Mendes et al. (2017). Our analyses showed that most of the chemical constituents of the all essential oils were sesquiterpenes, which more of 70% in their composition and smaller amounts of monoterpenes, varying between 1.4% and 22.9% (Table 1). Other chemical characterizations of essential oils of P. guajava with the same terpenic nature were found in others works (Satyal et al., 2015;Souza et al., 2017).
In this study, the hydrogenated sesquiterpene (E)-caryophyllene was the main chemical constituent found in guava cultivars oils, appearing as a major component in three of the four oils evaluated. Differences in the chemical profile of essential oils are common in many works. The quantity and chemical composition of oils is influenced by several factors that can cause significant changes in the production of specialized metabolites. These can be determined by genetic characters, or by biotic and abiotic factors (Aslam et al., 2017;Souza et al., 2017). The stimuli resulting from the environment where the plant is located can redirect the metabolic route, leading to biosynthesis of different compounds, however genetic variations are very important factors for these variations in the composition of essential oils (Boaro et al., 2019
Phytotoxicity effects
The phytotoxic potential was tested of the essential oils of P. guajava cultivars by evaluating the inhibition of seed germination, germination speed index, and root growth of lettuce and sorghum. Phytotoxic analyzes in the model plants showed that for all oils, in general, the highest concentrations had the most significant effects on the evaluated variables, reducing them. C7 and C4 oils caused more evident effects. These oils significantly reduced the percentage germination of lettuce at 375, 750 and 3000 µg/mL compared to the water, and of sorghum at 3000 µg/mL compared to both controls (water and glyphosate). SEC caused effects in percentage germination of lettuce only at 3000 µg/mL, and sorghum at 750 µg/mL, concerning glyphosate and water, respectively (Figure 1a, 2a, 3a, 4a). of Lactuca sativa and Sorghum bicolor. The lowercase letters above boxplots indicates significant differences. Boxplots followed by the letter a are statistically identical to water, boxplots followed by b are statistically identical to glyphosate and boxplots not followed by letters are statistically different from water and glyphosate by Dunnett's test (P < 0.05).
Regarding the GSI, all concentrations of the oils of the tested cultivars negatively interfered in the GSI of the lettuce seeds, presenting statistical similarity with the glyphosate, except for 187.5 µg/mL of C7 and 1500 µg/mL of SEC. In lettuce, SEC oil caused the highest inhibition in the GSI with a reduction of 69.35% at 3000 µg/mL, whereas sorghum showed significant reduction only at 3000 µg/mL of C4 and 375 µg/mL of PAL, compared to the water (Figure 1b, 2b, 3b, 4b). Lactuca sativa and Sorghum bicolor. The lowercase letters above boxplots indicates significant differences. Boxplots followed by the letter a are statistically identical to water, boxplots followed by b are statistically identical to glyphosate and boxplots not followed by letters are statistically different from water and glyphosate by Dunnett's test (P < 0.05).
In RG evaluation, the roots of lettuce treated with essential oils of C7 and C4 presented drastic reduction. The effect of the concentrations was comparable to the glyphosate, except for the lowest concentration (187.5 µg/mL) of C4. In sorghum, C7 oil interfered negatively in the root growth at 3000 and 1500 µg/mL, while C4 oil caused a reduction of 47.29% at 3000 µg/mL (Figure 1c, 2c). The seedlings of lettuce and sorghum also suffered negative effects on root growth from the oils of PAL and SEC. At a concentration of 3000 µg/mL, the PAL and SEC oils caused a severe reduction of root growth in lettuce (31.67%) and sorghum (56.45%) in comparison to the water (Figure 3c, 4c). Boxplots followed by the letter a are statistically identical to water, boxplots followed by b are statistically identical to glyphosate and boxplots not followed by letters are statistically different from water and glyphosate by Dunnett's test (P < 0.05).
The chemical composition of essential oils provides clues about their phytotoxic effects. Based on the obtained results of bioassays involving germination and root growth of L. sativa and S. bicolor, we found that the oils affected the variables GP, GSI, and RG these models, principally in the highest concentration. These effects may be associated with the terpenic nature of essential oils. According to Harborne and Tomas-Barberan (1990), these substances in contact with the soil inhibit the development of other plants, causing decomposition, volatilization, leaching, and exudation of compounds present in the vegetable tissues.
Figure 4. Effect of Cortibel VII (C7) essential oil on (a) germination, (b) germination speed index (GSI), and (c) root growth
of Lactuca sativa and Sorghum bicolor. The lowercase letters above boxplots indicates significant differences. Boxplots followed by the letter a are statistically identical to water, boxplots followed by b are statistically identical to glyphosate and boxplots not followed by letters are statistically different from water and glyphosate by Dunnett's test (P < 0.05).
Source: Authors.
Among the terpenoids, the sesquiterpenes were the most abundant chemical constituents found in the essential oils of guava cultivars. They are considered a source of active compounds with important biological activity that can act as herbicides (Duke et al., 2000). (E)-caryophyllene, hydrocarbon sesquiterpene present in all essential oils evaluated, except for C4, have allelopathic effects reported for seedlings of Brassica campestris and Raphanus sativus (Ruilong et al., 2009). Phytotoxic and cytotoxic effects of the essential oil of Psidium species on L. sativa and S. bicolor were associated with the presence of (E)caryophyllene (Vasconcelos et al., 2019).
Caryophyllene oxide, which is a product of the oxidation of (E)-caryophyllene, oxygenated sesquiterpene found in all cultivars, was reported for its allelopathic potential, significantly affecting the germination and development of L. sativa (Dias et al., 2009). The sesquiterpenes to affect plant growth through oxidative stress in conjunction with effects on physiological processes (Duke & Oliva, 2004). Effects on mitochondrial respiration, distribution, and organization of microtubules are also reported (Araniti et al., 2016;Ibrahim et al., 2013).
Monoterpenes are identified as compounds present in essential oils of plants with greater allelopathic potential, as Eucalyptus genus, influencing seed germination, and inhibition of root growth (Amri et al., 2013). The low germination, GSI, and reduction of RG observed for seeds treated with essential oils of C4 and C7 in this study can be justified by the presence of these substances. Essential oils with higher monoterpene content are known for their high ability to suppress weeds (Fagodia et al., 2017). The hydrogenated monoterpene limonene, found mostly in cultivar C4, is known to blocking the nitrogen cycle and inhibiting cytochrome respiration, seed germination and growth in neighboring plants (Maffei et al., 2011). Limonene is used as a leading compound in various formulations of commercial herbicides, such as GreenMatch O, GreenMatch EX, and Avenger® (Dayan et al., 2009). Vokou et al. (2003) suggest that the herbicide effect of essential oils is due to the combined reactions of various chemical compounds, which can act in an additive, synergistic, and antagonistic way. In this sense, the herbicide activity found in the essential oils of P. guajava cultivars can be attributed mainly to the presence of sesquiterpenes and monoterpenes.
Considering the importance of essential oils due to the great applicability they possess, it is essential to know the chemical compounds present in them, since they are responsible for the phytotherapeutic and biological properties. In larvicidal assays with Aedes aegypti, performed with the essential oils SEC, C4, C6, PAL and PET, the cultivar SEC presented itself as the most efficient for this purpose, which was attributed to a large amount of sesquiterpene present in the oils (Mendes et al., 2017). When comparing this to the results of this study for the cultivar SEC, was observed that the germination of lettuce and sorghum as well as their root growth were affected only when the highest oil concentration (3000 µg/mL) of the oil was used. So, while SEC on the one hand presented high larvicide activity acting on A. aegypti in past studies, our results show that the phytotoxic activity of SEC is less favorable than those of other genotypes. This proves that performing these tests on the potentials of each essential oil, are important to direct them to the most appropriate use.
For all the oils tested the effects were more evident in L. sativa, a eudicot, than S. bicolor, a monocot. Differences in sensitivity among target species are commonly reported in studies that verify the allelopathy and phytotoxicity of plants (Hazrati et al., 2017;Vasconcelos et al., 2019). These differences are related to the variation of the mechanisms of absorption, translocation, and the place of action of substances among different target species, which may explain the differences in selectivity among the species tested in this work (Oliveira Jr., 2011).
Cytotoxicity effects
Cytotoxicity analyses were performed to investigate the toxic effect of the essential oil of P. guajava cultivars on the cell cycle of lettuce and to determine the mode of action involved in the inhibition of the germination variables (GP and GSI) and root growth of lettuce and sorghum. Based on MI, as well as NA and CA were founded low cytotoxicity effects of the essential oils of P. guajava cultivars when compared to the positive control. The exposure to the highest concentrations of the evaluated oils caused the greatest damage to the meristematic lettuce cells (Table 2). 8.01 ± 0.22 0.00 ± 0.00 ab 17.91 ± 4.16 a MI% = mitotic index, CA% = chromosome alterations out of the total of cells, NA% = nuclear alterations out of the total of cells. Cultivars: C4 (Cortibel branca LG), C7 (Cortibel 7), PAL (Paluma) and SEC (Século XXI). * Means in the columns followed by the letter a are statistically identical to water, means followed by b are statistically identical to glyphosate and means not followed by letters are statistically different from water and glyphosate, by Dunnett's test (p< 0.05). Source: Authors.
In general, MI was the variable most affected by the essential oils, differing statistically from the negative and positive controls at the concentration 3000 µg/mL. We could observe a decrease in MI with increasing PAL concentrations, showing a decline at 750 (25.47%), 1500 (27.22%), and 3000 µg/mL (33.41%) concerning the negative control. For NA, the negative and positive controls showed little difference, with zero or low frequency of alterations. For CA, only the highest concentration of cultivar C4 showed the statistical difference to the negative control, with an increase of approximately 54% in alterations. The other tested essential oils were statistically equal to the two controls (C7) or only to the negative control (PAL and SEC). This result may be related to the reduction of the macroscopic variables found in the phytotoxic analyses.
According to Harashima & Schnittger (2010), cell division and plant growth are associated with cell proliferation, where changes in the cell cycle act directly on the percentage of germination, germination speed index, and root growth. The reduction of MI is the result of the blockade of the mitotic division, preventing the beginning of the prophase and, consequently, the cell division (Sousa et al., 2009). The MI is a parameter used to indicate the cytotoxic effect of substances (Leme & Marin-Morales, 2009). MI reduction was also observed in meristematic cells of L. sativa treated with the essential oil of Psidium cattleianum, P. myrtoides, P. friedrichsthalianum and P. gaudichaudianum (Vasconcelos et al., 2019).
Sesquiterpenes and monoterpenes are allelochemical compounds reported for causing the reduction of mitotic activity and formation of lipid cells in plants, causing inhibition of germination and root growth in several species (Vaughn & Spencer, 1993). In plants, these substances derived from secondary metabolism act as phytoalexinssubstances synthesized anew by plants, and responsible for chemically combating the growth and propagation of parasitic bacteria and fungi (Barbosa et al., 2007). Table 3 shows the frequency of chromosomal alterations found in the meristematic cells of L. sativa. We observed that the exposure to essential oils caused the occurrence of chromosome loss, chromosomal breaks, adherent chromosomes, cmetaphases and bridges ( Figure 5). A significant increase in the frequency of alterations, however, was observed only at the highest concentrations. Cultivar C4 caused the highest frequency of adherent chromosomes at a concentration of 1500 µg/mL and of bridges at 3000 µg/mL, differing from the negative control. In C7, the highest concentration of the oil ( Lost% = chromosome loss, Breakage% = chromosomal breaks, Adherent% = adherent chromosomes, C-met% = c-metaphases and Bridge% = chromosome bridges. Cultivars: C4 (Cortibel branca LG), C7 (Cortibel 7), PAL (Paluma) and SEC (Século XXI).*Means in the columns followed by the letter a are statistically identical to water, means followed by b are statistically identical to glyphosate and means not followed by letters are statistically different from water and glyphosate, by Dunnett's test (p< 0.05). Source: Authors.
The presence of chromosomal alterations shows the genotoxic effect while the nuclear alterations show the mutagenic effect, these alterations modify the structure and/or quantity of chromosomes (Fiskesjö, 1985). In this study, it was possible to observe a higher incidence of chromosomal alterations of the bridge type, c-metaphase, and adherent chromosomes, demonstrating the genotoxic effect of essential oils.
Chromosomal bridges are clastogenic alterations that indicate some action in the structure of the DNA. They usually occur in anaphase and telophase and are related to terminal breaks (telomere loss) in both chromatids of a chromosome followed by the union of the same (Leme & Marin-Morales, 2009;Matsumoto et al., 2006). The adherent chromosomes and cmetaphases are alterations that indicate the aneugenic mode of action. The adherent chromosomes reflect a change in the chromosomal structure, leading to the loss of the normal characteristics of condensation and the formation of agglomerates.
This change is irreversible and can lead to cell death, thus evidencing a strong cytotoxic effect (Babich, 1997;El-Ghamery et al., 2003). The c-metaphases are alterations that point to the action toxic compounds in the spindle fibers, paralyzing the mitotic cycle in metaphase. This alteration is characterized by scattered and condensed chromosomes with very well-defined centromeres in the interior of the cell (Fiskesjö, 1985;Leme & Marin-Morales, 2009).
Essential oils of cultivars C4 and SEC presented both modes of action, aneugenic and clastogenic since was observed a higher frequency of alterations of type bridges, adherent chromosomes, and c-metaphase compared to the negative control.
The essential oils of C7 and PAL presented a higher frequency of chromosomes in c-metaphase, characterizing them as aneugenic essential oil. Given the above, the terpene constitution of essential oils and the interactions between these compounds inducing several chromosomal abnormalities, which are responsible for inhibition of germination, germination speed index and root growth of the model plants.
Conclusions
This study showed the allelopathic potential of P. guajava cultivars essential oils in plant bioassays lettuce and sorghum. The effects were more evident in L. sativa than S. bicolor, demonstrating greater sensitivity of lettuce to the tested compounds. The exposure of the tested species to the essential oils caused the reduction of germination, root growth, and mitotic index in L. sativa. In general, the highest concentrations (3000 µg/mL) of the oils caused more evident effects in the analyses performed.
We also observed an increase in the percentage of chromosomal changes, such as bridges, c-metaphase and adherent chromosome in L. sativa meristematic cells. These alterations reveal the aneugenic and clastogenic mode of action associated with the oils. Phytotoxic, cytotoxic and genotoxic activity of essential oils may be associated with the terpene constitution of essential oils and the interactions between these compounds, mainly by the presence of caryophyllene oxide, (E)-caryophyllene and limonene. Given the above, the essential oils of guava cultivars are potential sources for the development of natural and sustainable herbicides due to their inhibitory activity in plants. | v3-fos-license |
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} | pes2o/s2orc | Mirasol PRT System inactivation efficacy evaluated in platelet concentrates by bacteria-contamination model
Background/Aim. Bacterial contamination of blood components, primarily platelet concentrates (PCs), has been identified as one of the most frequent infectious complications in transfusion practice. PC units have a high risk for bacterial growth/multiplication due to their storage at ambient temperature (20 ± 2°C). Consequences of blood contamination could be effectively prevented or reduced by pathogen inactivation systems. The aim of this study was to determine the Mirasol pathogen reduction technology (PRT) system efficacy in PCs using an artificial bacteria-contamination model. Methods. According to the ABO blood groups, PC units (n = 216) were pooled into 54 pools (PC-Ps). PC-Ps were divided into three equal groups, with 18 units in each, designed for an artificial bacteria-contamination. Briefly, PC-Ps were contaminated by Staphylococcus epidermidis, Staphylococcus aureus or Escherichia coli in concentrations 102 to 107 colony forming units (CFU) per unit. Afterward, PC-Ps were underwent to inactivation by Mirasol PRT system, using UV ( = 265–370 nm) activated riboflavin (RB). All PC-Ps were assayed by BacT/Alert Microbial Detection System for CFU quantification before and after the Mirasol treatment. Samples from non-inactivated PC-P units were tested after preparation and immediately following bacterial contamination. Samples from Mirasol treated units were quantified for CFUs one hour, 3 days and 5 days after inactivation. Results. A complete inactivation of all bacteria species was obtained at CFU concentrations of 102 and 103 per PC-P unit through storage/investigation period. The most effective inactivation (105 CFU per PC-P unit) was obtained in Escherichia coli setting. Contrary, inactivation of all the three tested bacteria species was unworkable in concentrations of ≥ 106 CFU per PC-P unit. Conclusion. Efficient inactivation of investigated bacteria types with a significant CFU depletion in PC-P units was obtained – 3 Log for all three tested species, and 5 Log for Escherichia coli. The safety of blood component therapy, primarily the clinical use of PCs can be improved using the Mirasol PRT system.
Introduction
The use of various inactivation techniques clearly reduces pathogen occurrence in collected blood.The Mirasol pathogen reduction technology (PRT) system is based on the treatment by ultraviolet (UV) illuminated/activated riboflavin (RB), resulting in inactivation of white blood cells (WBC) and pathogens at the molecular level due to irreversible photochemically induced damage of nucleic acids.These photochemical mechanisms inhibit nucleic acid replication and decrease incidence of potential transfusion side effects or complications [1][2][3][4][5] .
Generally, the risk of transfusion-associated infections -applying bacteria contaminated platelet concentrates (PCs) is about 1,000 times greater than the hazard of transfusion-related HIV, hepatitis C or B virus and human Tlymphotropic virus transmission 6,7 .The most important sources of bacterial contamination of collected blood are the donor skin [8][9][10][11] or asymptomatic donors -low-level or transient bacteremia in chronic bacterial infections, as well as a recovery from a disease [12][13][14][15] .Seldom, the source of bacteria can be a nonsterile equipment for collection or devices for processing of harvested blood units [16][17][18] .The prevalence of bacterial contamination is relatively high in PCs -from 0.14% to 1.41% -since their storage temperature (22 ± 2°C) favors bacteria growth/multiplication 6,19 .Consequently, PCs are the most common cause of transfusion-associated bacterial morbidity and mortality.However, the rate of blood contamination is higher than the incidence of bacterial infections because their clinical manifestation depends on numerous factors, such as patient's general condition, antibiotic therapy, quantity and type of bacteria, etc [20][21][22] .
Strategies to reduce the risk of transfusion-associated bacterial infections include superior donor selection 23 , improved preparation and disinfecting of venepunction field 24- 27 , redirecting the initial blood stream into satellite bag at the start of collection 25,26 , improved processing procedure safety and reduced storage time 28,29 , reevaluation/optimization of thresholds and criteria for transfusion supportive treatment 30,31 , as well as the use of different pathogen inactivation systems 1,[5][6][7]32 .
The aim of this study was to evaluate the Mirasol PRT system efficacy in artificial bacteria-contamination model and to predict the importance of its application in prevention of potential infectious complications of PC clinical use.
Methods
The study included 216 units of buffy coat derived PCs; the volume was 62.4 ± 8 mL in average.The units of PC were separated from whole blood collected by a CPD/SAGM quadruple bag system (Macopharma, France) within 6 hours after donation, using a T-ACE II blood processor (Terumo, Japan).According to the ABO blood groups, PC units were pooled into 54 pools (PC-Ps; four PCs per PC-P unit).After that PC-Ps were divided into three equal groups, with 18 PC-P units in each (mean PC-P volume was 256.6 ± 14 mL).PC-Ps were stored at ambient temperature (22 ± 2°C) for 2 hours and then were filtered using an Imugard III-PL (Terumo, Japan).
The PC-P units were artificially contaminated by three different bacteria species.In brief, into the units of the first PC-P group Staphylococcus epidermidis (isolated from the skin), in the second group Staphylococcus aureus (ATCC 25923), and in the units of the third group Escherichia coli (ATCC 25922) were inoculated.The initial bacteria concentration for all the three species was 0.5 McF (1.5 × 108 CFU / mL).Initial suspensions were diluted (six different dilutions were applied) and inoculated into the units of PC-P groups regarding all the three species in the same way.Therefore, the final counts of inoculated CFUs were 102 to 107 per PC-P unit.
Before bacterial contamination, samples were taken (1st sample; sterility control) from the PC-P units and investigated by a BacT/Alert Microbial Detection System (Biomerieux, France).After contamination, from PC-P units samples were taken also to confirm contamination success (2nd sample; contamination checking).All PC-P units underovment inactivation by the Mirasol PRT system (Caridi-anBCT, USA) -that is using UV ( = 265 -370 nm) activated RB according to the manufacturer's instructions.Concisely, a sterile solution contains RB (500 μmol / L) in a 0.9% sodium chloride solution (pH range: 4.0-5.0).A volume of 35 ± 5 mL of this solution is added to PC-P units to produce a final concentration 57-60 μmol / L. The illuminator delivers the required UV light dose (6.24 J / mL) to the contents of an illumination bag (Mirasol Platelet Illumination/Storage set), based on product volume and measured flux rate 3 .
The units are then returned to platelets shaker up to the moment of the investigations that followed.Finally, the samples from the inactivated units one hour, 3 days and 5 days after the Mirasol inactivation and storage at 20 ± 2C (3rd, 4th and 5th samples) were investigated for CFU units.
Results
The results of the PC-P testing before and after the contamination with bacteria Staphylococcus epidermidis (six different concentrations), and after inactivation of pathogens using the Mirasol PRT system, are presented in Table 1.
The samples of contaminated PC-Ps with Staphylococcus epidermidis in the concentration of 10 4 CFU per PC-P were also sterile after the Mirasol PRT inactivation process and during a storage period, while in bacterial concentration of 10 5 CFU per PC-P, only one PC-P was sterile.
Testing relating to contamination of PC-Ps with bacteria Staphylococcus aureus and bacteria Escherichia coli in different concentrations is shown in Tables 2 and 3, respectively.
In the samples from PC-Ps contaminated with Staphylococcus aureus in the concentration of 10 4 CFU per PC-Ps, we proved the presence of the said bacteria after the storage period of three and five days (4th and 5th samples, respectively), despite the negative results of the first sample taken one hour after the Mirasol PRT inactivation.
The highest degree of pathogen reduction has been made in PC-Ps contaminated by Escherichia coli inoculation.In concentrations of bacteria ≤ 10 5 CFU per PC-Ps, the sam- ples were sterile during the whole storage period.However, pathogen inactivation was not successful with bacterial concentrations 10 6 CFU per PC-Ps.
The results show that all PC-P units (n = 54) were sterile before testing (1st sample), as well as that the contamination of units by all the three bacteria species in all concentrations -from 10 2 to 10 7 -was confirmed (2nd sample).There was a complete inactivation of bacteria in concentrations of 10 2 and 10 3 CFU per PC-P (the degree of reduction was 2 and 3 Log) during the storage period (3rd, 4th and 5th samples) for all the three types of bacteria.
Summarily, in our study using the Mirasol PRT system bacterial depletion rank was 3-5 Log for all the three of bacteria species.
Discussion
The bacteria presence in PCs is often the result of their inadequate removal from the skin of donors (venepunction field), not diagnosed donor's bacteremia and possible blood contamination during collection and processing [33][34][35][36] .Bacterial contamination of PCs, associated with adverse transfusion reactions showed that most commonly isolated Grampositive bacteria from donor's skin (Staphylococcus epidermidis and Staphylococcus aureus) were found in more than 70% of published cases of sepsis associated with PC transfusion 6,35,37 .Contrary to this, some published data showed that Gram-negative bacterias, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus and Serratia marcescens are most commonly isolated pathogens in transfusion associated sepsis.Fatal outcome was the result of infection with Gram-negative bacterias in 63% of cases in comparison with 37% of fatal outcome after infections with Gram-positive bacterias 6,35,37 .
To assess the efficacy of bacterial reduction by the Mirasol PRT system, two types of experiments known as "high spike bacterial titer" and "low spike bacterial titer" tests were performed 1 .Both methods involve inoculation of the known number of bacteria before inactivation of pathogens, and subsequently prove the presence or quantification of remaining bacteria (CFU) and calculate degree of their reduction.The aim of the experiments with high-titer bacteria inoculation was to determine the full potential of the Mirasol PRT system in the terms of reduction of a large number of bacteria in PCs.Contrary, in studies with inoculation of low, but clinically significant titer of bacteria, after the Mirasol inactivation of pathogens (0.5-2 Log CFU per mL), evaluation of PCs usefulness for transfusion was performed using standard systems to detect contamination during the whole storage period 1,38 .
Based on these facts, our pathogen inactivation model examined the Mirasol treatment efficacy in PCs, previously contaminated with different bacterial species most frequently associated with bacterial adverse transfusion complications -Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli.Inoculation prepared with a various bacterial concentrations (range: 10 2 to 10 7 CFU per PC-P unit).Checking the maximum capacity of the Mirasol PRT system for the degree of pathogen inactivation was testing by inoculation of high bacterial concentrations in the PCs.Evaluation of the Mirasol efficacy in the prevention of potential infectious complications after transfusion of contaminated PC units was performed due to inoculation of lower bacteria's concentrations -mimicring the conditions regularly seen in clinical practice.
For the period of storage bacteria can growth/multiply quickly, as in our study with PC-Ps contaminated with Staphylococcus aureus species in the concentration of 10 4 CFU per PC-P in two PC-Ps.Despite the reduction of bacteria's number after the Mirasol PRT system inactivation to undetectable degree (negative result in the 3rd sample), there was a multiplication of the remaining viable bacteria to detectable levels (confirmed in two PC-P units at 3rd and/or 5th days).Concerning the literature data, fresh PCs are contaminated with less than 100 bacteria per product 1,20 .The number of inoculated bacteria can vary from low concentrations (100-1,000 times higher than clinically relevant concentrations) to high, when their number is approximately 10,000-100,000 times bigger than in typical clinical conditions.In our model, we achieved the degree of pathogen reduction from 3-5 Log which represents an additional high-level safety for patients receiving PCs.Impossibility to complete inactivation of viable bacteria number in concentrations 10 6 CFU per PC-P unit has no importance, because in clinical practice we do not regularly see such a large number of bacteria in fresh blood products.Finally, the obtained degree of pathogen reduction/inactivation in our research model was in accordance with the studies of other authors 1,4,38 , as well as the manufacturer's instructions.
The advantage of the Mirasol PRT system, unlike other systems developed to inactivate pathogens in blood products, is in the fact that after illumination of product with UV light during 6-10 minutes (6.24 J / mL), these products are immediately ready for clinical use.Therefore, there is no need for subsequently removing RB and its metabolites from blood products, since it is a vitamin, already present in the body of the recipient.
Conclusion
In this study efficient pathogen inactivation (CFU depletion) was obtained in investigated PC-P units -3 Log for all the three tested bacteria species and 5 Log for Escherichia coli.Thus, the safety of blood component therapy -predominantly the clinical use of PCs -can be significantly improved (lower morbidity/mortality rate) by using the Mirasol PRT system.
Table 2 Inactivation efficiency of the Mirasol PRT after platelet concentrations contamination with Staphylococcus aureus
*1st sample -
Table 3 Inactivation efficiency of the Mirasol PRT after platelet concentrate contamination with Escherichia coli
*1st sample - | v3-fos-license |
2017-06-19T16:46:39.665Z | 2014-04-05T00:00:00.000 | 18626682 | {
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} | pes2o/s2orc | A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study
Background Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. Results The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. Conclusion We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.
A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study 1st revision, 26/02/2014 At established time-points after i.v. injection GNP or vehicle (Phosphate Buffered Saline, PBS) administration animals (n=3 per group, as mentioned in methods: "MRI studies -in vivo studies") were sacrificed by cervical dislocation. Required tissues (liver, kidney, spleen, brain and tumor among others) as well as urine and blood samples were collected and stored at -80ºC. Samples were analyzed by
Superparamagnetic iron oxide nanoparticle (SPION) synthesis:
The negative contrast agents evaluated in this work consisted in different TEGor DMSA-coated SPIONs. Water-dispersible TEG-coated SPIONs were produced through a synthesis pathway described by Cai and Wan [2], with slight modifications [3]. In these experiments, a mixture of iron acetyl acetonate [Fe(acac)3] and triethylene glycol was heated at 180ºC, leading to the partial decomposition of the reactants and the formation of an intermediate alkoxy-acetylacetonate-Fe 3+ . After that, the heating of this mixture at 280ºC produced the reduction and subsequent decomposition of these complexes leading to the nucleation and final growth of the iron oxide nanoparticles. The resulting particles were washed with a mixture of ethyl acetate and ethanol, collected with the help of a magnet and transferred to a phosphate buffered saline (PBS) solution. This so-obtained colloidal dispersion presented particle size homogeneity (∼ 5 nm) and good particle size distribution, with a mean hydrodynamic aggregate of 16 nm measured using dynamic light scattering (DLS) in a 90 Plus apparatus (Brookhaven). DMSA-coated SPIONs were obtained by replacing TEG coating for DMSA molecules, using a ligand-exchange reaction process described previously [4]. In this process, the A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study
T Studies
A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study NA, 1; NR, 1; TAT, 3 min 12 sec. In both cases, the animals were anesthetized and handled as described for "in vivo studies". After that, animals were sacrificed and contrast administered as described in the "ex vivo post-mortem studies" section in the main text. After this, the T2 weighted image acquisition was repeated as above.
Processing and post-processing of MR data
The processing and post-processing or MR data were done essentially as described in the main text. The only remarkable difference is that for negative contrast agents, all slices with noticeable RCE effect were taken into account for RCE calculation.
Iv vitro results
The r2 relaxivity values for the negative CAs were measured at 1. Ferumoxtran-10 (20 nm) [6]. As expected, the increase of the SPIONs aggregate size markedly increases r2 at 1.4 T from 48.0 to 119.6 mM -1 s -1 , which is coincident with the results previously reported by Muller and collaborators [7] related to the variation of the relaxivity of superparamagnetic iron oxide MRI contrast agents in function of their aggregate size.
Ex vivo postmortem results
A representative T2-weighted image for SPIONs, and typical ROIs selected for analysis, are shown in Figure 3,
Discussion
The negative contrast agents showed differences between the r2 in vitro | v3-fos-license |
2018-04-03T00:46:47.257Z | 1993-08-25T00:00:00.000 | 24152456 | {
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} | pes2o/s2orc | Isotope effects and alternative substrate reactivities for tryptophan 2,3-dioxygenase.
Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catalyzes the first step in the oxidative degradation of tryptophan. The reaction is believed to proceed by addition of O2 across the 2,3-bond of the indole ring, followed by decomposition of the resultant dioxetane to give N-formylkynurenine. A primary D2O isotope effect of 4.4 on Vmax/Km was observed at the pH optimum, pH 7.0. This implies that abstraction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for L-[2-3H]tryptophan at this pH. The secondary isotope effect signals the formation of the C-O bond at C-2. As the rate of proton abstraction increased with increasing pH, the D2O isotope effect decreased to 1.2 at pH 8.5 and the secondary isotope effect increased to 0.92. The rate-determining steps therefore change with increasing pH, and bond formation at C-2 becomes more rate-limiting. The secondary isotope effect did not change significantly with varying O2 concentration so that substrate binding is primarily ordered with O2 binding first. The specificity of the enzyme towards substituted tryptophans shows that substitution of the phenyl ring of the indole is sterically unfavorable. Steric hindrance is highest at the 4- and 7-positions, while the 5- and 6-positions are less sensitive. 6-Fluoro-L-tryptophan was more reactive than tryptophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C-O bond formation at C-2.
Isotope Effects and Alternative Substrate Reactivities for
Tryptophan 2,S-Dioxygenase" (Received for publication, October 21, 1992, and Tryptophan 2,3-dioxygenase (EC 1.13.1.12) is a hemoprotein which catalyzes the first step in the oxidative degradation of tryptophan. The reaction is believed to proceed by addition of 0 2 across the 2,3-bond of the indole ring, followed by decomposition of the resultant dioxetane to give N-formylkynurenine.
A primary DzO isotope effect of 4.4 on V,aJKm was observed at the pH optimum, pH 7.0. This implies that abstraction of the indole proton is at least partially rate-determining. An inverse secondary isotope effect of 0.96 was observed for ~-[2-'H]tryptophan at this pH. The secondary isotope effect signals the formation of the C -0 bond at C-2. As the rate of proton abstraction increased with increasing pH, the DzO isotope effect decreased to 1.2 at pH 8.5 and the secondary isotope effect increased to 0.92. The rate-determining steps therefore change with increasing pH, and bond formation at C-2 becomes more rate-limiting. The secondary isotope effect did not change significantly with varying Oa concentration so that substrate binding is primarily ordered with O2 binding first. The specificity of the enzyme towards substituted tryptophans shows that substitution of the phenyl ring of the indole is sterically unfavorable. Steric hindrance is highest at the 4-and 7-positions, while the 5-and 6-positions are less sensitive. 6-Fluoro-~-tryptophan was more reactive than tryptophan, and the increased reactivity can be explained by an electronic effect that enhances of the rate of C -0 bond formation at C-2. Tryptophan 2,3-dioxygenase (EC 1.13.1.12) catalyzes the addition of oxygen across the 2,3-double bond of the indole ring, oxidatively cleaving it to N-formylkynurenine. As an iron-dependent dioxygenase, it is a member of the small class of enzymes that includes lipoxygenase and cyclooxygenase. Most studies of tryptophan 2,3-deoxygenase have concentrated on the enzyme from rat liver and to a greater extent on two related enzymes, Pseudomonas acidovorans tryptophan dioxygenase and indoleamine dioxygenase, a less specific mammalian enzyme that is induced by interferon-y .
Rat liver tryptophan dioxygenase is tetrameric with 4 identical subunits (Maezono et al., 1990). Purified enzyme retains 2 mol of noncovalently bound heme per tetramer, and exogenous heme stimulates activity, suggesting a stoichiometry of one heme/subunit . Tryptophan dioxygenase has allosteric as well as catalytic binding sites * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "duertkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 6137; Fax: 919-990-6147.
3 TO whom correspondence should be addressed. Tel.: 919-990-for tryptophan at neutral pH . a-Methyl-DL-tryptophan is not a substrate but is an allosteric effector. When the pH is increased to 8.0, a conformational change is observed by ultracentrifugation, and allosteric kinetics are no longer observed.
Equilibrium binding studies with Pseudomonas tryptophan dioxygenase and indoleamine dioxygenase (Sono, 1986;Sono et al., 1980;Makino et al., 1980a) have provided evidence that the ferrous form of these enzymes is the active form, and the ternary complex of enzyme, Oz, and tryptophan is catalytically competent (Sono, 1989;Taniguchi et al., 1979;Ishimura et al., 1970). The reduction potential of the heme of rat liver tryptophan dioxygenase is relatively low, -0.11 V at pH 7 and -0.28 V at pH 8.4 (Makino et al., 1980b). The present investigation serves to extend characterizations of the enzymesubstrate complex by using alternative substrate reactivities and isotope effects to provide information about intermediates along the reaction pathway.
Materials-~-[2-'~C]Tryptophan was purchased from Research
Products Int. ~-[3'-'~C]Tryptophan was purchased from New England Nuclear Research Products. [2-3H]Tryptophan was synthesized by catalytic tritiation of the N-1 trifluoroacetyl methyl ester derivative of 2-bromotryptophan (Du Pont-New England Nuclear). The labeled protected tryptophan was resolved and deprotected with chymotrypsin and carboxypeptidase A. Synthesis of the protected 2bromotryptophan and deprotection were according to Phillips and Cohen (1986). Fluorotryptophan analogues were resolved by the same derivatization and enzymatic deprotection scheme. The resulting Lisomers were purified by HPLC' on a C18 column in 5 mM potassium phosphate, pH 4, using a gradient from 0 to 50% acetonitrile. Enantiomeric purity was confirmed by derivatization with Marfey's reagent (Marfey, 1984). Specificity of tritium labeling was demonstrated by preparing [2-'H]tryptophan in parallel and characterizing the product by deuterium NMR. Additionally, tritium was shown to be released quantitatively as formate after incubation of labeled substrate with tryptophan dioxygenase and hydrolysis of the N-formylkynurenine product (see below). 7-Fluoro-~-tryptophan was a generous gift from Dr. Robert Phillips, Athens, GA. Other substituted tryptophans and L-tryptophan were purchased from Sigma. N-(-9-Fluorenyl)methoxycarbonyl-alanine (Fmoc-Ala) was from Applied Biosystems, San Jose, CA. Sprague-Dawley rats were purchased from Charles River.
Purification of Tryptophan 2,3-Diorygenase"The procedure of was adapted for the induction and purification of tryptophan dioxygenase from rat liver with minor modifications. Prednisolone sodium phosphate was used to induce the enzyme at a single dose of 50 mg/kg body weight, which was given intraperitoneally 5.5 h before excising livers. DEAE-cellulose was substituted for DEAE-Sephadex and the final gel electrophoresis step was omitted. Instead, DEAE-cellulose fractions were concentrated using Centriprep-10 protein concentrators (Amicon) and desalted on a G-25 column. The DEAE-cellulose-purified tryptophan dioxygenase was loaded onto a Mono Q column (Pharmacia LKB Biotechnology) The abbreviations used are: HPLC, high performance liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl. equilibrated with 20 mM HEPES, pH 7.0, containing 10 mM Ltryptophan. Activity was eluted with a linear gradient from O to 1.0 M KC1 in the equilibrating buffer. Purified tryptophan dioxygenase was stored at -80 "C in the presence of 10 mM L-tryptophan. This preparation had no contaminating kynurenine formamidase activity.
Routine radiometric assays were based on the fact that N-formylkynurenine is readily hydrolyzed to kynurenine and formic acid in the presence of 3% perchloric acid (Ozaki et al., 1986). The assay solution contained 4.9 D M L-tryptophan, 20 mM sodium formate, 2 mM sodium ascorbate, 0.46 p~ hematin, and ~-[2-"C]tryptophan (typically 400 dpm/nmol) in 0.1 M KHzP04, pH 7.0. The reaction was initiated by addition of enzyme. After 30 min at 37 'C, 100 pl of 6% perchloric acid was added and the samples were incubated an additional 30 min. 1.0 ml of 10% charcoal (w/v) suspended in 3% perchloric acid containing 1.0 M formic acid was added, and the samples were vortexed and then centrifuged for 5 min at 15,000 rpm to remove unreacted tryptophan. A 0.6-ml aliquot from each tube was counted to determine the extent of labeled formate formation.
HPLC analysis of reaction mixtures was performed on a C18 column using gradient elution with acetonitrile/water buffered with either 5 mM KH~POI, pH 4.0 , or 10 mM ammonium acetate, pH 7.0 (Takikawa et al., 1988). At pH 4, three cartridge C18 columns (4.6 X 33-mm each, 3-rm particles, Perkin-Elmer) were used in series at a flow rate of 1.5 ml/min. The columns were eluted for 5 min at 9% acetonitrile followed by a 10-min linear gradient to 32% acetonitrile. At pH 7, a 4.6 X 100-mm Brownlee ODS column (5-p particles, SCI-CON, Winter Park, FL) was eluted with a 10-min linear gradient from 0 to 5% acetonitrile, a 5-min linear gradient to 10% acetonitrile, and a 5-min linear gradient to 80% acetonitrile followed by 5 min at 80% acetonitrile. Quantitation was accomplished with an LC-235 diode array detector (Perkin-Elmer) connected to a model 171 radioisotope flow-through detector (Beckman Instruments, Inc., San Ramon, CA).
3H/14C
Isotope Effect Experiment-Tryptophan dioxygenase was desalted by centrifugation through a G-25 Sephadex column equilibrated in assay buffer containing 3 mM a-methyl-DL-tryptophan. The assay mixture contained 0.1 mM L-tryptophan, 3 mM a-methyl-DLtryptophan, 50 mM sodium formate, 4 mM sodium ascorbate, 0.92 g M hematin, 0.08 pCi of ~-[2-"C]tryptophan plus 0.25 pCi of L -[~-~H ] tryptophan per 100 pl. For assays at pH 7.0, a single reaction mixture was prepared in 0.1 M potassium phosphate. Aliquots were quenched by addition to 1% perchloric acid, and incubated for 1 h to hydrolyze formylkynurenine. Measurements at pH 8.4 were performed in 0.1 M Tris.HC1, at both 250 and 5 p~ Oz. 0, concentrations were maintained by saturation with air or 0.4% 0, in Nz, respectively. The reaction mixtures, including enzymes, were made up without ascorbate in glass vials sealed with two rubber septa. After the samples had been equilibrated to the desired 0, concentration, the reactions were started by addition of a small volume of ascorbate through a syringe. Reactions were quenched by freezing on dry ice. Samples for all conditions were analyzed by HPLC, and 1-min fractions were collected and counted for 20 min.
Tryptophan Analogues as Tryptophan Dioxygenase Substrates-Tryptophan analogues were assayed at a concentration of 1.8 mM in the presence of [2-"C]tryptophan, 0.92 p~ hematin, 2.0 mM sodium ascorbate, and 0.17 mg/ml Fmoc-alanine (the internal standard), with or without 3 mM a-methyl-DL-tryptophan. After incubation at 37 "C, the samples were frozen on dry ice for later analysis by HPLC.
Compounds were incubated with enzyme in triplicate and the amount of products formed was compared to a control without enzyme. HPLC analysis for experiments without a-methyl-DL-tryptophan were performed using a single Perkin-Elmer C18 cartridge with the pH 4 elution buffer and a modified elution profile: 8 min at 0% acetonitrile followed by a 10-min gradient to 24% acetonitrile, a 4-min gradient to 64% acetonitrile, and a 5-min gradient to 80% acetonitrile. When a-methyl-DL-tryptophan was included in the reaction mixture, a Brownlee C18 column was used at pH 4 with the following elution profile: 20 min at 0% acetonitrile followed by a 10-min linear gradient to 80% acetonitrile.
Reaction Rates in DZO-Buffers contained 100 mM potassium phosphate and 100 mM Tris in either HzO or D20 at pH 6.5, 7.0, 7.5, 8.0, and 8.4. pH meter readings in DZO were corrected for the known shift of 0.4 units from the true pD (Schowen and Schowen, 1982). 3 mM a-Methyl-DL-tryptophan was included in all the reaction mixtures in order to saturate allosteric interactions with the enzyme . The enzyme was concentrated and exchanged into buffers of the appropriate pH containing 50% HzO and 50% DzO by desalting on a P-10 column. Either 5 or 10 gl were added to reaction mixtures to give a final volume of 100 pl so that the isomeric purity of the water was >95% in the reaction mixtures. The rate of oxidation of L-tryptophan was determined using the charcoal precipitation assay described above. The rates of 5-fluoro-~-tryptophan and 6-fluoro-~-tryptophan oxidation relative to L-tryptophan were determined by HPLC analysis as described above.
RESULTS
Substrate Specificity-Reactivities of 4-, 5-, 6-, and 7-substituted tryptophans relative to tryptophan are given in Table I. A number of other tryptophan analogues were tested but turned over at (1% of the rate for tryptophan: 7-aza-DLtryptophan, 5-hydroxy-cu-methyl-~~-tryptophan, 1-thio-DLtryptophan, 5-hydroxy-~~-tryptophan, and N-methyl-DLtryptophan. Relative V / K values for tryptophan analogues were measured directly by comparing the rate of turnover of each analogue relative to tryptophan when the two substrates were incubated together in the same reaction mixture (Abeles et al., 1960). Note that rates for L-isomers were determined in the presence of a-methyltryptophan, an allosteric effector. In all cases, substrates with methyl or bromo substituents were poorer substrates than tryptophan. This agrees with previous studies (Civen and Knox, 1960) and most probably reflects steric hindrance, since these substituents have only a small electronic effect on the pK. of tryptophan (Yagil, 1967). This effect is greatest at positions 4 and 7 and smallest at positions 5 and 6.
The 5 -, 6-, and 7-fluorotryptophans also showed a wide range of reactivities. In parallel with the methyl-substituted analogues, 7-fluorotryptophan was least reactive, and the 6fluoro analogue was most reactive. The general trend (7 < 5 < 6) follows that predicted by steric effects, even for a substituent as small as fluorine. 6-Fluoro-~-tryptophan was actually a better substrate than tryptophan, however. This may well represent an electronic effect of fluorine that is large enough to dominate steric effects at this position.
When comparing the reactivity of substrate analogues in a two-substrate reaction, the relative V,,,/K,,, values for one substrate versus its analogues can change when the concen- tration of the second substrate is varied. Variations in relative V,,,,,/K, values depend on the order of substrate binding ("Appendix"). Accordingly, the K, for O2 was calculated by fitting oxygen electrode data for 0, consumption to the integrated Michaelis-Menten equation in order to establish a meaningful range for varying 0, concentration. The K, was 7.2 ~L M at pH 7.0 and 6.9 mM tryptophan. The relative V,,,/ K, values for 6-fluoro-~~-tryptophan compared to tryptophan were measured at 0, concentrations above and below the K, and were 2.14 +_ 0.06 at 5 FM 02, compared to 2.13 +-0.06 at 250 ~L M 02. These results are consistent with ordered binding with O2 binding first.
DZO Isotope Effects-The initial velocities for tryptophan oxidation by tryptophan dioxygenase are presented in Fig. 1 as a function of pH in H20 and DzO. Velocities were measured below the K, for tryptophan and reflect V,,,/K, values.
These reactions were all performed in the presence of 3 mM a-methyl-DL-tryptophan to minimize ambiguities due to allosteric effects . Rates of tryptophan oxidation in H20 were optimum at pH 7.0. The pH rate profile in D 2 0 differed substantially, however. The reaction in D20 was 5.25-fold slower than the reaction in H,O at low pH, pH 6.5. When the pH was increased, the D20 isotope effect decreased to only 1.19 at pH 8.4. The pH optimum in D20 was pH 8.4 or higher. The relative rates of turnover of 6fluoro-L-tryptophan versus \2-'4C]tryptophan were also determined over a range of pH values in both H,O and DzO (Fig. 2). The 6-fluor0 substituent effect did not change with pH in D20 and was small, approximately 1.5. In HzO, the substituent effect was similarly small at low pH but increased to 2.8 at pH 8. Changes in the substituent effect will reflect changes in rate-limiting steps with pH or solvent, and steps sensitive to 6-fluor0 substitution apparently predominate at high pH in H20. The data for 6-fluorotryptophan are also plotted as absolute rates in the inset for Fig. 2. The substrate analogue shows a solvent isotope that is comparable in magnitude to that for tryptophan and is similarly sensitive to pH, although the maximum isotope effect persists up to pH 7.5 for this substrate.
3H Isotope Effects-The 3H isotope effect on V,,,,,/K, for 3(V/K) is the rate of turnover of [2-14C]tryptophan relative to 12-"Hltryptophan and is the isotope effect on V,,,aJK,,,. Standard errors are shown and the number of determinations is given in parentheses. The isotope effects were measured in the range from 9 to 52% conversion and were independent of the extent of reaction in this range.
[2-3H]tryptophan was determined in competition with ~-[ 2 -'%]tryptophan (Table 11). In the initial experiment at pH 7.0, the observed isotope effect was 0.96 & 0.01. At pH 8.4, the isotope effect was significantly larger, 0.92 & 0.01. The variation of isotope effects with substrate concentration can provide information about order of substrate binding if isotope effects below and above the substrate K, are compared (Klinman et al., 1980). The 3H isotope effect for tryptophan dioxygenase did not change significantly when measured at 5 PM O2 compared to a normal 0, concentration of 250 pM, again indicating ordered addition with 0, binding first.
DISCUSSION
Based on solvent and substrate isotope effects, alternative substrate reactivities, and pH rate profiles, the mechanism of Fig. 3 is proposed for tryptophan 2,3-dioxygenase. It is assumed that oxidative cleavage of the indole ring procedes via formation and decomposition of a dioxetane intermediate. Our results provide information regarding the steps leading to dioxetane formation as outlined below.
Large D20 isotope effects were observed for tryptophan at low pH: 5.2 at pH 6.5 and 4.4 at pH 7.0 (Fig. 1). A similarly large isotope effect of 6.1 was observed for 6-fluorotryptophan at pH 7.5 (Fig. 2, inset). There are numerous possible sources of solvent isotope effects (Klinman, 1978;Schowen and FIG. 3. The proposed reaction mechanism for the oxidation of tryptophan by rat liver tryptophan 2,3-dioxygenase. The reaction is shown to proceed by abstraction of the indole proton, addition of oxygen to carbon 3 of the indole ring, and cyclization of oxygen to the second carbon of the indole ring to form the key dioxetane intermediate. Species enclosed in brackets are thermodynamically allowed but may not be discrete intermediates. The numbering convention for the indole ring is provided for convenience. Schowen, 1982). It is common, for example, that pK,, values will shift to approximately 0.5 units higher in D20, and this may be observed as a shift by as much as 0.5 units to higher pH in enzyme pH rate profiles. The solvent isotope effect for tryptophan dioxygenase, however, does not appear to be related to a simple shift of pH rate profiles. Thus, a shift of the curve for tryptophan turnover in D20 by 0.5 units to lower pH would have no effect on the observed isotope effect at pH 7.0. In addition, the pH rate profile for 6-fluorotryptophan in D20 is very similar to that for tryptophan, while the pH optimium in H 2 0 is pH 7.0 for tryptophan and pH 8.0 for the fluoro derivative. The DzO pH rate profiles for the two substrates, therefore, do not appear to represent simple and equal shifts of the profiles in H20. In the absence of a major contribution by a simple shift in pK,, the size of the observed isotope effects is large enough to be considered a primary kinetic isotope effect for transfer of an exchangeable proton. Considering the nature of the tryptophan dioxygenase reaction, the most straightforward interpretation of the effect would be that it represents deprotonation of the indole nitrogen.
A secondary isotope effect was observed for ~-[Z-~H]-tryptophan. This isotope effect increased from 0.96 at pH 7.0 to 0.92 at pH 8.4. The secondary isotope effect is inverse and is indicative of a change in hybridization from sp2 to sp3 at position 2, i.e. carbon-oxygen bond formation at C-2. The magnitude of the secondary isotope effect at pH 8.4 is sufficiently large to indicate that bond formation is essentially complete in the rate-limiting step at high pH (Klinman, 1978). Since the 3H isotope effect increased with pH as the solvent isotope effect decreased, proton transfer and bond formation at C-2 must represent different steps in the reaction mechanism. Furthermore, the proton transfer step must precede C -0 bond formation. This is because secondary isotope effects will generally be largest if they precede the rate-determining step and will be masked only if they follow the slow step.
The fact that the secondary isotope effect decreased by only a factor of two between pH 8.4 and 7 indicates that C-0 bond formation at C-2 is still partially rate-limiting at the pH optimum. This in turn implies that proton abstraction from the indole nitrogen is only partially rate-limiting at pH 7, and the intrinsic solvent isotope effect may therefore be larger than that observed by as much as a factor of two.
Varying the 0, concentration from 5 to 250 PM did not significantly affect the secondary isotope effect, and this can be taken as indication of a predominantly ordered binding pattern with 0, binding first (Klinman et aL, 1980). We also found that the rate of turnover of 6-fluorotryptophan relative to tryptophan did not change with varying O2 concentration. The interpretation of the effect of O2 concentration on the relative rates of alternative substrate turnover is similar but not identical to that for competitive isotope effects. An analysis is provided in the "Appendix." The fact that O2 binds first indicates that 0, binds to heme and implies that the function of the heme is to localize and activate O2 rather than tryptophan. This order of binding is contrary to that proposed for the closely related enzymes indoleamine dioxygenase (Ishimura et al., 1970) and tryptophan dioxygenase of P. acidouoram (Koike and Feigelson, 1971) based on equilibrium binding studies. The reason for the discrepancy may be either that the related enzymes have different kinetic mechanisms or that the equilibrium results are misleading in their prediction of kinetics.
Free energy changes for selected steps of the tryptophan dioxygenase reaction are provided in Table I11 and allow us to both strengthen and extend the above conclusions. Tryptophan is a weak acid (Equation 1, Table 111) and we must consider whether it is thermodynamically feasible that the tryptophanyl anion is an intermediate in the reaction. The accessibility of high energy intermediates in enzyme reactions has been discussed by Jencks (1980) and recently by Gerlt and Gassman (1992). The barrier to proton abstraction from tryptophan by a base on the enzyme with pK. = 7 would be 13.6 kcal/mol. To this thermodynamic barrier must be added a kinetic barrier which should be no more than the 3 kcal/ mol barrier for diffusion-controlled transfer of a proton from a nitrogen base in solution. The total barrier of 16.6 kcal/mol is very close to the predicted allowable limit of 16.7 kcal/mol (Gerlt and Gassman, 1992) for the tryptophan dioxygenase reaction with kc, = 10 s-l . We O2 is the corresponding nitrogen anion. The free energy is based on pK. = 24.5, calculated from the pK. of aniline (pK, = 27; Dolman and Stewart, 1967) with a correction for the electron-withdrawing effect of the dioxetane oxygens (Fox and Jencks, 1974). dDi~~ociation of oxygen from the heme of TDO calculated from Kd = 2 ~I M for the Trp-Pseudomonas TDO complex, consistent with the K,,, for oxygen for rat liver TDO (Ishimura et al., 1970;Sono et al., 1980). e The free energy of oxidation of Trp to the dioxetane, Trp. 02, in the gas phase was calculated from heats and entropies of formation (Benson, 1976;Richardson, 1989). The gas-phase free energies were corrected to aqueous solution using AM1-SM2 calculations (Cramer and Truhlar, 1992).
/The free energy for 1-electron oxidation of Trp was calculated from a reduction potential of 1.05 V versus NHE for Trp at pH 7 (DeFelippis et al., 1989). 1-Electron reduction of the heme-oxygen complex. Complexation of O2 to an iron-porphyrin complex raises the reduction potential of the 0 2 / O ; couple by 0.52 V (Sawyer and Valentine, 1981;Tsang and Sawyer, 1990).
Concerted addition of 0 2 to Trp to form the anion of the dioxetane (Equations 2 + 3 + 4). conclude that the tryptophanyl anion is a viable intermediate and note that it is probably not fortuitous that the observed rate of reaction is equal to that predicted from the thermodynamics. Iron-catalyzed formation of dioxetanes has been proposed to be a concerted singlet biradical reaction (Sheu e t aL, 1990), and we can address the question whether formation of the dioxetane in the tryptophan dioxygenase reaction proceeds by concerted addition of 0 2 across the 2,3-bond. Formation of the C -0 bond at C-2 occurs in a step subsequent to and discrete from proton transfer. Furthermore, if deprotonation of nitrogen is catalyzing addition of 02, then reprotonation must occur in a step subsequent to addition of O2 (Fig. 3).
1-Election oxidation of
The anilinium anion formed upon concerted addition of O2 would be a strong base with a pK, of approximately 24.5 (Equation 2, Table 111). The thermodynamic barrier to the overall reaction of deprotonation and addition of oxygen to form the anion of the dioxetane is calculated to be 27.7 kcal/ mol (Equation 7, Table 111). This is higher than the allowable limit of 16.7 kcal/mol, and the concerted pathway would be inaccessible by a barrier of at least 11 kcal/mol. In contrast, stepwise addition of O2 across the 2,3-bond would permit activation of tryptophan to oxidation by proton abstraction but would avoid formation of more unstable anionic intermediates. We therefore suggest that addition of O2 proceeds via the 3-peroxy intermediate (steps 1-3, Fig. 3). in brackets to indicate that protonation of the imine may be a discrete step or may be concerted with C-0 bond formation. This mechanism parallels closely the chemistry of electrochemical and photosensitized oxidations of tryptophan (Nguyen et at., 1986;Nakagawa et al., 1977a, 197713) in terms of formation of a 3-peroxy intermediate and subsequent acidcatalyzed nucleophilic addition at (2-2. In the model reactions, however, the nucleophil that adds to C-2 is solvent or the aamino group of tryptophan, and the thermodynamically less favorable dioxetane formation is not observed. The reactivity of 6-fluorotryptophan is also consistent with the proposed mechanism. The 6-fluor0 analogue is a better substrate than tryptophan, and this is taken to represent an electronic effect. The effect of fluorine does not represent a simple effect on the pK, of tryptophan since the effect is suppressed in DzO, where proton transfer is most rate-limiting. The effect of fluorine is greatest at high pH where ring closure to form the dloxetane becomes more rate-limiting, and we propose that the effect of fluorine is to activate the intermediate imine to nucleophilic attack (step 5, Fig. 3).
Finally, we show C-0 bond formation at C-3 proceeding in steps of proton abstraction, 1-electron transfer, and bond formation. It is equally likely that C-0 bond formation is concerted with proton abstraction, and the proposed intermediates are enclosed in brackets to emphasize this possibility. Thermodynamic data, however, indicates that these intermediates would be allowed. The thermodynamic barrier calculated for the l-electron oxidation of tryptophan to the free radical by heme-bound oxygen is 15.9 kcal/mol (Equation 8, Table 111). It is not clear how large the additional kinetic barrier would be at the active site, but it is likely that it would be smaller than the barrier in solution of 2-3 kcal/mol (Perrin, 1984). The total calculated kinetic barrier is, therefore, 18-19 kcal/mol or less. Within the necessary uncertainties in predicting reduction potentials at the active site, this is very close to the maximum allowable barrier of approximately 17 kcal/mol, consistent with the formation of radical intermediates that would essentially instantaneously collapse by carbon-oxygen bond formation (step 3).
APPENDIX
Kinetic expressions for ordered binding mechanisms are provided in Table IV. Rate constant ratios defining k,,,/K, for tryptophan are given for the limiting cases of high and low 0 2 concentrations. This approach has been used to predict the behavior of competing isotopically labeled substrates (Klinman et al., 1980). The isotope effects are a simplified case in which the assumption can be made that any difference in rates of turnover must arise from steps associated with a chemical reaction (b in this case). The treatment can be generalized to any two competing substrates, in this case 6fluorotryptophan versus tryptophan, with one important limitation.
If O2 binds first, then it can be said without qualification that the relative rates of turnover of two competing substrates will not change as the O2 concentration is varied. This can be seen in the ratio of rate constants at low versus high O2 concentrations. This ratio depends only on the binding constant for binding of O2 to free enzyme and will therefore be independent of the identity of the substrate. If tryptophan binds first, then any differences in rate between alternative substrates that are reflected in rate constants k2, ks, k4, or kg will be observed only at low 0 2 concentrations. This is parallel to the situation for competing isotopically labeled substrates, for which the differences in rate would be associated only with kg. However, in the more general case of any two competing substrates, observed differences in rate can also be associated with kl, and this rate constant is a factor in the rate expression at all O2 concentrations. In the general case, therefore, is only possible to say that relative rates may change and that the direction of the change may be to increase or decrease with changing O2 concentrations, depending on the contribution of the rate of binding, kl, to the difference in rates.
For 6-fluorotryptophan versus tryptophan we would argue that the difference in kcJK,,, is not significantly determined by kl since the relative rates vary both with pH and DzO versus H20 as solvent. We argue that, in the present case, the increased reactivity of 6-fluorotryptophan is an effect on a chemical step of the reaction rather than simply on the rate of binding. The data for 6-fluorotryptophan at high and low 02, therefore, support a kinetic mechanism in which O2 binds first. | v3-fos-license |
2020-10-14T05:06:42.045Z | 2020-09-25T00:00:00.000 | 222311697 | {
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} | pes2o/s2orc | Interactive Effects of Chemical Pretreatment and Drying on the Physicochemical Properties of Cassava Flour Using Response Surface Methodology
Calcium chloride and citric acid (0.6–3.4%w/v) were separately applied in the pretreatment of two South African cassava landraces (white and red) processed into flour at drying temperatures of 45–74°C. Optimisation using the response surface methodology showed ash (0.79–4.42%) and crude fibre (2.77–5.12%) increased as the drying temperature (DT) and concentration of pretreatment (COP) increased. Starch content (78.06–84.71%) was not influenced by the processing variables. Both pretreatments improved the lightness and whiteness index of cassava flour. Optimal processing conditions of 70°C DT and 3%w/v COP were the same for the proximate composition of cassava flour from all experimental groups.
Introduction
The world's most important staple root crop is cassava (Manihot esculenta Crantz), which is also known as tapioca or manioc [1]. It is a woody shrub belonging to the family Euphorbiaceae. The tuberous roots, rich in starch, are the main storage organ in cassava and the major part of the plant that is mostly consumed [2,3]. Approximately one billion people rely on cassava for food daily, predominantly in the tropical and subtropical regions of Latin America, Asia, and Africa [4]. Cassava flour is a major product of the root that has found various applications for both domestic and industrial use. The flour is a dry powdery product obtained from the root with simple process technology [5].
Cassava root handling and processing are faced with a challenge of rapid postharvest physiological deterioration (PPD) [6,7]. Rapid PPD is a complex physiological and biochemical process that begins with vascular streaking characterised by blue-black discoloration. The blue-black discoloration process is caused by microbial activity leading to a total deterioration of the root [8,9]. The use of chemicals regarded as safe, such as calcium chloride, citric acid, and ascorbic acid, in food processing to control enzymatic browning and discoloration is a common practice. There is available literature on the use of ascorbic acid and citric acid in the treatment of white yam flour [10]; the use of sulphite, calcium chloride, and citric acid in the treatment of potato flour [11,12]; and the use of calcium chloride treatment for the control of enzymatic browning of minimally processed cassava chips [13,14]. However, there is sparse literature on the use of safe chemical pretreatment in the processing of cassava flour.
Processing of cassava flour involves several units which can be varied as the quality of flour is determined by the process it undergoes [15]. Response surface methodology (RSM) is a mathematical and statistical technique used for defining the effect of more than one processing variable on a response (parameter) of interest. Pornpraipech et al. [16] used RSM to determine the drying behaviour of cassava chips using two cutting shapes (rectangular and circular) evaluated under different temperatures (60, 80, 100, and 120°C). The effect of drying temperature and time on the thermal and physical properties of cassava flour was investigated by Omolola et al. [17]. Nemaungani et al. [18] in their preliminary findings showed the improved colour quality of solar-dried cassava flour as a result of calcium chloride and citric acid pretreatment. Presently, there is no information on the interactive effects of chemical pretreatment and drying temperature on cassava flour using RSM. Therefore, in this research, citric acid and calcium chloride (0.6-3.4%w/v) are separately applied in the pretreatment of flour from two South African cassava landraces (white and red) under drying temperatures of 45-74°C. RSM is employed to determine the linear, interactive, and quadratic effects of these processing variables on the colour properties (L * , a * , b * , chroma, colour difference, whiteness index, and brownness index) and proximate components (starch, ash, moisture, and fibre content) of cassava flour.
Sourcing and Preparation of Cassava Root.
Two landraces of cassava (white and red) were sourced from the Institute of Tropical and Subtropical Crops-Agricultural Research Council (ITSC-ARC), Levubu, Limpopo Province, South Africa (22.946°S, 30.485°E). The roots were harvested fourteen months after planting and sorted before washing with tap water generously to remove adhering dirt and avoid contamination during processing. Within 24 h after harvest, processing commenced after ascertaining that the roots were in a fresh state, before the onset of deterioration [14].
Design of Experiment.
Before the conduct of the experiment, Design Expert (DE) software version 11 generated the experimental conditions for processing the flour samples. The conduct of the experiment was done using the central composite design with two independent variables: concentra-tion of pretreatment (COP) and drying temperature (DT). Table 1 shows the levels of the independent variables. Two pretreatments, citric acid (CA) and calcium chloride (CC), were used separately with the same variation in DT giving rise to four experimental groups. The four experimental groups are as follows: citric acid pretreated flour from the red landrace (CAR), calcium chloride pretreated flour from the red landrace (CCR), citric acid pretreated flour from the white landrace (CAW), and calcium chloride pretreated flour from the white landrace (CCW). The dependent variables are the proximate components (starch, ash, moisture, and fibre content) and colour properties (L * , a * , b * , chroma, colour difference, whiteness index, and brownness index) of cassava flour. Each experimental run generated by the DE software was done in triplicate.
2.3. Processing of Pretreated Cassava Flour. The method described by the Federal Institute of Industrial Research [19] was employed, with modifications, in processing the flour. Modifications involved the application of different concentrations of chemical pretreatment and drying temperatures ( Figure 1) for each experimental sample as generated the determination of the following colour properties of the samples: L * (lightness/darkness), a * (redness/greenness), and b * (yellowness/blueness). The colorimeter was calibrated with a standard white (L * = 93:71, a * = -0:84, and b * = 1:83) and black plate before use. Chroma, colour difference (ΔE), whiteness index (WI), and brownness index (BI) were calculated as follows [20]: where x = ða * + 1:75L * Þ/ð5:645L * + a * − 3:012b * Þ. 3 International Journal of Food Science 2.5. Near-Infrared Spectroscopy (NIRS) Analysis of Cassava Flour. Percentage ash, crude fibre, moisture, and starch contents of the samples were measured using an NIR analyser (DA 7250 Perten Instruments, Sweden). The analyser's open-faced sample dish was filled with cassava flour and placed in the NIR analyser. The samples were scanned by infrared rays emitted by the machine, and the software on the PC displayed the readings obtained. The NIR analyser was previously standardised by a selection of calibration and validation samples and reference data obtained by routine laboratory analysis [21].
2.6. Statistical Analysis and Optimisation. Design Expert software version 11 was employed in the analysis of variance (ANOVA) of the linear, interactive, and quadratic effects of the model parameters on cassava flour properties. Regression models, coefficient of determination (R 2 ), p values, F values, response surface plots, contour plots, and optimisation conditions were generated by the software for all four experimental groups. Statistical data analysis was done with the IBM SPSS Statistics software version 25 (IBM Corp., NY, USA) to ascertain the significant difference between means of the experimental and reference/control samples within each experimental group at a 95% confidence level. One-way ANOVA and separation of means were done using Duncan's multiple range test.
Effect of Chemical Pretreatment and Drying
Temperature on the Ash Content of Cassava Flour. The ash content of cassava is an indication of its nonvolatile content and mineral richness [3]. All four experimental groups CAR, CCR, CAW, and CCW had similar ash contents within the range of 0.79 to 4.42%, and they varied significantly within the groups. Significance in variation implies that the processing conditions affected the ash content of flour. Some values of ash content obtained in this study are similar while others were above the range (0.33-1.04%) reported by Ukenye et al. [22] for cassava root. The values (0.74-1.43%) reported by Aniedu and Omodamiro [23] for cassava flour are within the range obtained in this study. Response surface plots for the ash contents of all four experimental groups had a similar shape ( Figure 2) in which the ash contents increased with the concentration of pretreatment and drying temperature. The findings in this study disagree with those of Montagnac et al. [3] who stated that cassava processing significantly reduces the ash content of the roots and Ayetigbo et al. [24] who postulated that severe processing involving the application of high temperatures and chemicals may significantly decrease ash in cassava diets. The increase in ash content as COP increased could be attributed to elements from pretreatment solutions used in soaking fresh cassava chips in water, before drying, which may be retained in the samples after processing. This observation was buttressed by ANOVA of the model parameters F and p values in Table 2 which indicate that the linear, interactive, and quadratic effects of the experimental factors significantly influenced the ash content of cassava flour. Regression models generated by RSM (Table 3) and the coefficient of determination (R 2 ) of the ash content were relatively high between 0.9446 and 0.9710, which show that the model fits.
Effect of Chemical Pretreatment and Drying
Temperature on the Moisture Content of Cassava Flour. High moisture content in foods aids the growth of microbes [25]; hence, drying is applied to eliminate moisture. The cassava flour samples processed under varying conditions had moisture contents between 7.43 and 10.50%. The values in this study are within the percentage moisture range of 3.59 to 11.53 reported by Onitilo et al. [26] for flour from white-and yellow-fleshed cassava roots. The moisture content of the yellow-fleshed cassava root was higher than the white-fleshed roots. This was not the case in this study as Materials, like flour, having above 12.5% moisture possess less storage stability than those with less moisture content; hence, a moisture content of not more than 12.5% is generally specified for flours [27]. The amount of moisture contained in all the samples is below the 12.5% moisture level specified for flours. Therefore, it can be deduced that the pretreatment and drying temperature applied in this study are suitable to produce cassava flour with storage stability and potential for prolonged shelf life.
Effect of Chemical Pretreatment and Drying
Temperature on the Fibre Content of Cassava Flour. Textural behaviour and in vitro digestibility of cassava flour are influenced by its residual fibre [24]. The crude fibre content was between 2.77 and 5.12%. The least and highest crude fibre contents were exhibited by samples of experimental runs 1 (50°C/1%w/v) and 4 (70°C/3%w/v) in all the experimental groups. It gives a clue that the crude fibre content increased with DT and COP. The response surface plots for all experimental groups ( Figure 4) align with this observation. Aniedu and Omodamiro [23] reported lower values (0.62-1.63%) for flour processed from white-flesh and yellow-flesh cassava varieties. The authors reported that the fibre content in cassava flour from the white-flesh variety is generally higher than that of the yellow-flesh variety. Ukenye et al. [22] also reported relatively higher fibre content (0.62-4.92%) in the roots of white-flesh cassava than in those of the yellow-flesh varieties. Differences in fibre content maybe attributed to differences in varietal composition and age of harvest [3]. The International Journal of Food Science process flow in this study did not involve the removal of fibre from the roots (Figure 1) as is done in starch extraction and in some cassava flour processing methods. The omission of this step (removal of fibre) could be responsible for the relatively high fibre content in these samples. ANOVA of model parameters shows that the linear effect of drying temperature and the quadratic effect of concentration of pretreatment significantly influenced (p < 0:05230) the fibre content of the flours. The high fibre content obtained in this study may positively influence the dietary fibre content available in the flours. Consumption of an ample quantity of dietary fibre decreases the risk of diseases such as obesity, constipation, gallstones, diabetes, and coronary heart diseases [28]. This is an indication that the landraces and processing conditions are suitable for producing cassava flour advantageous to the health of consumers. Table 2 and the coefficient of determination (0.2120-0.7792) in Table 3 were relatively low compared to other proximate components of the flour. The ANOVA of model parameters produced p values below 0.05 for the model and linear effect of COP for samples pretreated with citric acid from the white landrace (CAW). This is evident in the response surface plots ( Figure 5) of CAW, the R 2 of 0.7792, and the F value of 15.15 which was higher than all other experimental groups. All other p values were above 0.05 indicating that the model did not fit, while the linear, interactive, and quadratic effects of the processing variables did not have a significant effect on the starch content of the flours. Table 6. The coefficient of determination was between 0.4241 (ΔE-CAR) and 0.9981 (b * -CCW). L * values of the cassava flour samples were between 91.37 and 93.65 with the control sample significantly lower (p < 0:05) than the experimental samples in all four groups. This shows that pretreatment of cassava flour from both landraces with citric acid or calcium chloride enhances the lightness of the flour. Soaking and grating were reported to cause a significant (p < 0:05) increase in the L * values of cassava flour [29]. The significance of the quadratic effect of COP on the L * values (not reported) in all experimental groups and the increase of L * values along the COP axis of the response contour plots (not reported) buttresses the fact that pretreatment improved the lightness of the flour. These findings are in line with the report of Nemaungani et al. [18] on citric acid and calcium chloride pretreated flour processed from solar-dried cassava chips.
Effect of Chemical Pretreatment and Drying
The measure of redness/greenness (a * value) and blueness/yellowness (b * value) of the flour samples ranged from -0.52 to 0.97 and from 4.55 to 8.24, respectively. Hasmadi et al. [30] reported similar a * values (-0.11 and 0.63) and b * values (4.55 and 8.24) for flour processed from two cassava varieties grown in Sabah, Malaysia. The ranges of a * (-1.75 to -0.74) and b * (6.42 to 9.92) values reported by Chiwona-Karltun et al. [29] for cassava flour subjected to different cassava genotypes are also similar to the values obtained in this study. Analysis of variance shows that the a * values of the con-trol sample in citric acid pretreated flours were significantly lower (p < 0:05) than the experimental samples within the respective groups (Tables 7 and 8). Control samples CCR and CCW exhibited significantly higher b * values, chroma, and browning index within their respective experimental groups. This implies that calcium chloride may have a reducing effect on the b * value, chroma, and browning index of the flours.
Change in colour (ΔE) is a function of the difference between the L * , a * , and b * values of the experimental samples and the control. Colour change was the highest (9.54-9.69) in cassava flour processed from the white landrace pretreated with citric acid (CAW) and the lowest (1.11-1.81) in cassava flour processed from the red landrace pretreated with citric acid (CAR). This gives a hint on the effect of landrace on the colour properties of cassava flour. As for the whiteness index (WI), the trend was similar to that of the L * value. The control in all experimental groups had the least WI. The values of L * (87.67-93.57), a * (-0.27-1.1), b * (8.4-11.83), chroma (8.4-11.87), and WI (82.88-89.42) reported in the work of Omolola et al. [17] for cassava flour are similar to those in this study. The findings of Falade and Ayetigbo [31] on the effect of citric acid modification on colour properties of yam starch agree with the trend in this study. Falade and Ayetigbo [31] reported that L * , a * , and WI increased while b * values decreased with citric acid modification of yam starches from four different cultivars.
Multiresponse Optimisation of Cassava Flour Processing.
Multiresponse optimisation of proximate components and colour properties of cassava flour was conducted, separately, using the Design Expert software. The following optimisation goal for the former was set: starch content in range, minimised moisture content, and maximised ash and crude fibre contents. Overlay plots for optimal processing conditions for cassava flour from all four experimental groups with the desired qualities are shown Optimisation of colour properties had the following targets: minimised BI and maximised WI and L * values. The two maximised colour parameters correspond to consumers' preference for cassava flour. Omolola et al. [17] stated that whiteness is the characteristic and acceptable Table 6: Regression models relating model parameters and colour properties of cassava flour.
Conclusion
Application of response surface methodology was effective in investigating the linear, quadratic, and interactive effects of calcium chloride or citric acid pretreatment (0.6-3.4%w /v) and drying temperature (45-74°C) in cassava flour processing. The study showed that the processing conditions yielded cassava flours with good shelf stability, increased ash content, and high fibre content which is good for the health of consumers. An increase in lightness and whiteness index of the flour indicates that both calcium chloride and citric acid pretreatment has mitigating efficacy against enzymatic browning associated with PPD and processing of cassava flour. The chemical pretreatment and drying temperatures had no significant effect (p > 0:05) on the starch content of cassava flours. Response surface plots, multiresponse overlay optimisation plots, and regression models generated from this study assist in eliminating the preexisting gap of the interactive effect of pretreatment and drying temperature on proximate composition and colour properties of cassava flour.
Data Availability
Data underlying the findings in this study can be made available to readers on request. Request can be made to [email protected].
Conflicts of Interest
The authors declare that they have no conflict of interest. | v3-fos-license |
2018-04-03T01:28:28.886Z | 2015-09-26T00:00:00.000 | 29555666 | {
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} | pes2o/s2orc | Crystal structure of (4-hydroxypiperidin-1-yl)[4-(trifluoromethyl)phenyl]methanone
The title compound, C13H14NO2F3, crystallises with two molecules, A and B, in the asymmetric unit, with similar conformations. The dihedral angles between the piperidine and phenyl rings are 83.76 (2) and 75.23 (2)° in molecules A and B, respectively. The bond-angle sums around the N atoms [359.1 and 359.7° for molecules A and B, respectively] indicate sp 2 hybridization for these atoms. In the crystal, O—H⋯O hydrogen bonds link the molecules into separate [100] chains of A and B molecules. The chains are cross-linked by C—H⋯O interactions, generating alternating (001) sheets of A and B molecules.
The motivation for the biological trial arises as piperidine derivatives are an important class of heterocyclic compounds with potent pharmacological/ biological activities (Ramalingan et al., 2004;Ramachandran et al., 2011). Heterocycles with piperidine sub-structures are being used as synthons in the construction of alkaloid natural products (Lee et al., 2001). Piperidine derivatives exhibit a broad-spectrum of biological activities such as anti-bacterial and anti-cancer (Parthiban et al., 2005).
In the title compound,the C-N distances of piperidine ring in molecule .
S2. Experimental
The title compound was synthesized following a published procedure . In a 250 ml round-bottomed flask, 100 ml of ethylmethylketone was added to 4-hydroxypiperidiene (0.03 mol) and stirred at room temperature. After a span of about 5 min, triethylamine (0.03 mol) was added and the mixture was stirred for a time frame of 10 min. 4-Trifloromethylbenzoyl chloride (0.03 mol) was added and the reaction mixture was stirred at room temperature for about 2 h. A white precipitate of triethylammonium chloride was produced, which was filtered and the filtrate was evaporated to get the crude product. Two recrystallizations from ethylmethylketone solution gave colourless blocks of the title compound (yield: 87%).
S3. Refinement
Hydrogen atoms other then hydroxy H atoms were positioned geometrically and treated as riding on their parent atoms and hydroxy H-atoms were located from difference Fourier maps and refined with,C-H distance of 0.93-0.98 Å, with
Figure 1
The molecular structure of the title compound, with displacement ellipsoids drawn at the 30% probability level.
Figure 2
The packing of the molecules in the crystal structure. The dashed lines indicate the hydrogen bonds. where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.27 e Å −3 Δρ min = −0.26 e Å −3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger. Symmetry codes: (i) x+1/2, −y+1/2, z; (ii) x−1/2, −y+1/2, z; (iii) x, y−1, z; (iv) −x+2, −y, z+1/2. | v3-fos-license |
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} | pes2o/s2orc | How the AHR Became Important in Intestinal Homeostasis—A Diurnal FICZ/AHR/CYP1A1 Feedback Controls Both Immunity and Immunopathology
Ever since the 1970s, when profound immunosuppression caused by exogenous dioxin-like compounds was first observed, the involvement of the aryl hydrocarbon receptor (AHR) in immunomodulation has been the focus of considerable research interest. Today it is established that activation of this receptor by its high-affinity endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ), plays important physiological roles in maintaining epithelial barriers. In the gut lumen, the small amounts of FICZ that are produced from L-tryptophan by microbes are normally degraded rapidly by the inducible cytochrome P4501A1 (CYP1A1) enzyme. This review describes how when the metabolic clearance of FICZ is attenuated by inhibition of CYP1A1, this compound passes through the intestinal epithelium to immune cells in the lamina propria. FICZ, the level of which is thus modulated by this autoregulatory loop involving FICZ itself, the AHR and CYP1A1, plays a central role in maintaining gut homeostasis by potently up-regulating the expression of interleukin 22 (IL-22) by group 3 innate lymphoid cells (ILC3s). IL-22 stimulates various epithelial cells to produce antimicrobial peptides and mucus, thereby both strengthening the epithelial barrier against pathogenic microbes and promoting colonization by beneficial bacteria. Dietary phytochemicals stimulate this process by inhibiting CYP1A1 and causing changes in the composition of the intestinal microbiota. The activity of CYP1A1 can be increased by other microbial products, including the short-chain fatty acids, thereby accelerating clearance of FICZ. In particular, butyrate enhances both the level of the AHR and CYP1A1 activity by stimulating histone acetylation, a process involved in the daily cycle of the FICZ/AHR/CYP1A1 feedback loop. It is now of key interest to examine the potential involvement of FICZ, a major physiological activator of the AHR, in inflammatory disorders and autoimmunity.
Introduction
Although the cytochrome P450 (CYP) family of enzymes was initially thought to catalyze the metabolism of xenobiotics and thereby be involved in chemical carcinogenesis, it has since become clear that many of these enzymes also play physiological roles in the metabolism of a variety of endogenous compounds [1]. One of these endogenous compounds is 6-formylindolo [3,2-b]carbazole (FICZ), which binds to the aryl hydrocarbon receptor with the highest affinity yet reported. In stark contrast to the anthropogenic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other well-characterized ligands for the AHR, FICZ is also an excellent substrate for CYP1A1, CYP1A2, and CYP1B1, all encoded by genes regulated by the AHR [1]. Accordingly, FICZ participates in an autoregulatory feedback loop, which maintains its own steady-state concentration, like that of many hormones, at a low level (reviewed in [2]).
FICZ was discovered serendipitously in connection with experiments designed to produce dimerized photoproducts of planar biomolecules for testing as ligands for AHR. Adenine was subjected to ultraviolet radiation in the presence of tryptophan (Trp), a photo-sensitizing molecule, and it turned out that similar irradiation of solutions containing Trp alone produced some compound(s) that could compete efficiently with TCDD for binding to the AHR. Two products with molecular weights of 284 and 312 Daltons were identified and immediately assumed to be endogenous signal substances, since they bind to this receptor with higher affinity than any other known compound, including TCDD [3,4].
Subsequently, FICZ was shown to be formed upon exposure of Trp to H 2 O 2 alone, as well as via several enzyme-catalyzed pathways [5]. To date, FICZ has been detected by liquid chromatographymass spectrometry in aged batches of Trp [3], cell culture media [6,7], cultured non-hematopoietic cells [8], hematopoietic cells [9], yeast cells [10], and extracts of human skin [10,11], as well as in mouse colorectal tissue [12]. In addition, sulfoconjugates of phenolic metabolites of FICZ are present in human urine [13]. Thus, ubiquitous formation/presence of FICZ, albeit at low levels, in most tissues under normal conditions is highly probable. Still, establishing FICZ as an important endogenous AHR agonist has been controversial since many studies have described the AHR to be highly promiscuous and suggested AHR ligand binding by a myriad of both endogenous and exogenous molecules.
Although the AHR is not essential for survival, this receptor is involved in several physiological processes, including regulation of homeostasis and immunity at epithelial barriers such as the one formed by intestinal epithelial cells (IECs) (reviewed by [14]). Since the most potent immune responses in the body occur in the gut, considerable focus is now being placed on elucidating the molecular mechanism(s) underlying the role of the AHR in cells in the intestinal mucosa-including the IECs and various immune cells, such as B cells, T cell receptor γδ T cells (TCRγδ), T helper 17 cells (Th17), regulatory T cells (Treg), type 1 regulatory T cells (Tr1), innate lymphoid cells (ILC), macrophages (MQ), intraepithelial lymphocytes (IEL), dendritic cells (DC), and neutrophils (reviewed by [15]).
CYP1A1 plays an essential role in the intestinal immune system, controlling related steady-state processes, as well as responses to pathogenic insults. In the present review, I discuss new perspectives on the role of FICZ produced by the microbiota in gut immunity, with particular focus on the key role played by CYP1A1 in the dynamic regulation of FICZ-stimulated production of interleukin 22 and the temporal pattern of AHR signaling in the gut.
Activators of the AHR Promote Intestinal Immune Responses
The enormous numbers and huge variety of bacteria in the intestine, as well as the pronounced diurnal oscillations in both the composition and function of the gut microbiome, require an efficient barrier for protection of the host. Normally, abundant secretion of mucus and a vigorous, but closely regulated immune system maintain this barrier, but perturbations of gut microbiota (termed dysbiosis) are associated with several pathological states, including inflammatory bowel disease (IBD), metabolic syndrome, and colorectal cancer (CRC) (reviewed in [16]).
Recently, several important investigations have established that the AHR is required both for regulation of the homeostasis of the intestinal epithelial and associated immune cells, as well as for mounting appropriate responses to epithelial damage and invading pathogens [17][18][19][20][21][22]. In addition, this receptor appears to be involved in peristaltic and secretory reflexes [23,24].
In mice raised in a conventional manner, CYP1A1 is expressed by epithelial cells in the duodenum, jejunum and ileum [25], with the most pronounced up-regulation by TCDD occurring in the proximal parts of the small intestine (SI) [26]. Mice that are germ-free (GF) or have been treated with an antibiotic and are thus exposed to lower levels of factors produced by the microbiota, express the AHR, AHRR, and CYP1A1 genes at lower levels in their SI [25,[27][28][29]. The lack of functional AHR signaling in such animals may explain why they are more susceptible to colitis induced experimentally, e.g., by trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) [30,31]. Such findings highlight the involvement of commensal microbes in the intestinal immune system through their production of factors that activate the AHR.
Dietary Activators of the AHR
Even before Alan Poland described the AHR in 1976 [32], Lee Wattenberg and colleagues had reported an elevated level of CYP1A1 activity (at that time measured as benzo[a]pyrene (BaP) metabolism or aryl hydrocarbon hydroxylase (AHH) activity) in the liver and gastrointestinal tract (GI) of rodents fed standard chow [33]. They discovered subsequently that feeding rats and mice cereal-based chow and synthetic semi-purified diets fortified with phytochemicals isolated from the Brassicaceae family of plants, alfalfa or spinach resulted in a high basal level of highly inducible CYP1A1 activity in their gut, from the gastrointestinal epithelium of the SI to the colon (reviewed by [34]). This basal activity was most pronounced in the proximal region of the SI.
These findings, in combination with the early interest in phytochemicals as potential anticarcinogens, motivated a number of investigations designed to test whether cruciferous vegetables induce AHH activity. For example, indole-3-carbinol (I3C), a hydrolysis product of glucobrassicin produced by myrosinase, was found to increase CYP1A1 activity in the liver and intestine of rodents [35,36]. I3C itself binds to the AHR with low affinity, but under acidic conditions can give rise to indolo[3,2-b]carbazole (ICZ), which has high affinity [37].
Investigations designed to unravel the mechanisms by which dietary phytochemicals induce CYP1A1 in the gut and also influence gut immunity and protect against inflammatory disorders within the GI often involve comparisons between conventional chow based on grain and purified diets (AIN-76, AIN-93, or similar) supplemented with I3C. It is now known that a wide range of phytochemicals-including flavonoids, alkaloids, stilbenes and curcuminoids-bind to this receptor with no or low affinity but still activate CYP1A1, and we have proposed that compounds that inhibit the expression and/or, activity of CYP1A1 may attenuate clearance of endogenous FICZ, leading to indirect activation of the AHR [38]. This may explain why this receptor in the intestine, systemically, is activated by many dietary phytochemicals that are potent inhibitors of CYP1A1 activity [39,40].
Microbial Activators of the AHR
The symbiosis between resident microbiota and the host is beneficial to both in many ways. Under the anaerobic conditions in the gut, the microbiota modify many small molecules present in the diet and these then enter the host circulation and influence immunity and other distal functions in a manner that contributes to the well-being of the host. For instance, gut bacteria give rise to essential vitamins (e.g., A, group B and K), as well as serotonin (5-HT), short-chain fatty acids (SCFAs), and some secondary bile acids [41,42].
It is now evident that indole metabolites of Trp formed in the mammalian intestine play important roles in gut health. In particular, Trp metabolism by Lactobacillus species elicits an anti-microbial immune response including production of IL-22, which is vital for maintenance of mucosal barrier integrity within the SI. Numerous studies with GF rodents or animals treated with antibiotics have demonstrated that reduced microbial catabolism of dietary Trp results in higher concentrations of this amino acid in the serum and or feces and reduced levels of AHR-activating compounds [20,28,[46][47][48].
Impaired Trp transport systems also have negative consequences for the health of the gut. For instance, mice lacking the angiotensin-converting enzyme 2 (Ace2) take up Trp from the diet poorly and also display an altered intestinal microbiome and enhanced susceptibility to the development of colitis [49]. Similarly, the intestinal microbiome in mice that lack the caspase recruitment domain family member 9 (CARD9) is altered and these animals fail to metabolize Trp into activators of the AHR, exhibit defective expression of IL-22 and antimicrobial peptides (AMPs) such as RegIIIβ and RegIIIγ in the colon, and are more susceptible to colitis [19]. Furthermore, defects in the LAT1-CD98 complex, which transports aromatic amino acids, in immune cells limit the intracellular level of FICZ due to lower access to Trp [9]. In contrast, the elevated levels of Trp resulting from the reduced metabolism of this amino acid in knock-out mice that do not express indoleamine 2,3-dioxygenase 1 (IDO1) enhances formation of microbial indoles that activate the AHR [20,48].
A recent update identified I3P as one of the microbial catabolites of Trp that activate the AHR most potently [50]. In a competitive binding assay, I3P displaced TCDD from the mouse AHR with an IC 50 of 55 µM, i.e., more efficiently than IAl (IC 50 > 1 mM), but much less efficiently than FICZ (IC 50 2nM), which was included in this experiment as a positive control, but not considered to be a potential microbial catabolite. However, in another recent study, the levels of FICZ in colorectal mouse tissue were quantified and the results support the notion that this ligand is the most potent AHR-activating Trp catabolite in the intestine [12]. These highly preliminary data require confirmation in further studies.
The species of commensal bacteria that have so far been shown to produce indoles that activate the AHR include certain strains of Lactobacillus, Allobaculum, Peptostreptococcus, and Propionibacterium [20,48,51,52]. However, many other such species are likely to be identified, since the common bacterial inhabitants of the gut, belonging to several different phyla, convert Trp into I3P, Tra, or IAAl [42,44,53], all of which are precursors of FICZ and other AHR-activating indoles as well ( Figure 1). family member 9 (CARD9) is altered and these animals fail to metabolize Trp into activators of the AHR, exhibit defective expression of IL-22 and antimicrobial peptides (AMPs) such as RegIIIβ and RegIIIγ in the colon, and are more susceptible to colitis [19]. Furthermore, defects in the LAT1-CD98 complex, which transports aromatic amino acids, in immune cells limit the intracellular level of FICZ due to lower access to Trp [9]. In contrast, the elevated levels of Trp resulting from the reduced metabolism of this amino acid in knock-out mice that do not express indoleamine 2,3-dioxygenase 1 (IDO1) enhances formation of microbial indoles that activate the AHR [20,48].
A recent update identified I3P as one of the microbial catabolites of Trp that activate the AHR most potently [50]. In a competitive binding assay, I3P displaced TCDD from the mouse AHR with an IC50 of 55 µM, i.e., more efficiently than IAl (IC50 > 1 mM), but much less efficiently than FICZ (IC50 2nM), which was included in this experiment as a positive control, but not considered to be a potential microbial catabolite. However, in another recent study, the levels of FICZ in colorectal mouse tissue were quantified and the results support the notion that this ligand is the most potent AHR-activating Trp catabolite in the intestine [12]. These highly preliminary data require confirmation in further studies.
The species of commensal bacteria that have so far been shown to produce indoles that activate the AHR include certain strains of Lactobacillus, Allobaculum, Peptostreptococcus, and Propionibacterium [20,48,51,52]. However, many other such species are likely to be identified, since the common bacterial inhabitants of the gut, belonging to several different phyla, convert Trp into I3P, Tra, or IAAl [42,44,53], all of which are precursors of FICZ and other AHR-activating indoles as well ( Figure 1).
Figure 1.
A scheme describing the microbiota-mediated catabolism of tryptophan that leads to several compounds that can activate the aryl hydrocarbon receptor (AHR), including the high affinity ligand FICZ (illustrated by an F in a blue triangle). Stimulation with FICZ causes AHR to partner with the nuclear translocator of AHR (ARNT), bind to AHR response elements (AHREs), and stimulate expression of CYP1A1, which takes part in the metabolic clearance of FICZ. The oxidative coupling that generates FICZ from IAAl has been described in [5].
Another group of AHR activators produced by the gut microbiota encompass the SCFAs acetate, propionate and butyrate (BUT), of which the role of BUT in maintaining intestinal immune homeostasis is most well documented. BUT is derived from microbial fermentation of indigestible polysaccharides, in particular dietary fibers and resistant starch, which escape digestion by enzymes in the upper gut and are consequently present at relatively high levels (mM) in the lumen of the lower gut (reviewed in [54]). In addition to providing an important source of energy for colonocytes, BUT inhibits inflammation of the intestine and promotes the development and function of Tregs in a beneficial manner. Many of the anti-inflammatory properties of BUT reflect its inhibition of histone deacetylases (HDACs) and simultaneous activation of certain G-protein-coupled receptors on colonic Figure 1. A scheme describing the microbiota-mediated catabolism of tryptophan that leads to several compounds that can activate the aryl hydrocarbon receptor (AHR), including the high affinity ligand FICZ (illustrated by an F in a blue triangle). Stimulation with FICZ causes AHR to partner with the nuclear translocator of AHR (ARNT), bind to AHR response elements (AHREs), and stimulate expression of CYP1A1, which takes part in the metabolic clearance of FICZ. The oxidative coupling that generates FICZ from IAAl has been described in [5].
Another group of AHR activators produced by the gut microbiota encompass the SCFAs acetate, propionate and butyrate (BUT), of which the role of BUT in maintaining intestinal immune homeostasis is most well documented. BUT is derived from microbial fermentation of indigestible polysaccharides, in particular dietary fibers and resistant starch, which escape digestion by enzymes in the upper gut and are consequently present at relatively high levels (mM) in the lumen of the lower gut (reviewed in [54]). In addition to providing an important source of energy for colonocytes, BUT inhibits inflammation of the intestine and promotes the development and function of Tregs in a beneficial manner. Many of the anti-inflammatory properties of BUT reflect its inhibition of histone deacetylases (HDACs) and simultaneous activation of certain G-protein-coupled receptors on colonic epithelial cells, in particular GPR109A, which is expressed at very high levels by innate immune cells and on the epithelium [55][56][57][58].
Reports published as early as 1996 and 1999 demonstrated that both BUT and trichostatin A, another inhibitor of HDAC, successfully restore expression of the AHR in hepatoma cells deficient in induction of CYP1A1 mRNA, as well as de-repress CYP1A1 expression in fibroblasts non-responsive to ligands of the AHR [59,60]. Several subsequent investigations established that BUT alters expression of the CYP1A1, AHR, and AHRR genes and induces CYP1A1 activity both in different types of cells in vitro [61][62][63][64] and in experimental animals [28,65] by inhibiting HDAC activity. However, although Marinelli and colleagues could reproduce the HDAC-dependent activation of CYP1A1 by trichostatin A, they did not obtain the same effect with BUT, but instead proposed that this compound induced the transcription of AHR-dependent genes as an AHR ligand [66]. Moreover, two other reports documented an increase in the expression of CYP1A1 in cells exposed to BUT alone [62,63]. These latter findings could reflect the presence of FICZ or some other activator of the AHR in the cell culture medium, since, indeed, FICZ has been detected in cell culture media exposed to light [6].
The Mechanism(s) by Which FICZ, IL-22 and Butyrate Promote Gut Homeostasis
Although dietary and microbial indoles that activate the AHR and support colonization by commensal bacteria are necessary for intestinal health, a balance between tolerance to such beneficial bacteria and immunological responses to potential pathogenic species must be maintained [67,68]. In this context, accumulating evidence indicates that the intestinal epithelium requires a constant supply of moderate levels of IL-22, IL-17, and a granulocyte macrophage-colony stimulating factor (GM-CSF) to protect against undesirable microbial invasion [69].
In adult mammals, the group 3 ILCs (ILC3s) are involved in protecting against microbial pathogens, as well as in regulating the integrity of the intestinal barrier and the relative abundance of the populations of various commensal bacteria. At the same time, through their expression of the transcription factor RORγt and cytotoxicity receptor NKp46 (NKp46 + RORγt + ILC3s), these cells are considered to be the most important source of IL-22 in the SI lamina propria [69][70][71][72], where they respond to cues provided by the diet and the commensal microbiota, as well as to their own intrinsic circadian rhythm to produce IL-22 [73,74]. In this connection, a key observation was that the AHR is required for postnatal expansion of such intestinal ILCs [19,75,76]. Indeed, Qiu and colleagues have proposed that in mouse pups after weaning, the AHR responds to bacteria in the gut in a manner that leads to the development of RORγt + ILCs [19].
Furthermore, it is well documented that under steady-state conditions, phytochemicals present in conventional rodent chow promote and sustain IL-22-producing RORγt + ILCs [19,[75][76][77]. Accordingly, the microbial flora, the AHR, and ligands of this receptor that stimulate IL-22 production by intestinal ILC3s are important innate effectors of intestinal health. Figure 2 and the sections below describe how the FICZ that is not broken down in the IECs can support colonization of the gut by beneficial strains of bacteria. epithelial cells, in particular GPR109A, which is expressed at very high levels by innate immune cells and on the epithelium [55][56][57][58].
Reports published as early as 1996 and 1999 demonstrated that both BUT and trichostatin A, another inhibitor of HDAC, successfully restore expression of the AHR in hepatoma cells deficient in induction of CYP1A1 mRNA, as well as de-repress CYP1A1 expression in fibroblasts non-responsive to ligands of the AHR [59,60]. Several subsequent investigations established that BUT alters expression of the CYP1A1, AHR, and AHRR genes and induces CYP1A1 activity both in different types of cells in vitro [61][62][63][64] and in experimental animals [28,65] by inhibiting HDAC activity. However, although Marinelli and colleagues could reproduce the HDAC-dependent activation of CYP1A1 by trichostatin A, they did not obtain the same effect with BUT, but instead proposed that this compound induced the transcription of AHR-dependent genes as an AHR ligand [66]. Moreover, two other reports documented an increase in the expression of CYP1A1 in cells exposed to BUT alone [62,63]. These latter findings could reflect the presence of FICZ or some other activator of the AHR in the cell culture medium, since, indeed, FICZ has been detected in cell culture media exposed to light [6].
The Mechanism(s) by Which FICZ, IL-22 and Butyrate Promote Gut Homeostasis
Although dietary and microbial indoles that activate the AHR and support colonization by commensal bacteria are necessary for intestinal health, a balance between tolerance to such beneficial bacteria and immunological responses to potential pathogenic species must be maintained [67,68]. In this context, accumulating evidence indicates that the intestinal epithelium requires a constant supply of moderate levels of IL-22, IL-17, and a granulocyte macrophage-colony stimulating factor (GM-CSF) to protect against undesirable microbial invasion [69].
In adult mammals, the group 3 ILCs (ILC3s) are involved in protecting against microbial pathogens, as well as in regulating the integrity of the intestinal barrier and the relative abundance of the populations of various commensal bacteria. At the same time, through their expression of the transcription factor RORγt and cytotoxicity receptor NKp46 (NKp46 + RORγt + ILC3s), these cells are considered to be the most important source of IL-22 in the SI lamina propria [69][70][71][72], where they respond to cues provided by the diet and the commensal microbiota, as well as to their own intrinsic circadian rhythm to produce IL-22 [73,74]. In this connection, a key observation was that the AHR is required for postnatal expansion of such intestinal ILCs [19,75,76]. Indeed, Qiu and colleagues have proposed that in mouse pups after weaning, the AHR responds to bacteria in the gut in a manner that leads to the development of RORγt + ILCs [19].
Furthermore, it is well documented that under steady-state conditions, phytochemicals present in conventional rodent chow promote and sustain IL-22-producing RORγt + ILCs [19,[75][76][77]. Accordingly, the microbial flora, the AHR, and ligands of this receptor that stimulate IL-22 production by intestinal ILC3s are important innate effectors of intestinal health. Figure 2 and the sections below describe how the FICZ that is not broken down in the IECs can support colonization of the gut by beneficial strains of bacteria. When the CYP1A1-mediated clearance of microbially produced FICZ in the intestinal epithelium is inhibited by for example dietary phytochemicals, drugs, or inflammatory mediators, this compound will bind to the aryl hydrocarbon receptor (AHR) in RORγt positive group 3 innate lymphoid cells (ILC3s). This stimulates them to secrete the interleukin IL-22 that signals to intestinal epithelial cells (IECs) to emit antimicrobial peptides (AMPs) and mucins, which promote colonization by commensal bacteria.
Repressors of CYP1A1 Prevent the Clearance of FICZ
The importance of a functional AHR-dependent pathway in the intestinal epithelium for induction of CYP1A1 expression by a factor originating in the gut was first demonstrated by the findings by Ito and colleagues in 2007 [78]. In mice (fed standard chow), where the ARNT gene was disrupted, predominantly in IECs, the levels of CYP1A1 mRNA and corresponding enzymatic activities were markedly elevated in almost all other tissues. In another study performed with mice deficient in CYP1A1/1A2/1B1, endogenous AHR ligands that escape breakdown in the epithelial cell lining activate this receptor, as demonstrated with a Cyp1a1 fate-reporter [21].
In addition to such genetic silencing, several types of agents that could prevent the CYP1A1mediated breakdown of FICZ have been described. CYP1A1 activity can be inhibited by external factors consumed orally or formed by microbiota, including dietary phytochemicals such as α-naphthoflavone, β-naphthoflavone, galangin, chrysin, kaempferol, apigenin, baicalein, and quercetin [40], as well as I3C and its acid-condensation products 3,3-diindolylmethane (DIM) and ICZ [21]. In fact, FICZ can slow down its own metabolic degradation by inhibiting CYP1A1 activity [79]. Other inhibitors of CYP1A1 activity include a large number of anti-inflammatory, anti-depressant, anti-parasitic, anti-psoriatic, beta-blocking, and cytostatic drugs, and inhibitors of proton pumps [38,39] and several ubiquitous carcinogenic polycyclic aromatic hydrocarbons (PAHs), exemplified by BaP and 3-methylcholanthrene (with IC 50 values in the nM range) [80]. In the studies referred to above, the ability to inhibit 7-ethoxyresorufin O-deethylation activity was used as a measure of CYP1A1 inhibition, and the inhibitors were mostly substrates that competed with 7-ethoxyresorufin for binding to the enzyme. Thus, the inhibitors of CYP1A1 that has been shown to attenuate metabolic clearance of FICZ include a wide range of phytochemicals [81], environmental pollutants [82], metals, and oxidants [38,83,84].
Moreover, signals released in connection with microbial-associated molecular patterns (MAMPs) or pathogen-associated microbial patterns (PAMPs) can interact with pattern recognition receptors (PRRs) on the surface of IECs, DCs and MQs to trigger downstream signaling cascades. This promotes the production of mediators of inflammation or infection, such as IL-1β, IL-6, TNFα, and IFNs, which also leads to inhibition of CYP1A1 expression and/or activity (reviewed in [85]).
Theoretically, several molecular mechanisms can be involved in the downregulation of the expression and/or activity of CYP1A1, e.g., competitive or mixed inhibition of the enzyme, alterations caused by reactive oxygen species, or chromatin remodeling in the promoter region of this gene. Regardless of the mechanism involved, lower CYP1A1 activity permits higher concentrations of FICZ to reach innate lymphoid cells that express IL-22.
FICZ Induces Expression of IL-22 by ILC3s
IL-22 binds to the heterodimeric receptor IL-22Rα1/IL-10Rβ on IECs and induces a downstream signaling cascade that leads ultimately to phosphorylation of the transcription factor STAT3. This signaling supports the epithelial barrier against bacterial infections by stimulating secretion of AMPs, e.g., RegIIIβ, RegIIIγ, and members of the S100 family of proteins by enterocytes and Paneth cells in the SI and enterocytes in the colon of mice [86,87]. Paneth cells sense commensal bacteria in the gut and aid in broad regulation of both commensal and pathogenic bacteria that maintain intestinal homeostasis [88,89]. Furthermore, IL-22 controls tissue regeneration and repair through direct action on epithelial stem cells [90], as well as inducing epithelial goblet cells to secrete more components of mucus to form a thick gel-like layer impenetrable to many commensal bacteria, thereby limiting their potential to cause inflammation [91].
As mentioned above, RORγt + ILCs appear to be the principal source of IL-22 under steady-state conditions and their constitutive expression of this cytokine is apparently unaffected by the proinflammatory IL-23 cytokine, which is known to activate IL-22 production as part of the response to pathogens [92]. The strict dependence of the ILC3s and their secretion of IL-22 can be explained by the presence of AHR-responsive elements in the promoter region and intron 1 of the IL-22 gene [19]. Moreover, recruitment of the transcription factor RORγt to the IL-22 promoter is facilitated by the AHR [19,93].
Intriguingly, circadian regulation of the numbers of these cells and their expression of circadian clock genes, AHR and IL-22 was recently documented [73].
The fact that exposure of T cells to FICZ, under conditions that induce Th17-cells, potently up-regulates their level of IL-22 mRNA, was reported in 2008 [94,95]. Subsequently, expression of this same mRNA by differentiated mouse Th17 cells was found to be elevated in the presence of as little as 10 pM FICZ, and this induction was enhanced considerably by co-exposure to fluoranthene, pyrene, and phenanthrene, environmental PAHs that inhibit CYP1A1 [21,82].
It is now clear that FICZ stimulates the expression of IL-22 by a variety of different immune cells, including ILC3s, both in vitro [21,82,[96][97][98], including human intestinal lamina propria mononuclear cells from IBD patients [18], and in experimental animals [18,20,29,98,99]. In this context, Monteleone and colleagues (2011) found that a remarkably low amount of FICZ ameliorates colitis induced experimentally in mice [18]. A single intraperitoneal administration of 1 µg per mouse (50 µg/kg) lowered the mortality, enhanced the level of IL-22 in colonic samples, and ameliorated colitis induced by TNBS or DSS. As proof-of-concept, they also demonstrated that a neutralizing antibody against IL-22 largely prevented these anti-inflammatory effects of FICZ.
IL-22 Promotes Colonization by Commensal Bacteria
Normally, more than 1 × 10 13 bacteria, predominantly of the Firmicutes and Bacteroidetes phyla, symbiotically colonize the mammalian intestine. Microbial density is lowest in the SI and the microbiota demonstrate diurnal rhythmicity with respect to both their localization and production of metabolites that depend on light exposure, the time of food consumption and the type of food [16,[107][108][109].
IL-22 has been shown to be needed to promote the colonization of the GI by beneficial bacteria and gut homeostasis both in studies with mice lacking this cytokine and mice treated with antibodies against it [110]. Zenewich and co-workers found that healthy IL-22 KO mice have an altered colonic microbiome containing lower relative abundances of certain families of bacteria, including Lactobacillaceae, Bacteroidaceae, Clostridiaceae, and Peptococcaceae, and more of others. In addition, when colitis was induced experimentally into these animals, they developed more severe disease. Moreover, when their altered gut microbiota were transferred to wild-type (WT) mice in the same cage, these wild-type animals also exhibited enhanced susceptibility to experimental colitis [110].
Notably, bacteria that metabolize Trp are more abundant in the SI, where constitutive expression of IL-22 helps to shape and constrain the commensal community [20,111]. It has been proposed that colonization by bacteria that metabolize Trp and/or promote health in other ways is regulated by the availability of mucins, as well as by antimicrobial proteins that may increase the proportion of Lactobacillus [20,48,110].
In comparison to the diverse microbiota of mice reared on conventional grain-based chow, the immune phenotype of the microbiome of mice fed purified, phytochemical-free diets (often termed AHR ligand-free diets) is changed in a manner similar to that seen in AHR-deficient animals [28,112,113]. Like the AHR-deficient mice, animals fed purified, phytochemical-free diets exhibit enhanced susceptibility to severe colitis [113][114][115]. Furthermore, there are numerous reports that phytochemicals-including berberine [116], curcumin [117], galangin [118], resveratrol [119], and rutin [120]-can prevent microbial dysbiosis and stimulate colonization of the gut by beneficial anaerobic bacteria (reviewed by [121,122]). 13C, which is commonly used to activate the AHR and stimulates IL-22 expression, can also promote colonization by beneficial bacteria, both when administered in the diet [113] and injected intraperitoneally [123]. Accordingly, Schanz found that purified diets exert a profound negative impact on the composition of the microbiome of the murine SI and colon, lowering counts of Gram-positive bacteria belonging to the Firmicutes phylum, such as Clostridium butyricum, Faecalibacterium prausnitzii, Roseburia, and Lactobacillus species [113]. Importantly, this deleterious change could be reversed by addition of I3C to the diet. Interestingly, the beneficial effects of 13C even after intraperitoneal administration [123] exclude the possibility that DIM and ICZ, acid condensation products of this compound that are formed in the stomach [37], were the protective compounds.
Furthermore, BUT concentrations were higher in mice fed conventional chow diets than in mice fed purified diets [124], and butyrate-producing Roseburia spp. were more common in mice fed purified diets and administered I3C intraperitoneally [123], which illustrates the important role of phytochemicals in supporting the growth of BUT-producing bacteria. The lower portion of the SI (ileum) and colon, where microbial density is highest, contain large numbers of BUT-producing bacteria belonging to the Firmicutes phyla (e.g., the Bacteroides fragilis and Clostridium clusters IV and XIVa strains) [55,125]. Commensal bacteria that produce BUT and utilize mucins as an energy source are capable of penetrating the inner mucus layer and stimulating the underlying IECs to produce mucin peptides, as well as of supporting the production of different SCFAs. For example, acetate that contributes to the production of BUT must sometimes be supplied through co-colonization by primary fiber degraders that initiate the utilization of complex fibers [54].
The observations described above indicate that conventional chow and diets enriched in phytochemicals influence the composition of the intestinal microbiota in a manner that favors the production of both FICZ and BUT. As expected, this production of beneficial microbial metabolites is reduced in germ-free animals [20,28,[46][47][48].
BUT Fine-Tunes IL-22 Signaling
BUT helps maintain immunological homeostasis in the gut by inducing the differentiation of IL-10-producing Treg cells [55] and Tr1 cells [126], in addition to its indispensable counteraction of the pro-inflammatory responses associated with the signaling pathways involving activated nuclear factor-kB (NF-kB) and IL-6/STAT3/IL-17 [127][128][129]. IL-10-producing Tregs play a particularly important role in limiting inflammatory responses and there are relatively high numbers of these cells in the lamina propria (LP) of the ileum and the colon, where the density of BUT-producing bacteria is also higher than in the SI [108]. BUT induces the differentiation and expansion of Tregs by inhibiting HDACs, which results in acetylation of the histones associated with the promoter region of the forkhead box P3 (FoxP3) gene [55]. Differentiation of FoxP3-negative Tr1 cells is enhanced by this same inhibition in combination with signaling through GPR109A [126].
When the density of bacteria that produce BUT is low, inhibitors of CYP1A1 activity can elevate the steady-state level of FICZ to which the LP is exposed, thereby stimulating the production of IL-22 by RORγt + ILC3s (Figure 3a). When BUT-producing bacteria are abundant, inhibition of HDAC by BUT enhances the expression and thereby activity of CYP1A1 (for references see Section 2.2). Crucially, this results in more extensive clearance of FICZ, preventing this substance from continuing to stimulate IL-22 production by ILC3s (Figure 3b).
Diurnal Rhythmicity in CYP1A1 Activity
The findings of Schiering and colleagues linked an absence of CYP1A1 activity in murine IECs to elevated numbers of ILC3s and Th17s in the colon, more IL-22 protein in cultures of colon explants, and an increased response to pathogens [21]. In contrast, the colon of mice whose IECs expressed CYP1A1 constitutively contained substantially fewer ILC3s and Th17s and less IL-22 protein, and these animals were more susceptible to enteric infection. These observations demonstrate that, at least in mice, CYP1A1 activity in IECs control gut levels of IL-22, and that to avoid inflammatory disorders, these levels must be regulated.
Interestingly, there are pronounced diurnal fluctuations in the composition of the intestinal microbiome [130], with as much as a 10-fold difference in the number of bacteria that adhere to the intestinal epithelium at night than day. Moreover, the amounts of SCFAs and other microbial products fluctuate in response to the nature and timing of the diet [130,131]. Consequently, microbial production of ligands derived from Trp and of BUT in the intestine varies during the 24-h day. Moreover, the level of mRNA encoding and activity of CYP1A1 in the liver and lungs of rodents also oscillate during the day [132,133], as do hepatic levels of AHR and ARNT mRNA and protein [133,134], as well as AHR mRNA in enteric ILC3s [73]. However, since few researchers in this field are aware of these fluctuations, few experimental studies include sampling at different times throughout the day.
In summary, a diurnal oscillation in the FICZ/AHR/CYP1A1 autoregulatory loop aids colonization of the gut by a symbiotic microbiome, helping to maintain tolerance to beneficial bacteria (Figure 3c).
Diurnal Rhythmicity in CYP1A1 Activity
The findings of Schiering and colleagues linked an absence of CYP1A1 activity in murine IECs to elevated numbers of ILC3s and Th17s in the colon, more IL-22 protein in cultures of colon explants, and an increased response to pathogens [21]. In contrast, the colon of mice whose IECs expressed CYP1A1 constitutively contained substantially fewer ILC3s and Th17s and less IL-22 protein, and these animals were more susceptible to enteric infection. These observations demonstrate that, at least in mice, CYP1A1 activity in IECs control gut levels of IL-22, and that to avoid inflammatory disorders, these levels must be regulated.
Interestingly, there are pronounced diurnal fluctuations in the composition of the intestinal microbiome [130], with as much as a 10-fold difference in the number of bacteria that adhere to the intestinal epithelium at night than day. Moreover, the amounts of SCFAs and other microbial products fluctuate in response to the nature and timing of the diet [130,131]. Consequently, microbial production of ligands derived from Trp and of BUT in the intestine varies during the 24-h day. Moreover, the level of mRNA encoding and activity of CYP1A1 in the liver and lungs of rodents also oscillate during the day [132,133], as do hepatic levels of AHR and ARNT mRNA and protein [133,134], as well as AHR mRNA in enteric ILC3s [73]. However, since few researchers in this field are aware of these fluctuations, few experimental studies include sampling at different times throughout the day.
In summary, a diurnal oscillation in the FICZ/AHR/CYP1A1 autoregulatory loop aids colonization of the gut by a symbiotic microbiome, helping to maintain tolerance to beneficial bacteria (Figure 3c).
When the Microbial Homeostasis in the Gut Is Disrupted
The fact that IL-22 production by ILC3s has both beneficial and deleterious effects has led several reviews to refer to IL-22 as a two-faced cytokine or a double-edged sword [67,69,74,111]. Under healthy conditions, IL-22 is constitutively expressed by ILC3s in the SI independently of IL-23 [92,111] and barely detectable in the colonic mucosa [91,104,135].
In order to keep pathogenic microbes such as Citrobacter rodentium under control in the colon, expression of AMPs by epithelial cells is induced via a process involving IL-23 signaling and early production of IL-22, with subsequent expression of IL-17 that acts synergistically with IL-22 [67,69,70,136]. Qiu and colleagues (2012) found that AHR-deficient ILCs lack the IL-23 receptor (IL-23R) and that AHR KO mice express IL-22 at reduced levels and, unlike wild-type mice, succumb to infection with C. rodentium. Administration of a plasmid encoding IL-22 protects such AHR KO animals from early mortality [19]. In line with this, ILC3s in the colon of patients suffering from IBD are dysregulated and express abnormally high levels of both IL-22 and IL-17 [69]. An important observation that has been repeatedly seen is that IBD patients display dramatically augmented microbial dysbiosis, in particular with a reduced abundance of bacteria that produce BUT [137][138][139]. A significant decrease of butyrate production has also been documented to occur in patients suffering from other autoimmune diseases such as type 2 diabetes [140], Behçet syndrome [141], and rheumatoid arthritis [65].
Together, the data documented in Table 1 suggest that, during infections, control of endogenous AHR signaling is required, because otherwise excessive and sustained production of IL-22 may exert deleterious effects on the colon that could lead to chronic inflammatory disorders and potential autoimmunity.
Conclusions
The observations described above indicate that dietary phytochemicals activate AHR-dependent immune processes, not as ligands to the AHR, but by influencing the composition of the intestinal microbiota in a manner that favors the production of both FICZ and BUT.
Unfortunately, the thousands of experimental studies on colitis that have been reported-involving the suffering and distress of large numbers of laboratory animals-had not yet led to effective treatment of patients with IBD. Novel strategies based on our knowledge concerning the involvement of FICZ in intestinal immunity may lead to more effective control of this disease, as well as of other autoimmune diseases, since FICZ can activate tolerogenic T cells (reviewed in [2]), in addition to the stimulation of systemic IL-22 signaling described in this review. However, as mentioned above, the biological functions of IL-22 are complex and therefore more mechanistic information is needed.
The most striking insight that comes from earlier work in our own laboratory in combination with the literature reviewed here is that rhythmic variations in the levels of FICZ and IL-22 are required for the maintenance of gut homeostasis.
•
When CYP1A1 activity is too low (resulting in high levels of FICZ), defenses against commensal and pathogenic microbes are boosted.
• On the other hand, when CYP1A1 activity is too high (low FICZ levels), the host becomes susceptible to infections. • Diurnal fluctuations in CYP1A1 activity fine-tune the activity of IL-22.
Therefore, a more detailed understanding of the diurnal regulation of crosstalk between FICZ and commensal bacteria that produce BUT, both when the intestinal barrier is functioning normally and during periods of infection, could pave the way for novel therapies for IBD, other autoimmune diseases, and possibly, for CRC as well.
Funding: This research received no external funding.
Acknowledgments: I wish to thank Ulf Ran nug for fruitful discussions and help in reviewing the manuscript.
Conflicts of Interest:
The author declares no conflict of interest. T cell receptor TGF-β tumor growth factor beta TNBS trinitrobenzene sulfonic acid Tra tryptamine Tr1 type 1 regulatory T cells Treg regulatory T cells Trp tryptophan UC Ulcerative colitis WT wild type | v3-fos-license |
2020-11-18T14:07:00.778Z | 2020-11-16T00:00:00.000 | 226990500 | {
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} | pes2o/s2orc | Vav2 catalysis-dependent pathways contribute to skeletal muscle growth and metabolic homeostasis
Skeletal muscle promotes metabolic balance by regulating glucose uptake and the stimulation of multiple interorgan crosstalk. We show here that the catalytic activity of Vav2, a Rho GTPase activator, modulates the signaling output of the IGF1- and insulin-stimulated phosphatidylinositol 3-kinase pathway in that tissue. Consistent with this, mice bearing a Vav2 protein with decreased catalytic activity exhibit reduced muscle mass, lack of proper insulin responsiveness and, at much later times, a metabolic syndrome-like condition. Conversely, mice expressing a catalytically hyperactive Vav2 develop muscle hypertrophy and increased insulin responsiveness. Of note, while hypoactive Vav2 predisposes to, hyperactive Vav2 protects against high fat diet-induced metabolic imbalance. These data unveil a regulatory layer affecting the signaling output of insulin family factors in muscle.
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Only significant outliers were excluded from the analysis. In figure 7c, one Vav2 L33A/L332A animal was excluded (0 min time-point). In figure S16c, one CD-fed WT was excluded in the 0 min time-point. In figure 7k, the 120 min time-point of a WT animal was excluded (lower than the 90 min time-point for the same mouse and significantly lower than the other animals). In figure 1a, one WT mice was excluded due to its low lean mass and abnormal metabolic parameters. In figure S11c, a 2-month-old Vav2 L332A/L332A animal was excluded due to its high adiposity content. In Fig. 8A, a WT animal had to be excluded from the final analysis and sacrificed due to health problems.
The number of independent replicates for each experiment is indicated at the corresponding figure legend in the manuscript. In general, at least three independent replicates were performed.
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For all animal studies, the investigators were blind to group allocation. Blinding was not applicable to the rest of experiments. Commercially available antibodies (see above) have been validated by the manufacturer for the application (immunoblot, immunoprecipitation or immnunocytochemistry) and species. This information is available at each manufacturer's website and can be obtained through the catalog numbers indicated above. The homemade Vav2 antibody has been validated by us in overexpression, knockdown and knockout experiments. | v3-fos-license |
2020-04-16T09:22:09.111Z | 2020-04-11T00:00:00.000 | 216496549 | {
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} | pes2o/s2orc | Chitoheptaose Promotes Heart Rehabilitation in a Rat Myocarditis Model by Improving Antioxidant, Anti-Inflammatory, and Antiapoptotic Properties.
Background Myocarditis is one of the important causes of dilated cardiomyopathy, cardiac morbidity, and mortality worldwide. Chitosan oligosaccharides (COS) may have anti-inflammatory and cardioprotective effects on myocarditis. However, the exact molecular mechanism for the effects of functional COS on myocarditis remains unclear. Methods Anti-inflammatory activities of COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) were measured in lipopolysaccharide- (LPS-) stimulated RAW264.7 cells. A rat model with myocarditis was established and treated with chitopentaose, chitohexaose, chitoheptaose, and chitooctaose. Serum COS were measured by using high-performance liquid chromatography (HPLC) in all rats. Myocarditis injury, the levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), inflammatory factors, and apoptotic factors were also measured. Pearson's correlation coefficient test was used to explore the relationship between the levels of ROS/RNS and cardiac parameters. Results Among all chitosan oligosaccharides, the COS > degrees of polymerization (DP) 4 showed anti-inflammatory activities (the activity order was chitopentaose<chitohexaose<chitoheptaose<chitooctaose) by reducing the levels of interleukin- (IL-) 1β, IL-17A, and interferon- (IFN-) γ and increasing the level of IL-10. However, the serum level of chitooctaose was low whereas it showed significant therapeutic effects on myocarditis by improving cardiac parameters (left ventricular internal dimension, both end-systolic and end-diastolic, ejection fraction, and fractional shortening), inflammatory cytokines (IL-1β, IL-10, IL-17A, and IFN-γ), oxidative factors (ROS and RNS), and apoptotic factors (caspase 3, BAX, and BCL-2) when compared with chitopentaose, chitohexaose, and chitooctaose (COS DP > 4). The levels of ROS/RNS had a strong relationship with cardiac parameters. Conclusions Chitoheptaose plays a myriad of cardioprotective roles in the myocarditis model via its antioxidant, anti-inflammatory, and antiapoptotic activities.
Introduction
Myocarditis is an inflammation of the myocardium, which is one of the important causes of dilated cardiomyopathy [1] and cardiac morbidity and mortality worldwide [2]. The proinflammatory factors include the following: high-level interleukin-(IL-) 1β is associated with high myocarditis risk [3] and IL-1 receptor antagonist (IL-1ra) can prevent cardiac dysfunction [4]. Myocarditis involves many myocardial inflammatory diseases, such as acute myocarditis, chronic myocarditis, and inflammatory cardiomyopathies, and inflammatory diseases with cardiomyopathies [5]. Oxidative stress is also an important risk in the pathogenesis of myocarditis [6]. Inflammatory and oxidative stress can affect the left ventricular internal diameter in end diastole (LVIDd) and left ventricular internal diameter in end systole (LVIDs) [7].
The volume of the ejection fraction (EF) is often measured clinically to assess cardiac mechanics and function and significantly reduced in the progression of myocarditis [8]. The value of fractional shortening (FS) is widely used to assess left ventricular dysfunction and often decreased after myocarditis [9]. On the other hand, preserved EF [10] is associated with predominant inflammation and oxidant stress.
IL-17A is a CD4 + T cell-derived proinflammatory cytokine and plays an important role in the pathogenesis of myocarditis [11]. Interferon-(IFN-) γ is an important immune regulator in normal immunity and involved in the regulation of most immune and inflammatory responses. IL-10 is a type 2 cytokine, which inhibits proinflammatory cytokine formation. Lowering the IFN-γ level and increasing the IL-10 level will reduce the progression of myocarditis [12].
Reactive oxygen species (ROS) [13] and reactive nitrogen species (RNS) [14] are normally produced highly in myocarditis. Antioxidants can ameliorate cardiac apoptosis and dysfunction in an animal model with autoimmune myocarditis [15,16]. From the above information, antioxidant and anti-inflammatory therapy has become a very important approach in preventing myocarditis progress.
Chitosan oligosaccharides (COS) are homo-or heterooligomers of N-acetylglucosamine and D-glucosamine and active and have more physiological functions than chitosan with solubility in water. COS exert cardiac protective effects on the patients with coronary heart disease (CHD) by improving antioxidant capacity [17]. COS also possess anti-inflammatory properties by activating mitogen-activated protein kinase (MAPK) signaling [18]. However, the effects of COS on myocarditis and which oligosaccharide of COS plays a more important functional role still remain unclear. Normally, long-chain oligosaccharides (degrees of polymerization ðDPÞ > 5) may have an excellent function than short-chain ones [19]. The effects of COS with different DP on myocarditis were explored in a rat model.
Establishment of the Experimental Autoimmune
Myocarditis (EAM) Model. Before the present experiment, all procedures were approved by the animal research ethics committee of China-Japan Union Hospital of Jilin University (approval no. 2017CCZYY03017). Forty-eight 8-week-old specific pathogen-free (SPF) rats (200-220 g) were purchased from the animal center of Jilin University. Porcine cardiac myosin (PCM, Sigma Aldrich, M0531) was dissolved in 0.10 mol/L PBS solution (10 g/L). The solution was mixed with complete Freund's adjuvant (CFA, Sigma Aldrich, F5881) in an equal volume of 1 : 1 to make it a homogeneous emulsion. On the first day and the eighth day, 200 μL of myosin (1 mg) was injected subcutaneously into the bilateral inguinal and axillary sites of the 40 rats. The control group (n = 8) was immunized with myosin-free PBS and CFA emulsion. From day 1, all rats were housed at a temperature of 23 ± 1°C in a 12 h/12 h daylight/dark cycle and allowed indicated food and water ad libitum.
Animal Grouping.
After the establishment of an EAM model, the mice were orally treated with 100 μM different DP of COS (chitopentaose, chitohexaose, chitoheptaose, and chitooctaose in saline solution) daily for one month. Forty-eight rats were evenly divided into the CG group (control), MG group (EAM model), PG group (chitopentaosetreated EAM model), HexG group (chitohexaose-treated EAM model), HepG group (chitoheptaose-treated EAM model), and OG group (chitooctaose-treated EAM model) ( Figure S1). The mice were treated with equal-volume saline solution in the CG and MG groups.
Echocardiography Analysis of Cardiac Parameters.
After one-month intervention, all rats were anesthetized by intraperitoneal injection of 10% chloral (300 mg/kg). After depilation of the chest, echocardiography was performed on the left side of the supine position, and the experiments were performed in triplicate. Cardiac parameters were measured by echocardiography, including the left ventricular internal diameter in end diastole (LVIDd), left ventricular internal diameter in end systole (LVIDs), and ejection fraction (EF). Fractional shortening (FS) was calculated as ð½LVIDd − LVIDs/LVIDdÞ × 100.
2.6. HPLC Analysis of Serum COS. Theoretically, the larger size oligosaccharide will be more difficult to be absorbed into blood vessels. Therefore, it is necessary to measure the serum level of different DP of COS. After one-month COS intervention, serum levels of COS were measured in all rats ( Figure S1). One mL of blood was sampled from each rat tail in anticoagulant-free tubes and kept at room temperature for 1 h before the serum was isolated (centrifugation at 1,500 g for 10 min at 22°C). Serum levels of COS were analyzed by using HPLC (Varian 920-LC, HPLC-DAD/UV system, Varian, Inc., Palo Alto, CA, USA) [21] according to COS standards (chitopentaose, chitohexaose, chitoheptaose, and chitooctaose). The column was Metacarb 87H (300 × 7:8 mm, Varian, USA) with an RID detector; the flow rate was set at 0.6 mL/min, and the temperature was 80°C. A sample of 20 μL was injected into the HPLC.
Oxidative Stress Measurement.
After one-month COS intervention, the oxidative stress levels were measured ( Figure S1) by using reactive oxygen species (ROS) and reactive nitrogen species (RNS) via DCF DA and/or DAF-FM DA fluorescence. Cardiac tissues were digested into a single cell by trypsin. The single cells were resuspended in RPMI 1640 medium (Gibco) and adjusted to 1 × 10 6 and were incubated using DCF DA with DAF-FM DA for 15 min at 37°C in the dark. The cells were washed with fresh medium twice and transferred to PBS buffer (20 mM, pH 7.0). The fluorescence intensity was measured using Synergy H1 Hybrid Reader (BioTek Instruments, Winooski, VT, USA).
2.9. Analysis of Myocardial Histopathology. After one-month COS intervention, myocardial histopathology was analyzed ( Figure S1). The rat chest was opened to expose the anterior cardiac region, and the heart was separated and removed. After being repeatedly washed with cold PBS on ice, the heart was cut longitudinally into two parts along the coronal plane, half of which was fixed in 10% formaldehyde solution for HE staining and immunohistochemistry, and the other part was stored at -80°C for subsequent analysis of the mRNA and protein expression of caspase 3, BAX, and BCL-2 ( Figure S1). The myocardial tissue fixed in 10% formaldehyde solution was dehydrated with gradient ethanol and treated with xylene I/II, respectively, and then dipped in benzene/wax I/wax II and embedded in solid paraffin at 65~70°C. After cooling, the paraffin was cut into 2-3 μm slices and treated in warm water at about 56°C for 30 min at 70°C. The heart tissue of each group of rats was subjected to hematoxylineosin (HE) staining and myocarditis injury, and inflammatory status was observed. H&E stain was scored to assess infiltration based on the following scale: 0 = normal myocardium, 1 = mild myocarditis (<5% cross section of infiltration), 2 = moderate myocarditis (5-10% cross section of infiltration), 3 = marked myocarditis (10-25% cross section of infiltration), and 4 = severe myocarditis (>25% cross section of infiltration).
2.11. Western Blot. Ten mg heart tissues were ground into a fine powder using a mortar and pestle in liquid nitrogen. The ground powder was placed into the Qiagen TissueLyser II® for lysing (MD, USA). The mixture was centrifuged at 12,000 g for 10 min, and the supernatants were obtained for Western blot analysis. Total proteins were separated by 10% SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 5% nonfat milk in TBS-T buffer for 2 h, incubated with specific primary antibodies overnight at 4°C, washed with TBS-T, and incubated with peroxidaseconjugated antibodies. Protein bands were observed by using 3 Oxidative Medicine and Cellular Longevity chemiluminescence, and density was analyzed using ImageJ software and normalized with β-actin.
2.12. Statistical Analysis. The data were processed by SPSS17.0 software (SPSS Statistics, IBM, Armonk, NY, USA). The measurement data were expressed as mean ± S:D. and analyzed by using the t-test, and the count data were analyzed by the χ 2 test. The Wilcoxon rank-sum test was used to evaluate grade data. One-way (with 1 independent variable) analysis of covariance (ANCOVA) was used to explore the response of the dependent variable among all groups. Pearson's correlation coefficient test was used to explore the relationship between the levels of ROS/RNS and the values of cardiac parameters. The difference was statistically significant if p < 0:05.
Chitoheptaose Improved Cardiac Function Better Than
Other COS. After the model establishment, the values of LVIDd (Figure 3(a)) and LVIDs (Figure 3(b)) in the MG group were higher than those in the CG group whereas the percent of EF (Figure 3(c)) and FS (Figure 3(d)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes cardiac injury. Meanwhile, chitoheptaose showed significant therapeutic effects by reducing the values of LVIDd (Figure 3(a)) and LVIDs (Figure 3(b)) and increasing the percent of EF (Figure 3(c)) and FS (Figure 3(d)) when compared with other COS (p < 0:05).
Chitoheptaose Prevented Myocarditis Injury Better Than
Other COS. H&E staining of the whole heart showed that the myocardium was thickest and the cells with light blue may be necrotic in the MG group when compared with other groups, and chitoheptaose showed significant inhibitory effects on the thickness of the myocardium and necrosis situation ( Figure S2). H&E analysis showed that myocarditis injury was observed with damaged myomuscular fiber and abundant inflammatory cells in the MG group after the model establishment (Figure 4). COS intervention repaired the damage, prevented the injury in the HepG group, and reduced the amounts of inflammatory cells. Chitoheptaose showed significant therapeutic effects by reducing more pathological scores and inflammatory situation when compared with other COS (Figure 4, p < 0:05).
Chitoheptaose Showed Higher Anti-Inflammatory Effects
Than Other COS. After the model establishment, the serum levels of IL-1β ( Figure 5(a)), IL-17A ( Figure 5(b)), and IFN-γ ( Figure 5(c)) in the MG group were more than those in the CG group whereas the level of IL-10 ( Figure 5(d)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes inflammatory responses. Meanwhile, chitoheptaose showed significant anti-inflammatory properties by reducing the serum levels of IL-1β ( Figure 5(a)), IL-17A ( Figure 5(b)), and IFN-γ ( Figure 5(c)) and increasing the level of EF ( Figure 5(d)) when compared with other COS (p < 0:05).
Chitoheptaose Showed Higher Antioxidant Effects Than
Other COS. After the model establishment, the levels of ROS (Figure 6(a)) and RNS (Figure 6(b)) in the MG group were higher than those in the CG group. The results suggest the model establishment increases the oxidative stress. Meanwhile, chitoheptaose showed significant antioxidant properties by reducing the levels of ROS (Figure 6(a)) and RNS (Figure 6(b)) when compared with other COS (p < 0:05).
Chitoheptaose Intervention Reduced
More the Relative mRNA Level of Apoptotic Factors Than Other COS. After the model establishment, the relative mRNA levels of caspase 3 (Figure 7(a)) and BAX (Figure 7(b)) in the MG group were Oxidative Medicine and Cellular Longevity higher than those in the CG group whereas the level of BCL-2 (Figure 7(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes the apoptotic responses. Meanwhile, chitoheptaose showed higher antiapoptotic properties by reducing the relative mRNA levels of caspase 3 (Figure 7(a)) and BAX (Figure 7(b)) and increasing the level of BCL-2 (Figure 7(c)) when compared with other COS (p < 0:05).
Chitoheptaose Intervention Reduced More the Relative Protein Level of Apoptotic Factors Than Other COS. For
Western blot analysis, three samples were measured from each group. Figure 8 shows the Western blot analysis for sample 1 from each group. Supplementary Figure S3 shows the Western blot analysis of samples 2 and 3 from each group. After the model establishment, the relative protein levels of caspase 3 (Figure 8(a)) and BAX (Figure 8(b)) in the MG group were more than those in the CG group whereas the level of BCL-2 (Figure 8(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes apoptotic responses. Meanwhile, chitoheptaose showed higher antiapoptotic properties by HepG: chitoheptaose-treated group; OG: chitooctaose-treated group. n = 3 for each group. A p < 0:05 vs. the CG group, B p < 0:05 vs. the MG group, C p < 0:05 vs. the PG group, D p < 0:05 vs. the HexG group, E p < 0:05 vs. the HepG group, and F p < 0:05 vs. the OG group. 8 Oxidative Medicine and Cellular Longevity reducing the relative protein levels of caspase 3 (Figure 8(a)) and BAX (Figure 8(b)) and increasing the level of BCL-2 ( Figure 8(c)) when compared with other COS (p < 0:05).
Chitoheptaose Intervention Reduced the Expression of
Apoptotic Factors More Than Other COS. IHC analysis showed that the IHC scores were lowest in the NC group and highest in the PC group, suggesting that the method was conducted successfully (Figure 9(a)). After the model establishment, the relative expression of caspase 3 (Figure 9(a)) and BAX (Figure 9(b)) in the MG group was higher than that in the CG group whereas the expression of BCL-2 (Figure 9(c)) in the MG group was lower than that in the CG group. The results suggest the model establishment causes apoptotic responses. Meanwhile, chitoheptaose showed significant antiapoptotic properties by reducing the relative expression of caspase 3 (Figure 9(a)) and BAX (Figure 9(b)) and increasing the level of BCL-2 (Figure 9(c)) when compared with other COS (p < 0:05).
Whole heart staining of IHC also showed that the caspase 3 ( Figure S4A) and BAX ( Figure S4B) expression was highest in the MG group and significantly inhibited in the HepG group. In contrast, the BCL-2 expression was lowest in the MG group and significantly improved in the HepG group ( Figure S4C).
Discussion
The present findings demonstrated that COS improved cardiac function by increasing the antioxidant and antiinflammatory capacities and reducing the apoptotic risk in a rat model with myocarditis. COS can exert better physiological function than chitosan with excellent water solubility. Theoretically, the COS with high degrees of polymerization will be with high bioactivity than the low-degree ones. Thus, different COS (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, chitoheptaose, and chitooctaose) were explored by using the rat model with myocarditis. Just This study showed that cardiac parameters (LVIDd, LVIDs, EF, and FS) were improved significantly in the HepG group compared with the other groups ( Figure 3). The results suggest that chitoheptaose shows higher therapeutic effects . CG: control group; MG: model group; PG: chitopentaose-treated group; HexG: chitohexaose-treated group; HepG: chitoheptaose-treated group; OG: chitooctaose-treated group; NC: negative control group without primary antibody; PC: positive control group with the primary antibody for β-actin. n = 8 for each group. A p < 0:05 vs. the CG group, B p < 0:05 vs. the MG group, C p < 0:05 vs. the PG group, D p < 0:05 vs. the HexG group, E p < 0:05 vs. the HepG group, F p < 0:05 vs. the OG group, G p < 0:05 vs. the NC group (negative control group without primary antibody), and H p < 0:05 vs. the PC group (positive control group with primary antibody for β-actin).
than chitooctaose according to the improvement of cardiac parameters although chitooctaose has higher antiinflammatory activity than chitoheptaose. The reason may be that chitooctaose is more difficult than chitoheptaose to be absorbed into the blood vessels in the small intestine in the rat models because the former has the longer chain. Just as we proposed, the short-chain COS in serum reached the highest level in the PG group and the longchain COS in serum reached the lowest level in the OG group (Figure 2). On the other hand, apoptotic activity is generally following oxidative [22,23] and inflammatory responses [24,25]. Therefore, the effects of COS on the apoptotic activity were also investigated here. Just as the antioxidant and antiinflammatory properties, the COS showed similar antiapoptotic activity in different COS. Chitoheptaose showed higher antiapoptotic properties than other COS (Figures 7-9). The results suggest that oxidative stress, inflammation, and apoptosis may affect each other.
The correlation test showed that the oxidative stress levels (ROS and RNS) had a strong relationship with the cardiac parameters. The results suggest that chitoheptaose may exert its cardioprotective function by affecting oxidative stress. Further work is highly needed to approve the central role for the correction among inflammatory, oxidative stress, and apoptotic activities in the pathogenesis of myocarditis. The results indicated that the COS with DP 7 exerted the highest antioxidant and anti-inflammatory activity in the animal model, which suggested that their activity had a close relationship with the degree of polymerization of COS. Larger oligosaccharides (chitohexaose and chitoheptaose) have the highest capability for scavenging DPPH [26]. Chitoheptaose has higher capability for scavenging ROS and RNS when compared with other COS and effectively scavenges ROS and RNS generated by electron leakage and protects cells against apoptosis induced by ROS and RNS. Chitoheptaose treatment showed the highest protective effects on the EAM model among the COS chitopentaose, chitohexaose, chitoheptaose, and chitooctaose. The effects of chitopentaose and chitooctaose were minimal when compared with the EAM model only treated with saline solution (Figure 9). Larger COS become more potent, and the biological activity of COS is size-dependent. Chitoheptaose was most active compound as the COS with the DP > 4. However, much work is needed to explore the exact molecular mechanism.
There were some limitations in the present work. Only 4 cardiac parameters were investigated here, and the interaction of these parameters was too complex to be explored. The relationship between the levels of ROS and RNS and the values of the parameters of cardiac parameters was only analyzed by using Pearson's correlation coefficient test and not demonstrated by the experiments. Thus, the mechanisms were examined by using an animal model with CHD via related gene overexpression or silence in future work. The effects of the interaction among different COS on heart therapy are very complex, and it is difficult to understand its exact functional molecular mechanism. The deep molecular mechanism should be explored in the subsequent experiment.
Data Availability
All data are available from the corresponding author on reasonable request. | v3-fos-license |
2020-09-17T13:06:18.341Z | 2020-09-15T00:00:00.000 | 221747401 | {
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} | pes2o/s2orc | Sustained absorption of delamanid from lipid-based formulations as a path to reduced frequency of administration
Delamanid is a poorly water-soluble drug currently being used for the treatment of tuberculosis. The high frequency of dosing leads to poor adherence for patients who live in lower economic and nomadic populations. Non-digestible self-assembling lipids as a formulation approach for poorly water-soluble drugs have previously been shown to extend the window of absorption through gastric retention. We hypothesise that this approach could lead to the reduction of dosing frequency for delamanid and thereby has potential to improve adherence. Formulations of delamanid were prepared in selachyl alcohol and phytantriol as non-digestible self-assembling lipid vehicles, and their behaviour was compared with reconstituted milk powder, as a digestible lipid-based formulation, and an aqueous suspension. The self-assembly of selachyl alcohol and phytantriol in aqueous media in the presence of delamanid was studied using small angle X-ray scattering and produced the inverse hexagonal (H2) and inverse bicontinuous cubic (V2) liquid crystal structures, respectively. The times at which maximum delamanid levels in plasma were observed (Tmax) after oral administration of the phytantriol, selachyl alcohol and reconstituted milk powder formulations of delamanid to rats were 27 ± 3, 20 ± 4 and 6.5 ± 1.0 h, respectively, compared with the aqueous suspension formulation with a Tmax of 3.4 ± 1 h, which confirms the hypothesis of an extended duration of absorption after administration in non-digestible self-assembling lipids. The digestion products of the triglycerides in the milk formulation increased the solubilisation of delamanid in the gastrointestinal tract, leading to an increase in exposure compared with the aqueous suspension formulation but did not significantly extend Tmax. Overall, the non-digestible nanostructured lipid formulations extended the duration of absorption of delamanid well beyond that from milk or suspension formulations. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s13346-020-00851-z) contains supplementary material, which is available to authorized users.
Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis remains one of the leading causes of mortality worldwide with over 95% of deaths occurring in low-and middle-income countries [1]. Current first-line treatments for TB typically consist of 6-9 months of oral administration of multiple drugs taken daily or several times a week; and drugs for second-line treatment are administered when resistance to the first-line TB drugs has been diagnosed. Delamanid (previously known as OPC-67683, see Fig. 1 for chemical structure) is a nitrodihydro-imidazooxazole derivative known to exhibit potency against multidrug-resistant tuberculosis (MDR-TB) strains [2,3], which can be used in a combination drug treatment for MDR-TB. The recommended dose of commercially available delamanid (Deltyba) in adults is 100 mg daily (two tablets twice a day) for a 6-month period of treatment administered Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13346-020-00851-z) contains supplementary material, which is available to authorized users.
with an appropriate combination drug [4]. Given the complexity of dosing and its likely impact on patient adherence, oral dosage forms for sustained release of drug can potentially be developed to minimise the frequency of dosing and to prevent excessive fluctuations in plasma drug concentrations [5].
Lipids are common excipients that have been used as oral formulations for the delivery of poorly water-soluble drugs [6]. There has been recent interest in lipid systems that can form and maintain ordered liquid crystal structures [inverse bicontinuous cubic phases (V 2 ) and inverse hexagonal phases (H 2 )] in the gastrointestinal (GI) environment as a means of extending the duration of drug absorption. While triglyceride-based lipids transiently form these ordered structures in the aqueous environment of the GI tract during digestion [7,8], other lipids form these structures intrinsically in water and the structures persist on exposure to aqueous gastrointestinal environments. Generally, lipids that are readily and rapidly digestible in the GI tract exhibit rapid drug release while slowly digested or non-digestible lipids that form these structures have been shown to provide a delayed drug release profile [6,9]. Examples of non-digestible lipids that form ordered phases are phytantriol (PHY) and selachyl alcohol (SA) [10,11], while glyceryl monooleate (GMO) is a digestible liquid crystal-forming lipid [10,12]. Phytantriol self-assembles in aqueous environments to form a V 2 cubic phase while SA forms an H 2 phase. The non-digestibility of the lipids provides for persistence of the ordered structure in the GI tract, which correlates with gastric retention. The combination of these properties provided an increase in oral bioavailability of the poorly water-soluble drug cinnarizine when administered in PHY or SA, compared with administration in a digestible liquid crystalforming lipid (GMO) [10,11].
In this study, we aim to investigate whether prolonged absorption of delamanid could be achieved following administration with the non-digestible lipids PHY and SA, and how it may be linked to the lipid self-assembly behaviour (Fig. 1). The impact of addition of delamanid on the liquid crystalline structures formed by PHY and SA was studied using small angle X-ray scattering (SAXS). The pharmacokinetic behaviour of delamanid released from PHY and SA was subsequently compared with a milk-based formulation as a digestible triglyceride-based comparator.
Self-assembly behaviour of phytantriol and selachyl alcohol: SAXS studies Bulk PHY and SA (about 30 mg) were added to 1 mL of water, 0.1 M HCl solution (simulated gastric condition) or bile salt micelle solution (simulated small intestinal condition) with and without delamanid (drug/lipid ratio of 1:10 w/w, selected as a compromise between analytical sensitivity and volume of administration in later in vivo studies), and the samples were incubated at 37°C for 48 h. The bile salt micelle solution (4.70 mM NaTDC and 0.98 mM DOPC), based on infant fasted conditions [13], was prepared in Tris buffer (50 mM trizma maleate, 5 mM calcium chloride dihydrate, 150 mM sodium chloride and 6 mM sodium azide) at pH 6.5, described in significant detail previously [14]. The self-assembled structures of PHY and SA in the three aqueous solutions were characterised using the SAXS/WAXS beamline at the Australian Synchrotron (ANSTO, VIC, Australia) [15]. Samples were loaded onto a 96-well temperature-controlled plate and sealed using Kapton (polyimide) tape. The plate (held at 37°C) was mounted in the path of the X-ray beam (photon energy: 12 keV and wavelength: 1.033 Å), and measurements were taken with a 1 s acquisition time. A Pilatus 1 M (170 mm × 170 mm) detector, located approximately 1540 mm from the sample position, was used to generate two-dimensional SAXS patterns, which were reduced to scattering functions plotted as intensity (I) versus scattering vector (q = (4π/λ)sinθ where λ = X-ray wavelength and 2θ = scattering angle) using Scatterbrain software version 2.71 developed in-house at the Australian Synchrotron. Suspensions of delamanid in water and the simulated gastric and intestinal solutions containing no PHY or SA were also analysed. Samples were loaded into 1.5 mm outer diameter glass capillaries (Charles Supper, Westborough, MA, USA) and placed on a temperature-controlled capillary holder maintained at 37°C. SAXS measurements were performed using the parameters described above.
Solubility of delamanid in lipid-based formulations
The solubility of delamanid in PHY, SA and milk (9% fat to match the dose of PHY/SA, prepared by reconstituting milk powder in Tris buffer) was measured to estimate drug loading for the in vivo pharmacokinetic studies. To determine the equilibrium solubility of delamanid in PHY and SA, excess delamanid was added into microcentrifuge tubes containing molten PHY and SA (prepared by incubating the lipids at 37°C). The lipid + drug mixtures were vortexed and incubated at 37°C on a roller mixer. Samples were removed after 4, 7 and 10 days of incubation, and centrifuged at 16,162g for 30 min. Approximately 100 mg of the upper lipid layer was transferred into ultracentrifuge tubes, and the samples were recentrifuged at 434,902g for 60 min at 37°C. About 20 mg of the lipid supernatant layer was collected and dissolved in 1:1 tetrahydrofuran:acetonitrile volume ratio. The samples were further diluted with acetonitrile (1:1 volume ratio) prior to injection into a Shimadzu (Shimadzu, Kyoto, Japan) HPLC system consisting of a CBM-20A system controller, an LC-30AD solvent delivery module, a SIL-30AC auto-sampler and a CTO-20AC column oven coupled with an SPD-M30A diode array UV detector set to integrate at 254 nm. Chromatographic separation of delamanid was performed at 35°C on a Waters Symmetry C 18 column (3.5 μm, 100 Å, 4.6 × 75 mm). Delamanid was assayed using a binary gradient elution with a flow rate of 0.5 mL/min: 30-90% B for 10 min, 90-30% B for 3 min and 30% B for 4 min. Buffer A was 1% v/v glacial acetic acid in water, and buffer B was 1% v/v glacial acetic acid in acetonitrile. Delamanid stock solutions (in ACN) were used to provide a standard reference range of 0.1-40 μg/mL in ACN. The injection volume was 10 μL and the retention time for delamanid was 6.9 min.
To determine the equilibrium solubility of delamanid in high fat (9% w/v) reconstituted milk, excess delamanid was added to the milk. The drug + milk mixtures were vortexed and incubated at 4°C with constant stirring. Samples (200 μL) were removed at specified time points (3 h, 24 h and 3 days) and w ere placed into ultracentrifuge tubes and ultracentrifuged at 434,902g for 60 min at 25°C. The resultant aqueous, lipid and pellet layers were collected separately followed by extraction of delamanid using acetonitrile (for the aqueous layer) or 1:4 v/v methanol/acetonitrile (for the lipid and pellet layers) containing diazepam as the internal standard for HPLC. The amount of delamanid partitioned into the individual digested phases was quantified using the HPLC method described in the "Quantification of delamanid in plasma using HPLC" section.
Bioavailability of delamanid in rats
Preparation of formulations for oral administration Delamanid was loaded into bulk PHY, bulk SA and milk, at a drug/lipid ratio of 1:10 w/w and a fixed dose of 10 mg delamanid per kilogram of rat. Aqueous suspensions of delamanid (containing 0.5% v/v sodium carboxymethylcellulose and 0.4% v/v Tween 80 in saline) were also prepared as control formulations. The drug aqueous suspensions and the lipid-containing formulations were equilibrated for 24 h at 37°C prior to dosing.
Animal procedures and sample collection
All animal studies were approved and conducted in accordance with the guidelines of the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee. Male Sprague Dawley rats (280-320 g) were used for the pharmacokinetic studies, and the rats were divided into 4 treatment groups with 4 rats per treatment: delamanid in aqueous suspension, PHY, SA and milk formulations. Rats were anaesthetised via inhalation of 5% isoflurane (and maintained at 2% for the duration of the procedure). The right carotid artery was isolated and cannulated with a 0.80 mm OD × 0.50 mm ID polyethylene tubing to allow serial blood sampling. Cannulas were filled and flushed with 10 IU/mL heparin in normal saline to maintain cannula patency. The cannula was tunnelled to back of the neck. The rats were then placed in a tether swivel system in individual wire bottom metabolic cages to recover overnight prior to dosing and blood sampling. The rats were then fasted for at least 12 h prior to dosing and 8 h after dosing, with water provided ad libitum. The lipid formulations and the saline suspensions were dosed via oral gavage (225 μL), with the dose accurately calculated by weighing the gavage and syringe before and after dosing. Serial blood sampling was performed at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 24, 36, 48, 60, 72 and 96 h (up to 48 h for milk) with 250 μL drawn at each time point. Blood samples were dispensed in 1.5 mL microcentrifuge tubes containing 10 IU sodium heparin, which were then centrifuged for 5 min at 6700g. Aliquots of plasma (50 μL) were collected in duplicates, and samples were kept at − 20°C until quantification of delamanid using HPLC.
Quantification of delamanid in plasma using HPLC
The collected plasma samples (50 μL) were spiked with 10 μL of diazepam solution (10 μg/mL in acetonitrile) as an internal standard followed by the addition of 90 μL acetonitrile. Samples were then centrifuged at 16,162g for 7 min at room temperature, and 95 μL of the supernatant were transferred to HPLC vials. Plasma standards were prepared by spiking delamanid stock solutions (in ACN) into plasma to give a standard reference range of 5-1000 ng/mL. The injection volume was 50 μL. Chromatographic separation of delamanid was performed using HPLC methods described in the "Solubility of delamanid in lipid-based formulations" section with slight modification to the elution gradient: 30-50% B for 7 min, 50% B for 0.5 min, 50-70% B for 2.5 min, 70% B for 1 min, 70-90% B for 1 min, 90-30% B for 1 min and 30% B for 4 min. The retention time for diazepam was 9.6 min and for delamanid was 10.3 min.
Determination of pharmacokinetic parameters
Concentrations of delamanid in plasma were dose-normalised to 10 mg/kg to account for variations in animal weight. Values of C max (maximum drug concentration in plasma) and T max (time to reach C max ) were determined from the normalised data. The truncated area under the curve (AUC) from 0 to the last measurable time points (AUC 0-tlast ) was determined using the trapezoidal rule. SPSS for Windows (Version 23) was used to statistically determine differences in the data using one-way ANOVA analysis of variance with Tukey's multiple comparison, assuming statistical significance when p value was ≤ 0.05.
Solubilisation of delamanid in milk during in vitro lipolysis
In vitro lipolysis of milk mixed with delamanid was performed to use the in vitro solubilisation data to interpret the bioavailability of delamanid. Delamanid (22.5 mg) was added to 2.5 mL of water containing 0.25 mL of 1 M HCl solution. The drug sample was vortexed thoroughly prior to the addition of 17.5 mL of reconstituted powdered milk (9% fat in the Tris buffer) containing bile salt micelles with final concentrations of 4.7 mM NaTDC and 0.98 mM DOPC. The drug + milk mixture (22.5 mg delamanid/1.58 g milk fat) was incubated in a thermostated (37°C) glass vessel under constant magnetic stirring. The pH of the mixture was adjusted to 6.500 ± 0.005 prior to the initiation of lipolysis with pancreatin lipase suspension (2.25 mL,~700 TBU/mL of digest). The pancreatin lipase solution was prepared from pancreatin extract using methods previously described [16]. The pH of the sample in the digestion vessel was maintained at 6.5 during digestion using 2 M NaOH solution titrated into the digesta by a pH STAT control module (Metrohm AG, Herisau, Switzerland). Samples (200 μL) were collected before (0 min) and after digestion (60 min) into ultracentrifuge tubes, and 2 μL of lipase inhibitor (0.5 M 4-BPBA in methanol) was added into each sample before ultracentrifugation at 434,902g for 1 h at 37°C. The resultant aqueous, lipid and pellet layers were collected separately after ultracentrifugation and extraction of delamanid from the three separate layers were performed using methods described in the "Solubility of delamanid in lipid-based formulations" section. Delamanid concentrations in each separated layer were determined based on the standard curve made by spiking delamanid stock solutions (in ACN) in digested milk with concentrations ranging from 0.1-250 μg/mL. The delamanid concentration was quantified using the HPLC method described in the "Quantification of delamanid in plasma using HPLC" section.
Results and discussion
Self-assembly behaviour of phytantriol and selachyl alcohol in the presence of delamanid Formation of lipid liquid crystal structures in the gastrointestinal tract is known to dictate the partitioning of poorly watersoluble drugs and their oral bioavailability [9]. Characterising the self-assembly properties of PHY and SA in the gastrointestinal condition and the impact of incorporated drug is therefore important in understanding how these lipids behave during their passage through the gastrointestinal tract. The phase behaviour of bulk PHY and SA after exposure to different aqueous solution conditions (water, simulated gastric and simulated small intestinal conditions) was characterised using synchrotron SAXS to confirm the formation of lyotropic liquid crystalline structures due to self-assembly of the amphiphilic molecules. The scattering profiles in Fig. 2 show that an inverse bicontinuous cubic phase (V 2 ) with a Pn3m space group was formed after hydration of PHY in excess water as observed in previous studies [11,19]. In comparison, SA selfassembled in water to form an inverse hexagonal phase [20] (H 2 ; relative peak ratios of 1, ffiffi ffi 3 p and ffiffi ffi 4 p ) [17], although H 2 phases with smaller lattice parameters relating to nonequilibrium structures were also present (see Table 1). These liquid crystal structures were not significantly impacted by the addition of delamanid. Similarly, no structural changes occurred following incubation of PHY and SA with and without delamanid at low pH (gastric) conditions, which was not unexpected considering the non-ionic nature of the lipids. Figure 2 shows the influence of bile salt micelles (NaTDC/ DOPC) on the lyotropic liquid crystal structures formed by self-assembly of PHY and SA. Changes in structure of the V 2 and H 2 phases occurred following addition of surfactants at an intestinal pH of 6.5. For PHY, the Pn3m phase remained stable but there was an increase in lattice parameter of the Pn3m phase from 64 to 87 Å (92 Å for PHY with delamanid). For SA, the H 2 structure transformed into to a Pn3m cubic phase upon addition of bile salt micelles (Table 1). Addition of bile salts to PHY and SA therefore resulted in a less negative lipid curvature that could arise due to interaction between the bile acids and the lipid components, increasing the average size of the polar headgroups, and/or by decreasing the rigidity of the lipid bilayers [21]. It should be noted that the amount of bile salt micelles added to the aqueous buffer was within the concentration range generally reported for simulated human fasted intestinal fluid [22] and may not reflect the in vivo conditions in rats, in which higher concentrations of bile acids up to about 50 mM have been observed [23]. The high concentrations of bile salts could therefore potentially further reduce the negative curvature of the lipids from Pn3m and H 2 towards complete incorporation of PHY and SA into the bile salt mixed micelles, completely disrupting the inverse liquid crystalline phases.
Pharmacokinetic studies of delamanid
Mean concentrations of delamanid in plasma after oral administration in saline solution, PHY, SA and milk are shown in Fig. 3. Prolonged and increased levels of exposure to delamanid could be observed for all the lipid-based formulations compared with the saline suspension, particularly when administered with SA and PHY. The extent of drug exposure (determined by the area under the curve in the plasma concentration-time plot) and the maximum concentration of delamanid (C max ) achievable at a specific time point (T max ) are summarised in Table 2.
Overall, the relative bioavailability of the lipid formulations, calculated by the ratio of the AUC in the lipid formulations to delamanid aqueous saline suspension after dose normalisation, was highest for SA followed by PHY and milk. Analysis of the pharmacokinetic parameters using ANOVA revealed statistical differences in the C max , T max and AUC 0-last between PHY, SA, and delamanid suspensions with p values ≤ 0.05. However, no statistical differences were observed in the T max and AUC 0-last (p values > 0.05) between delamanid in the aqueous suspension and milk, although the mean C max for delamanid in milk was significantly greater.
As shown in Table 2, the mean T max was shortest when delamanid was administered in aqueous saline and milk suspensions. This indicated a rapid absorption of delamanid from the saline and milk suspensions compared with the other two formulations, although the lower mean C max and AUC 0-last meant that delamanid was less bioavailable from the milk and aqueous suspension when compared with PHY and SA.
The solubility of delamanid in PHY and SA was experimentally determined to be~0.14 ± 0.01 and 0.41 ± 0.03 mg drug/g lipid, respectively, compared with 3.29 ± 0.04 mg/g milk lipid (overall solubility in reconstituted milk at 9% fat was~0.38 ± 0.01 mg/mL). The lower drug exposure from the milk formulation may therefore be attributed to the loss of drug solubilisation capacity following digestion of milk due to the formation of increasingly hydrophilic lipids during digestion. Digestion of the lipids in milk and the release of fatty acids, which have been previously shown to improve bioavailability of other poorly water-soluble drugs [24], were apparently insufficient to support the complete solubilisation of delamanid, despite the higher solubility of DEL in undigested milk compared with PHY and SA.
To further support the findings that digestion of milk did not result in improved drug solubilisation, in vitro digestion of delamanid in the reconstituted powdered milk (9% fat w/v) was performed and the amount of drug partitioned into the Fig. S1 showed that the amount of delamanid partitioned into the lipid and aqueous phases of milk before and after 60 min of digestion were not significantly different (p value > 0.05). The solubilisation capacity of delamanid in the digested milk at the 60 min time-point was also not statistically significantly different to the equilibrium solubility of delamanid in 9% fat milk. It was therefore likely that digestion of milk did not improve the solubility of delamanid; hence, there was no substantial improvement in oral bioavailability using the milk formulation.
The pharmacokinetic profiles for PHY and SA showed that absorption of delamanid occurred for an extended period of time, and a significant increase in the oral bioavailability was realised. Delamanid was formulated in PHY and SA bulk lipid at a 10:1 lipid:drug mixture, which was in excess of the measured equilibrium solubilities. This meant that excess drug crystals were present due to low drug solubility. It has been previously demonstrated that PHY and SA are retained in the stomach for extended periods of time (> 24 h for PHY) [10]. Previous studies with these lipids have typically had drug in solution in the lipid which resulted in T max values of 33.0 ± 5.0 and 23.5 ± 5.9 h, respectively [10]. However, one study incorporated gold nanoparticles in PHY that were also retained in the stomach for extended periods (> 8 h) compared with a digestible lipid system when measured by X-ray computer tomographic imaging [25]. Thus it is not unexpected that a suspension of drug in these lipids is also likely to be retained for an extended period of time in the stomach. The chemical stability of PHY and SA against digestive enzymes appears to prevent these systems from exiting the stomach as quickly as digestible lipid systems or aqueous suspensions. The gastric compartment then has the potential to act in a non-sink condition, requiring drug released from the lipid suspension matrix to leave the stomach before further dissolution and partitioning can occur into the gastrointestinal fluids. Thus, the long duration of release can be understood through likely gastric retention already well demonstrated for these selfassembling lipid systems, but for the first time here with drug in suspension rather than fully dissolved.
Perhaps of greater interest are the differences in C max and AUC for the SA formulation compared with the PHY formulation. There is more drug in solution in the SA formulation than the PHY formulation by virtue of the greater solubility of delamanid in the host lipid, so it is likely that some lipid erosion occurs together with the drug release described above, leading to more drug arriving in the intestine in a dissolved state and commensurately greater absorption. The kinetic profiles are very similar, indicating that there are likely differences in the amount of drug in solution and available for absorption and that this drives the differences in plasma concentrations of drug over time. While the formation of self-assembled structures using non-digestible lipids has been previously shown to be critical in observing the long duration gastric retention behaviour, the type of liquid crystal structure formed does not appear to play a major role in the process. The self-assembly of the lipids under intestinal conditions was the same (both formed the Pn3m cubic phase under intestinal conditions, Table 1) so phase behaviour in the intestine is unlikely to have played a major role in the differences in overall exposure observed between the PHY and SA formulations. The similar shape of the kinetic profiles also suggests that the differences in phase behaviour in the gastric conditions (inverse hexagonal phase vs cubic phase) did not appear to play a significant role either. Thus, it is the propensity to form a liquid crystalline structure with non-digestible lipids, rather than the lipid and structure per se that appears to be important.
The PHY and SA formulations show a clear ability to provide enhanced drug exposure over at least a 24 h period. In the context of reducing pill burden and the potential for coformulation with a second once-daily drug in combination with delamanid, the lipid formulations studied here have potential to enable improved administration regimes including once daily dosing, which would provide an important opportunity to simplify treatment for tuberculosis in challenging populations. The two non-digestible lipids investigated here do not yet have GRAS status, although phytantriol [26] is a commercial cosmetic ingredient and selachyl alcohol [27] is an endogenous lipid also used in cosmetics. The oral LD50 values of phytantriol and related compounds are high > 2000 mg/kg in rats, so it is unlikely that they would show unusual toxicity behaviour; however, this remains to be determined in future formal preclinical and clinical safety studies.
Conclusions
This study showed the ability of phytantriol (PHY) and selachyl alcohol (SA) to sustain the release of delamanid from lipid formulations when administered in rats. The simple approach incorporates the non-digestible nature of lipids with prolonged gastric retention. In contrast, oral administration of delamanid with digestible lipids in the form of milk did not show sustained drug release characteristics. Comparable drug exposure between milk and an aqueous saline formulation was seen, which also highlighted the limited effect of milk fat digestion on the absorption of delamanid. Our findings suggested the potential use of non-digestible lipids as formulation strategies to improve the oral bioavailability of delamanid and extend the duration of drug exposure in plasma, with the potential to improve patient compliance by reducing the frequency of treatment. | v3-fos-license |
2016-10-25T01:49:57.267Z | 2017-01-01T00:00:00.000 | 4377394 | {
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} | pes2o/s2orc | Multiple roles of GluN2B-containing NMDA receptors in synaptic plasticity in juvenile hippocampus
In the CA1 area of the hippocampus N-methyl-d-aspartate receptors (NMDARs) mediate the induction of long-term depression (LTD), short-term potentiation (STP) and long-term potentiation (LTP). All of these forms of synaptic plasticity can be readily studied in juvenile hippocampal slices but the involvement of particular NMDAR subunits in the induction of these different forms of synaptic plasticity is currently unclear. Here, using NVP-AAM077, Ro 25-6981 and UBP145 to target GluN2A-, 2B- and 2D-containing NMDARs respectively, we show that GluN2B-containing NMDARs (GluN2B) are involved in the induction of LTD, STP and LTP in slices prepared from P14 rat hippocampus. A concentration of Ro (1 μM) that selectively blocks GluN2B-containing diheteromers is able to block LTD. It also inhibits a component of STP without affecting LTP. A higher concentration of Ro (10 μM), that also inhibits GluN2A/B triheteromers, blocks LTP. UBP145 selectively inhibits the Ro-sensitive component of STP whereas NVP inhibits LTP. These data are consistent with a role of GluN2B diheretomers in LTD, a role of both GluN2B- and GluN2D- containing NMDARs in STP and a role of GluN2A/B triheteromers in LTP. This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.
Introduction
N-Methyl-D-aspartate receptors (NMDARs) are centrally involved in synaptic transmission, synaptic plasticity and learning and memory Collingridge, 2013, 1993;Volianskis et al., 2015). NMDARs are glutamate gated ion channels that form heterotetrameric complexes. They usually consist of two GluN1 subunits and two GluN2 subunits, of which there are four possible types (GluN2A, 2B, 2C, 2D) (Collingridge et al., 2009). At the majority of excitatory synapses, and notably the CA1 area of the hippocampus, the induction of long-term potentiation (LTP) and one form of long-term depression (LTD) requires NMDA receptor activation. Use of the NMDAR specific antagonist, D-AP5, demonstrated the receptor involvement in LTP (Collingridge et al., 1983) and de novo LTD (Dudek and Bear, 1992) in rodent hippocampal slices.
It is possible that there are age-dependent differences in experimental observations due to a developmental change in subunit composition of NMDARs with GluN2As being expressed less at younger ages (Barria and Malinow, 2005;Loftis and Janowsky, 2003;Monyer et al., 1994). This cannot, however, be the only explanation since at a given stage of development there is also controversy regarding the role of GluN2A and GluN2B subunits (Berberich et al., 2007(Berberich et al., , 2005Liu et al., 2004;Morishita et al., 2007).
Another complicating factor could be in the selectivity profiles of the pharmacological agents that are commonly used to investigate the role of the different NMDA receptor subtypes. The most commonly used antagonists have a narrow selectivity window or, in the case of ifenprodil-like GluN2B antagonists, a complex mode of action (Fischer et al., 1997;Hansen et al., 2014).
Recently we rigorously characterized three antagonists NVP-AAM077 (NVP), Ro 25-6981 (Ro) and UBP145 and showed that they could be used to identify the roles of GluN2A, GluN2Bcontaining diheteromers, GluN2A/B triheteromers and GluN2Dcontaining NMDARs in synaptic plasticity in the CA1 region of adult rat hippocampal slices (Volianskis et al., 2013a). We found that the predominant receptor required for the induction of LTP was the GluN2A/B triheteromer. In addition we found that a significant component of short-term potentiation (STP), an initial decremental phase of LTP that is observed following high frequency activation and low frequency test stimulation (Volianskis and Jensen, 2003), involved both GluN2B and GluN2D subunits (Volianskis et al., 2013a).
In the present study we have investigated the role of NMDAR subunits in LTP and de novo LTD in P14 animals using these three antagonists. In particular, we also sought to establish the role of GluN2B and GluN2D-containing NMDARs (GluN2B, GluN2D) in rats of this age.
Test stimuli (100 ms) were delivered at 0.033 Hz through bipolar nickel-chromium electrodes, which were placed in the CA1 area of the hippocampal slice to stimulate the Schaffer collateral fibres.
Field excitatory postsynaptic potentials (fEPSPs) were recorded using glass microelectrodes filled with 3 M NaCl solution (resis-tance~2e5 MU) and positioned in the stratum radiatum of the CA1. A 30 min baseline was recorded at a stimulus intensity that gave 60e70% of the maximal response. LTD was induced by low frequency stimulation (LFS, 1 Hz stimulation for 15 min) and LTP was induced using high frequency stimulation (HFS, 100 Hz for 1 s). The data were collected and analysed using WinLTP (Anderson and Collingridge, 2007).
Extracellular fEPSP recordings were amplified using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA), filtered at 1e3 kHz and digitised at 20 kHz (CA-1000, National Instruments). The early slopes of the fEPSPs were measured starting at the point of the fibre volley termination (0.2e0.5 ms). Post-LFS/HFS responses were normalised to baseline.
Data analysis
A single slice from one animal was used for each experimental group (hence n values refer to both the number of slices and the number of animals) and pharmacological experiments were randomized and interleaved with controls. Data are presented as mean values ± SEM. The LTD/LTP levels were estimated at the end of each single experiment (1 h post LFS/HFS) from 4 min averages, generating the mean values for each of the groups. The values from the single experiments were used for the statistical comparison. Significance of LTD/LTP was assessed using paired t-tests when comparing to the pre-LFS/HFS baseline. One-way ANOVAs with Bonferroni post-hoc tests were used to compare the normalised fEPSP slopes between the groups (SigmaPlot). Decay times of STP were analysed as described previously (Volianskis et al., 2013a;Volianskis and Jensen, 2003). Briefly, decay of STP was fitted using a mono-exponential fitting routine in GraphPad Prism and statistical comparisons between decay time constants (t) were done with extra sum-of-squares F-test (Prism). t values are presented together with their confidence interval (CI). Statistical differences were set at p < 0.05.
Inhibition of GluN2B receptors is sufficient for blockage of LTD
The results of experiments using GluN2A-and GluN2Dpreferring antagonists suggested that these subunits are not required for the induction of LTD. We therefore tested whether Ro 25-6981 (Ro, Fig. 2), which effectively blocks GluN2B-containing diheteromers selectively at a concentration of 1 mM, is able to block LTD. A 20 min pre-application of Ro had a variable effect on LTD; in 8 experiments it had no effect whereas in 7 experiments a significant block was observed (Fig. 2D). In contrast, when Ro was pre-applied for 30 min, complete inhibition of LTD was observed in all experiments. Representative single example experiments for Ro (filled symbols) and NVP (open symbols) are shown for a 20 min pre-incubation ( Fig. 2A) and a 30 min pre-incubation (Fig. 2B). This clearly shows the time-dependence of the block of LTD by Ro but not by NVP. The pooled data for Ro experiments, showing no overall significant effect with 20 min pre-incubation (15 ± 4% vs 26 ± 4%, n ¼ 15, p ¼ 0.3, Bonferroni correction) but complete inhibition with 30 min pre-incubation (2 ± 4%, n ¼ 5, p ¼ 0.03, Bonferroni correction) when compared to control experiments (26 ± 4%) are presented in Fig. 2C, D.
Effects of NMDAR antagonists on the induction of STP and LTP
In control experiments, tetanisation (100 Hz, 1 s, HFS) induced STP that declined with a t value of 5.2 min (CI 3.7e8.5 min) to a stable level of LTP (Fig. 3A, open circles, LTP ¼ 53 ± 5%, n ¼ 10, p < 0.05 when compared to the pre-HFS baseline, paired t-test).
Both STP and LTP were abolished by pre-application of 50 mM AP5 (Fig. 3A, filled circles, n ¼ 4). By the end of experiments using AP5, potentiation was 2 ± 8% and significantly different from the control LTP (p < 0.001, Bonferroni correction).
In contrast to AP5, a low concentration of NVP (0.1 mM) had no effect on the induction of STP or LTP (open triangles, Fig. 3A) and STP declined with a t value of 3.3 min (CI 1.9e11.1 min, p ¼ 0.32 vs control) to a LTP level of 54 ± 9% (n ¼ 4, p ¼ 1 vs control, Bonferroni correction). However, both STP and LTP were completely blocked by a high concentration of NVP (1 mM, n ¼ 5, filled triangles, Fig. 3A, Ro had concentration-dependent effects on synaptic potentia-
Discussion
The present study investigated the role of NMDAR subunits in LTD and LTP in P14 animals using GluN2A, 2B and 2D subunitpreferring antagonists. NMDARs are most commonly composed of two GluN1 and two GluN2 subunits and it is the identity of the GluN2 subunit, which contains the glutamate binding site, that confers the receptors with distinct biophysical properties, has no effect on LTD. (C) Summary of the experiments using 1 mM Ro showing that a 30 min pre-application time is necessary for complete blockade of LTD (filled squares, n ¼ 5) whereas a determining their affinity to binding glutamate, regulating the probability of channel opening and the decay kinetics of macroscopic currents and distinct pharmacological properties (Erreger et al., 2005;Monyer et al., 1994Monyer et al., , 1992Vicini et al., 1998). GluN1 subunits bind the co-agonists glycine or D-serine and their activation is obligatory to the channel function. The voltage sensitivity of the channel is due to the Mg 2þ block that gets relieved during depolarizing membrane potentials (Ault et al., 1980;MacDonald and Wojtowicz, 1980;Mayer et al., 1984;Nowak et al., 1984). The sensitivity of channels to the Mg 2þ block is also dependent on the identity of GluN2 subunits, which can be found in a functional receptor in either "homomeric" (i.e. both GluN2 subunits are identical) or in "heteromeric" (i.e. two, different GluN2 subunits are found in the receptor assembly) form. Thus, NMDARs, containing two identical GluN2 subunits (e.g. 2GluN1/2GluN2A) are frequently referred to as diheteromeric whereas receptors that include different GluN2 subunits (e.g. 2GluN1/GluN2A/GluN2B) are referred to as triheteromeric (Hansen et al., 2014;Paoletti and Neyton, 2007). The expression of NMDARs is regulated both regionally in the brain and throughout development and maturation of an animal (Buller et al., 1994;Monyer et al., 1994;Thompson et al., 2002;Watanabe et al., 1992). Thus, expression of GluN2As starts postnatally and then increases with development to steady adult levels in the hippocampus. In contrast, GluN2B subunits are expressed highly across the different developmental stages whereas a low expression of the GluN2Ds has been observed postnatally. GluN2C subunits are not expressed in rodent hippocampus, postnatally.
GluN2 subunit-preferring antagonists
In the present study, in addition to AP5, which is routinely used to block synaptic plasticity at the Schaffer collateral e CA1 synapse (Collingridge et al., 1983;Dudek and Bear, 1992), we used three subunit-preferring antagonists: NVP-AAM077 (Auberson et al., 2002), Ro 25-6981 (Fischer et al., 1997) and UBP145 (Costa et al., 2009;Irvine et al., 2012) to block GluN2A-, GluN2B-and GluN2Dcontaining receptors, respectively. We have previously characterized NVP, Ro and UBP in detail and used these antagonists to determine subunit composition of NMDARs involved in the induction of STP and LTP in adult hippocampus (Volianskis et al., 2013a). NVP and UBP145 bind to the glutamate-binding site of NVP (open triangles, n ¼ 4) has no effect on the induction of potentiation whereas 1 mM NVP blocks both STP and LTP (filled triangles, n ¼ 5). (B) STP is significantly reduced after pre-incubation with 1 mM Ro (filled squares, n ¼ 4) whereas LTP is not affected. 10 mM Ro (open squares, n ¼ 4) completely abolishes LTP. (D) 10 mM UBP (filled triangles, n ¼ 4) reduces STP but spares LTP.
NMDARs with differential potency at the receptors dependent on the identity of the GluN2 subunit, whereas Ro is a negative allosteric modulator of GluN2B-containing NMDARs with a complex mode of action (Karakas et al., 2011).
NVP is about 10-fold more potent at the GluN2A-containing NMDARs when compared to the GluN2B and can, at a concentration of 0.1 mM, discriminate between these receptor subtypes as shown previously in recombinant receptor assays (Feng et al., 2004;Frizelle et al., 2006;Volianskis et al., 2013a). NVP blocks GluN2Dcontaining receptors also, where it presents with intermediate potency (GluN2A > GluN2D > GluN2B, rank order potency). UBP145 is~10-fold more potent at the GluN2D subunits than at the other receptor subtypes (GluN2D > GluN2A ¼ GluN2B) and at a concentration of 10 mM it blocks~90% of recombinant GluN2Dcontaining receptors expressed in HEK293 cells (Volianskis et al., 2013a). Ro is the most selective of the three subunit-preferring antagonists that were used in this study. At concentrations of up to 1 mM it blocks diheteromeric GluN2B-containing receptors, with an IC 50 value in the low nanomolar range, although its potency is inversely dependent on the concentration of the agonist (Volianskis et al., 2013a). Furthermore, at low agonist concentrations, Ro can potentiate diheteromeric GluN2B-containing receptor response, a feature that is shared with ifenprodil and not seen at the other receptor subtypes (Fischer et al., 1997;Hansen et al., 2014;Volianskis et al., 2013a). At concentrations of 3e30 mM, Ro blocks triheteromeric NMDARs containing both GluN2A and GluN2B subunits, whereas at higher concentrations (>30 mM) it starts showing inhibitory effects at the GluN2A-containing diheteromers (Fischer et al., 1997;Hansen et al., 2014;Volianskis et al., 2013a). At concentrations of up to 30 mM Ro does not inhibit GluN2D subunits (higher concentrations of this antagonist have not been tested at the GluN2D subunit).
In summary, although NVP, Ro and UBP145 have limited selectivity towards the different NMDA receptor subtypes, a direct comparison of the actions of these antagonists at appropriate concentrations enables firm conclusions to be drawn about the involvement of these receptor subtypes in the induction of synaptic plasticity.
NMDARs in synaptic plasticity
Although there is no doubt about the involvement of NMDARs in generating synaptic plasticity in the CA1 area of the hippocampus Collingridge, 2013, 1993;Collingridge et al., 1983;Volianskis et al., 2015), considerable disparity remains in allocating selective functional roles for the specific NMDAR-subunits in the induction of synaptic plasticity. Some of the differences in the results might be explained by differences in experimental conditions, animal species and their developmental stage (Bartlett et al., 2007(Bartlett et al., , 2011Berberich et al., 2007Berberich et al., , 2005Hendricson et al., 2002;K€ ohr et al., 2003;Liu et al., 2004;Massey et al., 2004;Morishita et al., 2007). In addition, as mentioned previously, allosteric modulators such as ifenprodil and Ro can function as potentiators at low glutamate concentrations (Fischer et al., 1997;Hansen et al., 2014;Volianskis et al., 2013a), potentially confusing the results. Furthermore, various induction paradigms may engage NMDARs subtypes differently due to their distinct biophysical properties and localization.
In the current study we have used two of the most-common induction paradigms, i.e. low frequency stimulation (1 Hz for 15 min) and high frequency tetanisation (100 Hz for 1 s) to induce LTD and STP/LTP respectively.
NMDAR subunits in LTD
The role for GluN2B receptors in the induction of LTD was originally suggested by the observation that both Ro and ifenprodil can block LFS-induced LTD in slices from adult rat perirhinal cortex (Bartlett et al., 2007;Massey et al., 2004) and from 3 to 4 week old rat hippocampus (Liu et al., 2004). However, in other experiments, Ro and ifenprodil were unable to block LTD in rat hippocampal slices from 3 to 4 week old animals (Morishita et al., 2007), for reasons that are still unclear.
In the present study, we focused on LTD at a slightly earlier developmental stage, 2 weeks of age. Our observation that Ro completely blocked LTD at a concentration selective for GluN2Bcontaining diheteromers is consistent with the canonical view that this NMDAR-subtype can mediate LTD induction. However, blockade of this subtype is not invariably sufficient to inhibit LTD at this developmental stage (Bartlett et al., 2007) with other factors such as slice orientation playing a role (Bartlett et al., 2011).
We conclude therefore, that GluN2B receptors are required for LTD under some circumstances but their involvement may be compensated for under other conditions. Developmental changes in the expression of GluN2B receptors may be one determinant but other factors, such as the level of cholinergic modulation (Bartlett et al., 2011), may be involved. In the present study, we observed no effect on LTD with concentrations of NVP and UBP145 that are selective for GluN2A and GluN2D, respectively. This supports the idea, that GluN2B can be the major determinant of LTD.
NMDAR subunits in LTP
The role of NMDAR subunits in the induction of LTP is also highly controversial (e.g. Bartlett et al., 2007;Berberich et al., 2007Berberich et al., , 2005Li et al., 2007;Liu et al., 2004;Massey et al., 2004;Volianskis et al., 2013a). In the current study, LTP was completely blocked by either 50 mM AP5,1 mM NVP or 10 mM Ro. However, 1 mM Ro and UBP145 were ineffective. These data suggest that triheteromeric NMDARs, containing both GluN2A and GluN2B subunits, play an important role in the induction of LTP at this stage of development, as previously shown in adults (Volianskis et al., 2013a).
NMDAR subunits STP
STP, the transient enhancement in synaptic transmission that overlaps with LTP, has been shown to have a different NMDARdependence compared to LTP in adult rats (Volianskis et al., 2013a). More specifically, in slices prepared from adult rats, STP comprises two overlapping components; a fast component, termed STP 1 , that has the same sensitivity to antagonists as LTP, and a slow component, termed STP 2 , that is sensitive to both Ro and UBP145. It was therefore proposed that STP 2 involves both GluN2B and GluN2D subunits. A similar slow component of STP with high sensitivity to both Ro and UBP was observed in the present study. Therefore STP 2 shows no obvious developmental regulation. Its function remains to be determined although a role in working memory has been postulated (Volianskis et al., 2013a,b). In terms of STP 1 the parallel developmental regulation in both its sensitivity and that of LTP to NVP reinforces the view that these two forms of synaptic plasticity are closely associated with one another.
Concluding remarks
In this study on slices obtained from P14 hippocampus, using GluN2A, 2B and 2D subunit-preferring concentrations of NVP, Ro and UBP145, we have demonstrated that activation of GluN2Bcontaining receptors can be sufficient for the induction of LTD.
We have also shown that GluN2B-and GluN2D-containing receptors are involved in the induction of a component of STP. Finally, we have presented evidence that GluN2A/2B triheteromers are the dominant form involved in LTP. These data support the view that different NMDA receptor subtypes play distinct roles in various forms of synaptic plasticity. They also demonstrate that a single subunit, in this case GluN2B, is involved in multiple forms of synaptic plasticity at the same set of synapses. | v3-fos-license |
2020-04-18T11:17:51.684Z | 2020-04-16T00:00:00.000 | 215803347 | {
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} | pes2o/s2orc | Exosomal miRNAs in tumor microenvironment
Tumor microenvironment (TME) is the internal environment in which tumor cells survive, consisting of tumor cells, fibroblasts, endothelial cells, and immune cells, as well as non-cellular components, such as exosomes and cytokines. Exosomes are tiny extracellular vesicles (40-160nm) containing active substances, such as proteins, lipids and nucleic acids. Exosomes carry biologically active miRNAs to shuttle between tumor cells and TME, thereby affecting tumor development. Tumor-derived exosomal miRNAs induce matrix reprogramming in TME, creating a microenvironment that is conducive to tumor growth, metastasis, immune escape and chemotherapy resistance. In this review, we updated the role of exosomal miRNAs in the process of TME reshaping.
MicroRNAs (miRNAs) are a class of short ncRNAs with 19-25 nucleotides in length [15]. Through regulating gene expression, miRNAs regulate a variety of important biological functions, such as proliferation, apoptosis, differentiation, migration, invasion and drug resistance. Genetic or epigenetic changes in cancer cells can induce abnormal expression of miRNAs, thus causing abnormal expression of their target genes [16][17][18][19][20][21]. miRNAs function through 6-7 base complementary binding to target mRNA and inhibition of target gene expression at the level of protein [22][23][24]. From the literature, miRNAs can work as oncogenes to promote the formation and biological changes of TME [25][26][27][28]. For example, miR-9 and miR-200s induce normal fibroblasts (NFs) in TME to transform into CAFs and promote tumor metastasis [29,30], miR-526b and miR-655 promote angiogenesis and lymphangiogenesis in TME [31], and miR-340-5p and miR-561 induce formation of immunosuppressive microenvironment [32,33]. How these biologically active miRNAs are transmitted and function in cells and TME is an important breakthrough in the study of TME.
Recently, exosomes are considered to be the key mediators responsible for the heterogeneity of the TME and carry biologically active cargos, such as protein, metabolites, nucleic acids (e.g. ncRNAs), to shuttle between tumor cells and TME, thereby affecting tumor development [34][35][36][37]. Among the biologically active substances, tumor-derived exosomal miRNAs can induce TME heterogeneity while changes in TME promote tumor progression. This paradigm, similar to a positive feedback loop, makes the uncontrollable growth of the tumor [38][39][40][41][42][43]. In this article we updated the interaction of exosomal miRNAs and TME.
The overview of microenvironment and exosomes in cancer
The components of tumor microenvironment Growth, metastasis and treatment resistance of tumors are inseparable from the support of TME, a dynamic ecosystem containing multiple cell types and noncellular components. Some of the basic biological behavioral features of tumors, such as proliferation, migration, invasion, apoptosis inhibition, immune evasion, angiogenesis, and metabolic reprogramming are all affected by TME. The complex communication network in TME is the basis for the regulation of these biological functions, including autocrine and paracrine. Exocrine-mediated communication is an important emerging pathway in paracrine signal transduction [2].
Non-tumor cells in TME, such as fibroblasts, endothelial cells and immune cells, are affected by tumor-related active substances, and their original cellular functions undergo tumor-like changes, constantly adapt to new environments and promote tumor growth. Due to the influence of TME, NFs are activated into CAFs. CAFs are the most abundant stromal cells in TME, producing an ECM that differs from normal ECM in terms of stiffness and alignment, which support tumor cells migration [9]. Hypoxia in TME causes tumor to secrete angiogenic factors to act on endothelial cells and promote angiogenesis [44,45]. The immune cells in TME show diversity, and they block the immune response. The inflammatory molecules around the tumor cells also cause the system to fail to recognize and eliminate cancer cells [38,46,47]. These make TME a complex heterogeneous environment and often leads to an uncontrollable trend in the development of tumors [48,49].
The biosynthesis and function of extracellular vesicles and exosomes
Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles (40-1000nm in diameter) released by cells or detached from the plasma membrane [50,51]. EVs are generally divided into two categories: ectosomes and exosomes. Ectosomes are vesicles formed from the plasma membrane sprouting outwards, including microvesicles, microparticles and large vesicles with a size range of 50-1000nm in diameter. Exosomes are small extracellular vesicle (sEVs) in a size range of 40-160nm in diameter with an endosomal origin. EVs have biological activities and mediate intercellular communication [36]. During tumor progression, EVs derived from different cells (tumor cells, stromal cells, immune cells, etc.) play an important role and participate in the formation of TME [44,[52][53][54].
In this review, we mainly focused on the exosomes. However, because of absence of strict standards for exosome isolation and purification methods, the International Society for Extracellular Vesicles encouraged researchers to establish minimum requirements and strictly control the integrity, size, molecular cargo, and functionality of the vesicle population [38,[55][56][57], so that we narrowed the research of exosomes based on the widely accepted methods. Exosomes are small extracellular vesicles (40-160 nm in diameter) formed by dynamic exocytosis [58][59][60]. Exosomes originate from the luminal cavity or early intracellular bodies in the circulation pathway of the plasma membrane. These membranes or early intracellular bodies will sag inward to form intraluminal vesicles (ILV), which will further develop into multivesicular bodies (MVB) [61,62]. In general, multivesicular bodies are fused with lysosomes to be degraded, but some multivesicular bodies are fused to the cell surface under the traction of intracellular molecular motors and eventually secreted outside the cell, which called exosomes [36,63].
Exosomes are involved in the biology of many diseases. Exosomes can regulate the immune response and inflammation, possibly through transfer and presentation of antigen peptides, to induce expression of inflammatory genes in recipient cells [64,65]. In metabolism and cardiovascular diseases, exosomes induce metabolic disorders in adipocytes and islet cells [66,67]. Exosomes may impair the formation of neurotoxic oligomers and promote neurodegeneration [68][69][70]. More importantly, exosomes are associated with tumor growth, angiogenesis, metastasis, sensitivity to chemotherapy, and immune evasion [47,71,72].
miRNAs sorting to exosomes
Exosomes contain a variety of biologically active molecules, such as proteins, lipids and nucleic acids. miRNAs are one of them and play an important role in intercellular cellular transport and signal transduction [73][74][75]. Exosomes can transfer metabolites and promote communication between different cells through the exchange of exosomal miRNAs, and then play an immune response, tumor microenvironment remodeling and tumor metastasis during tumor progression [38,[76][77][78]. Many reports indicate that exosomes affect the biology of recipient cells by transferring miRNAs from donor cells to recipient cells, but the mechanism of how exosomes sorting miR-NAs has not been thoroughly solved. According to exosomes database (www.exocarta.org), 2838 miRNAs are listed in the latest update. Among the 2588 annotated miRNAs in the human genome, 593 miRNAs have been detected in exosomes [79]. Four potential mechanisms for sorting miRNAs into exosomes were proposed. The neural sphingomyelinase 2 (nSMase2) was the first molecule found to be linked with miRNAs packaging into exosomes. Overexpression of nSMase2 leads to an increased number of miRNAs loaded into exosomes. This suggests that the neural sphingomyelinase 2 (nSMase2)dependent pathway is associated with the sorting of exosomal miRNAs [80]. The second is based on the control of the sumoylated form of heterogeneous nuclear ribonucleoprotein (hnRNP). Sumoylated hnRNPA2B1 controls the sorting of exosomal miRNAs by recognizing the GGAG and GGCU base sequence of the 3 'end region of miRNAs [81,82]. The third is that most exosomal miR-NAs isolated from urine or B cells were uridylated at 3 ′ ends. The sorting of miRNAs to ILV may also require hydrophobic modification and GGAG base sequence at 3 ′ end of the miRNAs. This indicates that the 3 'ends of miRNAs may be involved in directing miRNAs into exosomes [83,84]. Finally, there are reports that Argonaute proteins (functional carriers of miRNAs) are related to the selection of exosomal miRNAs. Knocking out Argonaute 2 (Ago2) reduces the contents of certain exosomal miR-NAs, such as miR-142-3p, miR-150, and miR-451 [85,86]. In summary, some specific protein complexes and miR-NAs own structural characteristics have affects the miR-NAs' transfer to exosomes, but the complete sorting mechanism and process have not yet been elucidated and need further exploration.
The role of exosomal miRNAs in TME
During the progression of the tumor, primary tumorderived exosomal miRNAs can be transferred to nonmalignant cells in the tumor microenvironment to induce heterogeneity [50,[87][88][89]. At the same time, with the changes in biological activity of non-malignant cells in the tumor microenvironment, non-malignant cells can also secrete exosomal miRNAs to further regulate tumor cells or other microenvironmental components [40,90]. In most studies, the stromal cell receptors of cancer-derived exosomal miRNAs are cancer-associated fibroblasts (CAFs), endothelial cells and immune cells dynamically regulate each other in TME. Exosomal miRNAs on the heterogeneity of TME is mainly reflected in the fact that exosomal miRNAs can activate cancer-associated fibroblasts and thus reshape ECM, which is beneficial to the spread of tumor cells. Exosomal miRNAs promote endothelial cells to form tubes, and the formation of abundant vascular networks is conducive to the metabolism and survival of tumor cells. Exosomal miRNAs also mediate inflammatory cell infiltration and immune escape, which is conducive to colonization and proliferation of tumor cells. Through these macroscopic effects, exosomal miRNAs can make TME more suitable for tumor development [91]. Herein, we focused on the roles of exosomal miRNAs in following aspects.
Reshaping ECM to promote tumor progression
Extracellular matrix (ECM) is composed of protein and carbohydrates, with the functions of connection, support, water retention, anti-stress and protection. ECM supports the basic life activities of cells, such as proliferation, differentiation, and migration [92,93]. However, tumors are often accompanied by dysfunction of ECM. Tumor development is a complex process involving dynamic interactions between malignant cells and their surrounding stroma composed of cells and non-cellular components. Within the stromal, fibroblasts represent not only the major cell types, but also the main source of extracellular matrix (ECM) and soluble factors [94,95]. Normal fibroblasts exert multiple inhibitory functions against cancerinitiating and metastasis through direct cell-cell contact, paracrine signaling, and ECM integrity [96]. However, tumor-derived exosomal miRNAs can trigger a series of tumor-promoting signals, inducing normal fibroblasts (NFs) transformation into CAFs, which changes the original ECM physiological state, thus creating the optimal niche for the widespread growth of cancer cells [96,97].
In tumors, tumor cell-derived exosomal miRNAs are highly diverse and are capable of differentiating NFs into CAFs through a variety of signaling pathways. Exosomal miRNAs from cancer cells elicit a parenchymal signaling response at the receptor site and effectively inducing fibroblast activation, such as α-smooth muscle actin (α-SMA), fibroblast growth factor 2 (FGF2) and fibroblast activating protein (FAP) expression [98][99][100]. Matrix composed of CAFs is conducive to the proliferation and migration of tumor cells. In ovarian cancer, the cancerrelated exosomal miR-124 targets sphingosine kinase 1 (SPHK1) and upregulates α-SMA and FAP, which differentiates NFs into CAFs and regulates CAFs migration and invasion [101,102]. High expression of exosomal miR-27b-3p and miR-214-3p in myeloma cells triggers proliferation and apoptotic resistance of bone marrow fibroblasts via the FBXW7 and PTEN/AKT/GSK3 pathways. At the same time, miR-27-3p and miR-214-3p were up-regulated in fibroblasts co-cultured with myeloma, and activated expression of fibroblast activation markers α-SMA and FAP. The biological behavior of bone marrow fibroblasts is programmed to alter the myeloma microenvironment [103,104]. Exosomal miR-NAs in digestive system tumors also reshaped ECM in adjacent sites and promote tumor progression. Exosomal miR-27a derived from gastric cancer (GC) is transported to fibroblasts, and thus results in decreased expression of CSRP2, enhanced expression of α-SMA, and differentiation of fibroblasts into CAFs [105]. Exosomal miR-10b secreted by colorectal cancer cells can be transferred to fibroblasts, where it inhibits PIK3CA expression and PI3K/ Akt/mTOR pathway activity, promote expression of TGFβ and α-SMA, and enable fibroblasts to acquire the characteristics of CAFs [106,107]. These changes promote the proliferation, migration and invasion of tumor cells. Exosomal miRNAs have found similar effects in colorectal cancer (CRC). Exosomal miR-2149-5p, miR-6737-5p, and miR-6819-5p can inhibit the expression of TP53 in fibroblasts to promote tumor proliferation [108].
In addition, changes in ECM also affect angiogenesis, inflammatory response, and metabolic reprogramming. Phenomenon was shown in melanoma where highly expressed exosomal miR-155 inhibits the expression of SOCS1, activates the JAK2/STAT3 pathway, up-regulates the expression of FGF2, VEGFA and MMP9 in CAFs, and promotes the formation of blood vessels in the tumor [109,110]. In hepatocarcinoma (HCC), exosomal miR-21 is transferred to CAFs, directly targeting PTEN to activate PDK1/Akt signaling, up-regulating VEGF, MMP2, MMP9, bFGF, and TGF-beta and thus promoting angiogenesis [111,112]. Exosomal miR-1247 targets B4GALT3 and activates the beta1-integrin-NF-kappaB signaling pathway, which activates CAFs to secrete the inflammatory cytokines IL-6 and IL-8 and induce inflammatory infiltration [113]. Exosomal miR-9 and miR-105 are derived from breast cancer; the former promotes the activation of NFs into CAFs by affecting MMP1, EFEMP1 and COL1A1 [30], and the latter activates MYC signal transduction to induce metabolic reprogramming of CAFs, and adapts CAFs to different metabolic environments, promoting tumor growth [18]. Similar reports include miRNA-142-3p in EVs secreted by lung cancer cells, which promotes the cancer phenotype of lung fibroblasts [114] (Fig. 1 and Table 1).
These researches show that cancer-derived exosomal miRNAs can affect the physiological function of stroma. Conversely, a reciprocal exosomal miRNAs exchange from the stroma to cancer cells also modulates cancer progression. For example, CAFs-derived exosomal miR-148b in the matrix surrounding endometrial cancer can up-regulate DNMT1, leading to changes in EMT-related molecules like E-cadherin, N-cadherin, vimentin, and fibronectin and promoting cancer cell metastasis [65]. CAFs are resistant to cisplatin and deliver exosomal miR-196a, which binds to target CDKN1B and ING5, mediates the expressions of p27, CDK2, CDK4, Cyclin D1 and Cyclin E1 and thus induces cisplatin resistance to cancer cells [115]. CAFs-derived exosomal miR-522 reduces the contents of lipid-ROS in gastric cancer cells by inhibiting the expression of ALOX15, which leads to a decrease in the sensitivity of gastric cancer to chemotherapy [12]. Compared with NFs, CAFs have the characteristics of excessive proliferation and unique cytokines. This not only induces the formation of new blood vessels, but also promotes the entry of immune cells into TME, which greatly changes the physiological function of ECM to support tumor proliferation, metastasis and treatment resistance [116,117]. However, cells involved in ECM formation are not only fibroblasts, but also chondrocytes, osteoblasts, and certain epithelial cells. Exosomal miRNA remodeling of ECM can also be achieved by affecting the function of these cells. For example, studies have shown that cancer-secreted exosomal miR-940 promotes osteogenic differentiation of mesenchymal cells by targeting ARHGAP1 and FAM134A, and then induces osteogenic phenotypes in the bone metastasis microenvironment and promotes tumor metastasis [118]. But research on the interaction of exosomal miRNAs with these cells is not comprehensive. At the same time, the composition of ECM not only includes collagen (synthesized by fibroblasts, chondrocytes, osteoblasts and certain epithelial cells and secreted outside the cell), but also includes non-collagen glycoproteins, glycans and elastin. Whether exosomal miRNAs reshape ECM by affecting these ingredients remains to be proven.
Promoting angiogenesis to enhance proliferation and migration
Tumor growth depends to a large extent on the metabolism of cancer cells [119]. The disordered distribution of tumor blood vessels and the loss of normal vascular function lead to local hypoxia and impaired nutrient supplies. At the same time, the distance gradient between different vascular beds also leads to the imbalance of drug distribution and absorption [120]. These changes of vascular network promote the formation of internal microenvironment and intratumoral heterogeneity.
The exosomal miRNAs can be taken up by the vascular endothelial cells to change the original distribution and physiological functions of the blood vessels in the microenvironment. Exosomal miRNAs secreted by tumor cells have been reported to promote angiogenesis in TME. In nasopharyngeal carcinoma (NPC), exosomal miR-23a mediates angiogenesis by repressing TSGA10 and phosphorylation of ERK, which enhances tube generation ability of human umbilical vein endothelial cells (HUVECs) in vitro and in vivo [121,122]. Glioma stem cell-derived exosomal miR-21 stimulates VEGF/p-FLK/ VEGFR2 signaling pathway to promote angiogenesis in endothelial cells [123,124]. The exosomal miR-210-3p secreted by HCC cells is transferred to endothelial cells, targeting SMAD4 and STAT6 to promote angiogenesis, and it is found that the higher miR-210-3p in the serum of HCC patients is positively correlated with the microvessel density in HCC tissues [125]. EVs and sEVsmediated miRNAs transfer also promotes angiogenesis in TME. In NSCLC, EVs-mediated miR-142-3p transferred to endothelial cells and fibroblasts, inhibiting the expression of TGFβR1, PDGFR-β and p-SMAD2/3 to promote angiogenesis [114]. Human ovarian carcinoma cell line SKOV-3 secretes miR-141-3p in small extracellular vesicles (sEVs), which activates the JAK-STAT3 pathway in endothelial cells and promotes angiogenesis [126]. Besides, exosomal miRNAs that promote angiogenesis can also be derived from other cells. Exosomal miR-100 from human mesenchymal stem cells (MSCs) affects the mTOR/HIF-1α/VEGF signaling axis to promote angiogenesis in breast cancer [127].
The rich vascular network in TME is beneficial to the proliferation and metastasis of cancer cells. Exosomal miR-619-5p inhibits the expression of RCAN1.4, promotes angiogenesis, and facilitates the growth and metastasis of cancer cells [128]. Recent studies have shown that circulating exosomal miR-205 expression is elevated in OC patients and is related to microvessel density, and exosomal miR-205 induces angiogenesis via the PTEN-AKT pathway, and promotes tumor cell proliferation in vitro [129]. Changes in the vascular microenvironment are not only in the number of blood vessels, but also in vascular permeability, adhesion, and ability to form a ring. The colorectal cancer-derived exosomal miR-25-3p can down-regulate KLF2 and KLF4, and KLF2 affects the tube formation ability of HUVECs through the VEGFR2/p-Erk/p-Akt pathway while KLF4 activates ZO-1/Occludin/Claudin5 pathway to affect the growth of the aortic rings, which in turn changes the vascular microenvironment [130,131]. Under hypoxic conditions, lung cancer cell-derived exosomal miR-23a directly inhibits prolyl hydroxylase 1 and 2 (PHD1 and PHD2) and accumulates HIF-1α in endothelial cells, inducing angiogenesis, and exosomal miR-23a also inhibits ZO-1, increasing vascular permeability and transendothelial migration of cancer cells [132]. In human glioma, exosomal miR-9 promotes angiogenesis, vascular permeability and adhesion through the MYC/ OCT4 pathway [133] (Fig. 2).
Exosomal miRNAs influence on vascular network is not only promotion, but sometimes also play an inhibitory effect. Studies have found that exosomal miR-451 acts as a tumor suppressor and targets LPIN1 to induce apoptosis both in HCC cell lines and HUVECs. In addition, miR-451a suppresses HUVECs tube formation and vascular permeability [134]. NPC-derived exosomal miR-9 up-regulates MDK and activates the PDK/Akt signaling pathway to inhibit the formation of endothelial cells. High expression of MDK in NPC tumor samples is positively correlated with microvessel density, revealing the anti-angiogenic effects of exosomal miR-9 in the development of nasopharyngeal carcinoma [135]. Except for tumor-derived exosomal miRNAs, which inhibit angiogenesis, non-tumor cells have similar functions. miR-15a, miR-181b, miR-320c, and miR-874 in EVs released by human liver stem-like cells (HLSCs) possess an anti-tumorigenic effect by inhibiting tumor angiogenesis [136]. According to these reports, it can be found that exosomal miRNAs can regulate the vascular network in TME through multiple signaling pathways, but these molecular mechanisms have not been fully elucidated and need to be explored in the future.
Promoting the formation of immunosuppressive environment
In the TME, immune cells including lymphocytes, dendritic cells, and macrophages, regularly infiltrate tumor tissues and adjacent sites. Through multiple signal transduction pathways mediated by exosomal miRNAs, tumor cells can inhibit the maturation and differentiation of immune cells, thereby creating an immune microenvironment suitable for tumor growth [41,137,138]. At the same time, in hypoxia and low nutrient supplies in the microenvironment, tumor cells often secrete metabolic by-products, such as lactic acid, nitric oxide, reactive oxygen species, prostaglandins and arachidonic acid, leading to the formation of an inflammatory microenvironment [139,140]. Changes in the biological functions of various immune cells in microenvironment and the production of inflammatory mediators result in tumor cell escaping from immune surveillance.
Dendritic cells (DCs) are the most powerful professional antigen presenting cells in the body. Mature DCs can effectively activate the initial T cells and maintain the central part of the immune response [141,142]. Tumor-derived and endogenous exosomal miRNAs can regulate cross-presentation in dendritic cells and with other immune cells, this exomsomal miRNAs-mediated intercellular communication may affect the maturation of DCs [143,144]. In pancreatic cancer, exosomal miR-212-3p targets MHC class II TF RFXAP resulting in reduced expression of HLA-DR, -DP, and -DQ molecules and thus interfering with the function of DCs cells [145,146]. Exosomal miR-203 is able to reduce the expression of TLR4, TNF-α and IL-12 in DCs, affecting the activation of natural killer cells (NKs) [147]. Up-regulated exosomal miR-let-7i in tumor-derived exosomes (TEX) can be taken up by mDCs, resulting in changes in intracellular levels of IL-6, IL-17, IL-1b, TGFbeta, SOCS1, KLRK1, IFNγ, and TLR4, thereby suppressing the immune response [148]. miRNAs from regulatory T cells (Treg) can also affect the immune response, EVs-mediated miR-150-5p and miR-142-3p can be transferred to DCs to induce a cell-refractory phenotype, resulting in increased IL-10 and decreased IL-6 expression [149], exosomal miR-let-7d is transferred to T helper 1 (Th1) cells to inhibit Th1 cells proliferation and IFNγ secretion, and IFNγ secreted by Th1 cells (a subtype of Naïve CD4 T cells) plays a central role in anti-tumor immunity [150].
Tumor-associated macrophages (TAMs) are one of the most abundant immune cells in TME. TAMs play a huge role in the proliferation and migration of tumor cells and counteract the cytotoxic effect of T lymphocytes and NKs, facilitating cancer cells to evade immune surveillance [140,151]. TAMs have strong plasticity and can differentiate into immune-stimulating (M1-polarized) TAMs or oppositely immune-suppressive (M2-polarized) TAMs, respectively, having different biological functions [152]. TAMs in tumors often behave as M2 phenotype and are usually associated with poor prognosis [153]. A large number of studies have reported that exosomal miRNAs can regulate the phenotypes of TAMs. Exosomal miR-125b derivied from lung adenocarcinoma cells promotes macrophage repolarization toward an antitumor M1 phenotype [154]. Exsomal miR-125b-5p secreted by melanoma cells targets LIPA and increases the expression of M1 phenotype markers IL-1β, CCL1, CCL2, and CD80 [71]. Oppositely, exosomal miR-21 taken up by CD14 + human monocytes inhibits the expression of the M1 marker and increases the expression of the M2 marker. Knockout of miR-21 in Snail-expressing human head and neck cancer cells attenuated M2 polarization of TAMs, and miR-21 was found to be positively correlated with M2 marker MRC1 in head and neck cancer tissues [155]. In epithelial ovarian cancer (EOC), exosomal miR-222-3p can be transferred to macrophages, downregulating SOCS3, inducing phosphorylation of STAT3, and thus leading to polarization of the M2 macrophages [156]. In hypoxia, EOC-derived exosomal miR-21-3p, miR-125b-5p, miR-181d-5p, and miR-940 differentiate TAMs into M2 phenotypes and promote tumor progression [157,158]. Likewise, exosomal miR-301a-3p derived from hypoxic pancreatic cancer cells activates the PTEN/ PI3Kγ signaling pathway to trigger M2 phenotype polarization of macrophages [159,160]. Mutant p53 colon cancer cells-derievd exosomal miR-1246 induces M2 polarization of macrophages and reshapes the TME through increase the expression of IL-10, TGFβ, and MMPs [161] (Fig. 3).
Abnormal differentiation and function of myeloid cells is a hallmark of cancer. Among them, myeloid-derived suppressor cells (MDSCs) have the function of suppressing adaptive immunity and innate immune response, and play an important role in tumor immune escape [162][163][164]. Exosomal miRNAs affect the function of MDSCs by regulating the activity of transcription factors and transcription activators, thereby reshaping the immune microenvironment. In the research of glioblastoma, exosomal miR-10a targets RORA and affects the differentiation of MDSCs through the NFκB pathway, exosomal miR-21 targets PTEN and affects the activation of MDSCs via the p-STAT3/p-p65/p-Akt pathway [165]. Exosomal miR-155 istransmitted to monocytes, leading to nuclear translocation of NFkB and phosphorylation of STAT1, reprograming conventional monocytes into MDSCs [166]. Changes in the function of MDSCs affect the progression of the tumor itself. Recent research shows that exosmal miR-126a derived from MDSCs promotes angiogenesis and benefit breast cancer lung metastases [167].
The immunomodulation induced by exosomal miR-NAs is complex and dynamic. In TME, tumor cells interact with various types of immune cells and crosspromote immunosuppressive activity. Among them, exosomal miRNAs play a pivotal role in them, but the mechanism has not been elucidated. Thereby, the function of exosomal miRNAs in the reciprocal interplays between cancer cells and hosts immune system merits further investigation.
Perspectives of exosomal miRNAs
With the vigorous development of the biology of exosomes in tumors, more and more evidence indicates that exosomal miRNAs play an important role in tumor progression and TME reshaping. Compared with miRNAs released directly into the circulatory system, exosomal miRNAs are protected by lipid bilayer encapsulation and avoid degradation by ribonuclease in the blood. Notebalely, exosomal miRNAs are more bioactive pool of circulating miRNAs compared to those miRNAs transported with liposomes [41,168,169]. Considering the advantages of exosomal miRNAs and the widespread presence of exosomes in all biological fluids (blood, breast milk, semen, and urine), diagnostic and therapeutic technologies based on exosomal miRNAs have a bright future.
Some specific exosomal miRNAs have high diagnostic value in tumors, and detecting them is helpful for early diagnosis of tumors. For example, in prostate cancer, breast cancer, and oral squamous cell carcinoma, the expression of exosomal miR-1246 is closely related to pathological grades, distant metastasis and poor prognosis [170][171][172][173]. Circulating exosomal miR-375 is valuable for the diagnosis of ovarian, rectal and prostate cancer [174][175][176]. The combination of multiple exosomal ncRNAs can enhance the diagnostic and prognostic potential of exosomal miRNAs. For example, the combination of expression of plasma exosomal miR-30d-5p and let-7d-3p is valuable diagnostic markers for non-invasive screening of cervical cancer and its precursors [177].
Circulating exosomal miRNA-21 and lncRNA-ATB are related to the TNM stage of liver cancer and other prognostic factors, including the T stage and portal vein thrombosis [178].
Exosomal miRNAs, as a new tumor treatment method, are being widely explored. Based on the fact that exosomal miRNAs effectively bind to target mRNA and inhibit gene expression in recipient cells, related exosomal engineering techniques have been used to treat tumors by delivering tumor suppressor exosomal miRNAs. For example, delivery of exogenous miR-155 into DCs using TEX as a vector results in increased expression of MHCII (I/A-I/E), CD86, CD40 and CD83, promoting activation of DCs. Exosomal miRNA-155 significantly increases the levels of IL12p70, IFN-γ and IL10 and improves immune function [179]. By fusing Her2 affinity to the extracellular N-terminus of human Lamp2, and then using the modified exosomes to co-deliver 5-FU and miR-21 inhibitors (miR-21i), which targets colon cancer cells, effectively reverses the resistance of tumor cells and significantly enhances the toxicity of 5-FU resistant cancer cells [180].
Although exosomal miRNAs have made exciting progress in oncology, most of these results are experimental. Extension of exosomal miRNAs technologies to clinic remains challenging. There is no doubt that the function of exosomes is determined by their specific contents. A large amount of literature has reported that tumor-derived exosomal miRNAs can reshape TME and promote tumor progression, but little is known about the sorting mechanism of exosomal miRNAs. Although the basic framework of the endosome sorting complex required for transport (ESCRT) and Ago2 in MVB sorting has been reported in previous studies, it remains to be elucidate whether other novel sorting signals are involved in the release of exosomal miRNAs [86,[181][182][183][184].
The potential of exosomal miRNAs as diagnostic markers is unquestionable, but how to improve the sensitivity and specificity of exosomal miRNAs remains to be solved. The combination of different exosomal cargos, such as proteins, lipids, RNA and miRNAs for cancer diagnosis and prognosis can more comprehensively reflect the characteristics of tumors. At the same time, the scope of application of exosomal miRNAs also needs attention. The expression level of exosomal miRNAs is related to tumor types, clinical stages or other underlying diseases, and there are differences between individual patients. Therefore, how to use exosomal miRNAs accurately is also worth of considering.
The widespread use of exosomal miRNAs in clinical treatment remains challenging. First, exosomes-based therapeutic tools require more accurate and standardized exosomal purification methods, and the economic cost of mass-producing exsomes for clinical application cannot be ignored [56,57]. The second is that exosomal miRNAs-induced biological behavioral changes are often released through the cultivation of supra-physiological numbers of cell, and how many orders of magnitude of exosomal miRNAs are needed to achieve the corresponding efficacy in clinical applications remains to be determined.
Conclusion
Exosomal miRNAs, as a signaling molecule for communication between tumor cells and TME, play an important role in the formation and remodeling of TME, but its regulatory mechanism is still worth of further exploration. At present, most of the biological studies of exosomal miRNAs have been revealed by cell-culture systems in vitro. But the problems still remain whether exosomal miRNAs derived from supra-physiological numbers of cell reflect the biological conditions in vivo. It is necessary to conduct more experiments in vivo or in mammals.
With the increase of exosomes researches, people have gradually discovered that the exosomes obtained by traditional exosomal separation and purification methods (ultracentrifugation, density-gradient centrifugation, immune-affinity capture, and precipitation) not only contain sEVs, but also contain non-membrane structure vesicles (NVs). Components, double-stranded DNA (dsDNA) and histones, are more in the NVs rather than in exosomes or sEVs. Moreover, many of the most abundant miRNAs were more associated with extracellular NV fractions than with either parental cells or sEV fractions [56]. This indicates that we may need to reevaluate the composition of exosomes, and it is urgent to explore the generation and sorting mechanisms of exosomal miRNAs or miRNAs in other type of sEVs. | v3-fos-license |
2018-04-03T00:51:01.467Z | 2017-04-01T00:00:00.000 | 12128743 | {
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} | pes2o/s2orc | Tissue-protective activity of selenomethionine and D-panthetine in B16 melanoma-bearing mice under doxorubicin treatment is not connected with their ROS scavenging potential
Aim To evaluate molecular mechanisms of tissue-protective effects of antioxidants selenomethionine (SeMet) and D-pantethine (D-Pt) applied in combination with doxorubicin (Dx) in B16 melanoma-bearing-mice. Methods Impact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro- and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H2O2-specific) and dihydroethidium (DHE, O2--specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer. Results Selenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals. Conclusions Our results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.
Methods Impact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro-and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H 2 O 2 -specific) and dihydroethidium (DHE, O 2 --specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer.
Results Selenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals.
Conclusions
Our results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.
Tissue-protective activity of selenomethionine and D-panthetine in B16 melanomabearing mice under doxorubicin treatment is not connected with their ROS scavenging potential Golden chemotherapy standards (eg, anthracycline antibiotics, vinca alkaloids, inorganic platinum compounds) have been used for decades for treatment of advanced cancers (1). Despite different mechanisms of action (inhibition of DNA topoisomerase II, blockage of tubulin polymerization, DNA intercalation), their negative side effects are similar and include myelosuppression, nausea, mucositis, alopecia, renal damage, thus, leading to significant worsening of health conditions of cancer patients (2). Doxorubicin (Dx) is one of the most widely used chemotherapeutics possessing broad spectrum of anticancer activity. Its therapeutic potential is realized through various pathways, including DNA intercalation, DNA topoisomerase II inhibition, and induction of the oxidative stress in target cells (3). Numerous data indicate that Dx-mediated ROS production is the leading reason of an acute myelotoxicity and nephrotoxicity of this drug (4,5). However, the most dangerous consequence of Dx-induced oxidative stress is a delayed cardiac failure found in 18% cancer patients who received more than 551 mg/m 2 dose of this drug (6). Thus, novel approaches should be developed in order to enhance selectivity of action of the chemotherapies and diminish their toxic effect toward normal tissues of the organism.
Dietary antioxidant supplementations might be promising candidates for this role, though their use for treatment of various diseases remains a controversial topic for last decades. Initially popularized in mid-1970s by Nobel laureate Linus Pauling, antioxidants are now used by a majority of cancer patients, since it is considered that they decrease potential harmful effects of the chemotherapy and, thus, improve a quality of patients' life (7). This tendency is constantly growing even despite the results of numerous clinical trials showing that the antioxidants had no real impact on a survival of cancer patients, or some of them (vitamins A and E) even worsened their prognosis (8,9).
In the above mentioned trials, selenium was found to be the only dietary supplement demonstrating the anti-tumorigenic activity and, thus, it was considered to be a promising candidate for further application in chemotherapy (8). This might be of great importance for the regimens including Dx, as its combination with the antioxidant (eg, selenium) should improve therapeutic action of Dx and also lower drug-induced oxidative stress.
Up to now, there is little information regarding therapeutic efficiency of the organo-selenium compounds in cancer treatment. The main aim of this work was to study in more detail the mechanisms of tissue-protective and therapeutic activity in vivo of selenomethionine in comparison with D-pantethine -vitamin B 5 precursor. Previously, we have shown that both SeMet and D-Pt decreased the oxidative stress in tissues of healthy rats treated with the anthracycline antibiotic -doxorubicin (Dx) (10). Those studies were repeated on mice bearing NK/Ly lymphoma and revealed that both antioxidants used in a combination with Dx not only decreased the lymphopenia and monocytosis caused by Dx, but also led to an increase in animal survival time comparing to Dx treatment (11).
Since leukemias and lymphomas are considered to be the most sensitive tumors toward Dx action, the effect of using antioxidant supplementations can be hardly noticeable on the background of strong therapeutic effect of Dx alone. In order to see a more pronounced effect of the combined action of Dx and antioxidants, we addressed murine B16 melanoma that is considered to be one of the most resistant tumors regarding Dx action (12,13). In the present paper, therapeutic efficiency of the antioxidants in mice bearing B16 melanoma was studied, in particular, the impact of proposed regimen on animal survival, blood profile, hepatotoxicity, and nephrotoxicity. Additionally, identification of potential ROS-scavenging activity of SeMet and D-Pt in B16 melanoma cell line under Dx treatment in vitro was dissected by analysis of their influence on ROS production, namely, hydrogen peroxide and superoxide anions, and functional status of mitochondria.
Materials
Seleno-L-methionine (≥98% (TLC)) and D-pantethine were purchased from Sigma (St. Louis, MO), and doxorubicin hydrochloride was obtained from Pfizer (New York, NY). Antioxidants were dissolved in sterile 0.9% sodium chloride solution prior to per os treatment of animals or addition to cell culture.
Animal studies
Studies of the biological activity of selenomethionine, Dpantethine, and doxorubicin were conducted in 2016 at the animal facility of the Institute of Cell Biology, NAS of Ukraine (Lviv, Ukraine). 42 adult male С57/Bl6 mice with 25-28 g weight were kept under standard vivarium conditions with constant access to the full feed and drinking water. Tumor inoculation was conducted by a subcutaneous injection of B16F10 cell suspension (10 6 cells per animal) diluted with sterile phosphate buffered saline (PBS) in right rear paw of mice. The viability and number of cells stained with 0.1% Trypan blue were checked by cell counting in the hemocytometric chamber. The vitality of melanoma cells used for transplantation was not less than 98%. Animals were divided into 7 groups with 6 mice in each group ( Figure 1). Blood sampling for biochemical and cytomorphological studies was done at 20th day after tumor inoculation (and the 10th day after chemotherapy start).
Mice from experimental groups were administered selenomethionine (120 µg/kg, cumulative dose 1200 µg/kg) (groups 2, 5) or D-pantethine (50 mg/kg, cumulative dose 500 mg/kg) (groups 3, 6) per os every second day, starting from 10th till 30th day of tumor inoculation. Mice of zero group (healthy) and first group (B16 melanoma, untreated) received simultaneously the equivalent volume of 0.9% sodium chloride solution in a similar mode. Doxorubicin (1 mg/kg, cumulative dose 10 mg/kg) was injected i.p. every second day starting from the 10th to the 30th day of tumor inoculation to the animals of groups 4-6. Treatment of animals with antioxidants in groups 5-6 took place 1 h before Dx injection. The chemotherapy scheme was developed, based on NCI recommendations (15) and our previous results (10,11).
All in vivo experiments were conducted in accordance with the international principles of the European Convention for protection of vertebrate animals under a control of the Bio-Ethics Committee of the above mentioned institution (Protocol N 4/2016 from 5.06.2016 of the BioEthics Committee at the Institute of Cell Biology, NAS of Ukraine).
Myelotoxicity studies
For blood sampling, amputation of a small part of mouse tail was done, pumping of ~ 50 µL of blood in a test tube, followed by immediate disinfection of a wound with 70% alcohol. For counting of red blood cells, 5 µL of blood were dissolved in 5 ml of isotonic NaCl solution (1:1000 dilution), while for leukocyte 5 µL of blood was dissolved in 95 µL of 3% acetic acid solution (1:20 dilution). Erythrocytes were counted in 5 big squares (divided into 16 small ones) of the hemocytometric chamber, while leukocytes were counted in 100 big squares, grouped by 4, under the Evolution 300 Trino microscope (Delta Optical, Mińsk Mazowiecki, Poland). The number of erythrocytes and leukocytes was counted using standard formulas, described in (16).
For blood smear preparation, 3 µL of blood was put at the edge of a slide, and then spreaded for 1.5 cm using another narrow polished slide, placed at a 45° angle. The obtained smears were dried at room temperature, fixed with absolute methanol, and then rehydrated by subsequent washing in ethanol solutions with decreasing concentrations (96%, 75%, 50%, 25%, and 12.5%). Finally, the smears were washed with distilled water, stained with Giemsa dye using standard protocol and air-dried, after which they were ready for leukogram analysis.
Counting of leukocytes was performed under Evolution 300 Trino microscope (Delta Optical, Mińsk Mazowiecki, Poland) on 90 × oil immersion objective. Cell counting was always done using the same system -half of cell population was counted in the upper half part of the smear, and the other half was counted on the lower part of the smear. The percentage of certain types of white blood cells in each smear was determined after counting of at least 300 cells. The obtained values (due to differences of absolute numbers of cells in each counted smear) were normalized to 100%, and percent values of each leukocyte fraction were calculated as described in (17).
Nephrotoxicity studies
Creatinine level in blood serum of experimental animals was measure spectrophotometrically using Popper method based on Jaffe reaction (18). Briefly, blood serum samples were diluted 1:20 in working reagent solution (0.75M NaOH and saturated picric acid, mixed 1:1), and their optical density was measured at Thermo Spectronic spectrophotometer (Helios, Great Britain) at 510 nm wavelength after 30 (E 1 ) and 90 sec (E 2 ) following sample addition to working reagent solution. The obtained results were compared to etalon (creatinine solution, 440 µM) and the final creatinine content in blood serum samples was calculated using following formula.
All experiments were performed in triplicate and repeated 3 times. Statistical analysis of data was conducted in GraphPad Prisms Software (GraphPad Software, Inc) using Student's t test. Statistical significance was set at P ≤ 0.05.
ReSuLtS
The adverse effects of many anticancer drugs are the main drawbacks that accompany their use. Thus, application of specific non-drug agents that reverse these effects can significantly improve the treatment action of traditional anticancer drugs. Previously, we have shown that dietary compounds selenomethionine and D-pantethine partial- ly decreased hepatotoxicity and myelotoxicity of doxorubicin in NK/Ly lymphoma-bearing mice (11). We suggested that tissue-protective effect of these antioxidants might be explained either by their direct scavenging of Dx-induced ROS or by protecting mitochondria that are considered to be the major source of cell-produced ROS (19) during damaging effect of Dx. In order to confirm this hypothesis, in vitro studies of the combined action of Dx and antioxidants were performed. B16 murine melanoma was selected for several reasons: a) it is internally resistant to Dx action, thus, any enhancement of its cytotoxic activity toward tumor cells by the antioxidants will be well seen. This is in contrast to other cellular models, which are very sensitive to Dx action, and a moderate effect of other compounds can be missed; b) B16 melanoma can be used both in vitro and in vivo on C57/Bl6 mice, allowing immediate verification of in vitro data using the same animal tumor model.
Such a combined in vitro-in vivo approach should be of great importance for identification of potential clinical markers of drug-induced oxidative stress and mitochondrial dysfunction, as well as their modulation by studied antioxidants.
Selenomethionine and d-pantethine do not protect B16 melanoma cells from doxorubicin-induced mitochondrial damage and have a little modulatory action on the level of superoxide anions increased under doxorubicin action We have checked if the observed tissue-protective effects of D-Pt and SeMet can be explained by their ability to scavenge produced toxic reactive oxygen species (ROS). Thus, the impact of these antioxidants on the level of specific ROS (namely, hydrogen peroxide and superoxide anions) was studied in cultured B16 melanoma cells. For selecting optimal (eg, non-toxic) concentrations of SeMet and D-Pt, as well as for evaluation of LC 50 dose (lethal concentration of drug killing 50% cells) of Dx, Trypan blue cytotoxicity assay was used (Figure 2). One can see that B16 melanoma is characterized by an internal resistance to Dx (LC 50 5 µM, and LC 75 25 µM). SeMet and D-Pt do not possess cytotoxic action toward B16 cells even used in high doses (50 µM), while in low concentrations (5-25 µM), SeMet partially (by 10%-12%, P < 0.001) inhibited cytotoxic action of Dx toward studied tumor cells (Figure 2).
Cytotoxic effect of Dx toward B16 cells was accompanied by a significant time-dependent increase in the level of both hydrogen peroxide (measured by DCFDA fluorescence assay) and superoxide anions production (measured by DHE assay) (Figures 3 and 4). In particular, H 2 O 2 concentration increased 2-fold already at 3 h after Dx addition to cell culture, and further enhanced up to 4-fold at 24 h time point (Figure 3). The same fluctuations were observed for Dx-induced O 2 -radicals whose level increased 2-fold at 3 h, also reaching its peak at 24 h time point (Figure 4). These events tightly correlated with mitochondrial damage, measured by Rhodamine 123 accumulation assay ( Figure 5). Dx in LC 50 dose (5 µM) led to disruption of 10% of cellular mitochondria at 3 h, 15% -at 6 h, 20% -at 12 h, while at 24 h time point this number increased to 42%. Thus, Dx-induced mitochondrial damage leads to time-dependent increase of both hydrogen peroxide and superoxide anions production in Dx-treated cells.
Surprisingly, no major ROS scavenging effects were observed at the action of SeMet or D-Pt (Figures 3-5). On the contrary, SeMet (5 µM) increased hydrogen peroxide in 3 h after Dx addition to cultured cells (Figure 3). At later time points, (6 h, 12 h, 24 h), no significant difference in the action of Dx and its combination with SeMet or D-Pt on the level of hydrogen peroxide was observed (Figure 3). D-Pt also had no effects on the level of superoxide anions under Dx treatment, while SeMet partially decreased it at 24 h in case of using high (10 µM) dose of Dx ( Figure 4).
Finally, both studied supplements had no effect on functional status of mitochondria in B16 cells damaged by Dx ( Figure 5). Thus, SeMet and D-Pt possessed a little cytoprotective impact toward Dx cytotoxic action on the melanoma cells in vitro, and had no impact on mitochondrial dysfunction and subsequent oxidative stress induced by Dx in these cells.
Selenomethionine, in contrast to d-pantethine, causes partial inhibition of growth of B16 melanoma in mice and enhances a therapeutic action of dx
In previous studies (11), we demonstrated that SeMet and D-Pt increased both survival and quality of life on mice bearing NK/Ly lymphoma. In case of B16 melanoma, a direct comparison of animal survival time in different groups was not possible due to specificity of this solid tumor model in which rapid development of large necrotic nodules takes place. Thus, tumor-bearing mice had to be euthanized according to ethical reasons before their death caused by tumor-induced intoxication and cachexia. In particular, animals of control group were euthanized at 22nd day af-ter tumor inoculation when tumor volume reached 3 cm 3 ( Figure 6A). D-Pt possessed a weak tumor-inhibitive action, and the implanted B16 melanoma in D-Pt-treated animals reached its maximum allowable size only at the 33rd day after its inoculation. On the contrary, SeMet alone significantly inhibited B16 melanoma growth whose volume was only 1 cm 3 at the 33rd day after tumor inoculation, thus, suggesting a major therapeutic effect of this dietary supplement toward B16 melanoma ( Figure 6A).
Despite high internal resistance of B16 melanoma to Dx action revealed by us in vitro (Figure 2), it positively responded to Dx therapy in vivo ( Figure 6B), and at the 33nd day after tumor inoculation, average size of tumor nodules was less than 900 mm3. A combination of Dx and D-Pt had not revealed a cumulative effect on B16 melanoma growth, suggesting little therapeutic importance of D-Pt in this model. On the contrary, a combination of Dx and SeMet possessed a strong synergistic effect on B16 melanoma progression and efficiently inhibited its growth at the 33rd day after tumor inoculation (average tumor volume was 320 mm 3 , P < 0.05) ( Figure 6B). At the 60th day after B16 melanoma inoculation, these differences between Dx and Dx+SeMet groups were still visible, but not statistically significant due to high variability of sizes and necrotization of FiGuRe 6. Changes in tumor volume and body mass of animals with B16 melanoma treated with d-Pt and SeMet alone (A) or in combination with dx (B). tumor volume was measured every other day starting from 10th day after melanoma inoculation according to materials and methods section. Animal weight was measured every other day, starting from 1st day of tumor inoculation. FiGuRe 7. Changes in level of creatinine, activity of aspartate aminotransferase and alanine aminotransferase in B16 melanoma bearing animals treated with dx and antioxidant compounds, at the 20th day after tumor inoculation. **P < 0.01 related to control, unpaired t test. ***P < 0.001 related to control, unpaired t test.
FiGuRe 8. Comparison of number of erythrocytes and leukocytes in B16 melanoma-bearing animals, treated with dx and antioxidant compounds, at the 20th day after tumor inoculation. **P < 0.01 related to control, unpaired t test. ***P < 0.001 related to control, unpaired t test.
tumor nodules. That was the reason for conducting euthanization of tumor-bearing animals for the ethical reasons.
Selenomethionine and d-pantethine decrease nephrotoxicity and myelosuppressive effects of dx in mice with B16 melanoma We found that growth of B16 melanoma was accompanied by a severe cachexia revealed as a relatively weak increase in the total body weight, in contrast to big tumor volumes in control mice ( Figure 6). In addition, B16 melanoma-bearing animals were characterized by twice lower level of creatinine (P < 0.001) compared to healthy mice ( Figure 7). It is known that creatinine level in blood is tightly dependent on the fluctuations of muscle mass (20), thus, cachexia-derived loss of muscle mass might be the main reason for low creatinine found in blood of tumor-bearing animals. Dx therapy, despite inhibiting of tumor growth, was also found to be nephrotoxic, increasing 4-fold creatinine level compared to such level in B16-bearing animals and 2-fold -compared to healthy control group. It should be noted that both SeMet and D-Pt efficiently lowered creatinine level in blood of melanoma-bearing animals to the level observed in healthy animals ( ~ 140 µM). Thus, both studied antioxidants, despite their insignificant inhibitory effect on the growth of B16 melanoma, demonstrated an efficient protection against the nephrotoxic action of Dx.
B16 melanoma-bearing animals were also shown to be suffering from the tumor-induced hepatotoxicity, as revealed by a 3-fold increase in aspartate aminotransferase (AST) activity in blood serum of animals ( Figure 7). Dx therapy reversed it to the values observed in healthy animals, while the combination of Dx with SeMet or D-Pt had shown the effect identical to Dx action (Figure 7). There were no significant changes found in the level of another enzymealanine aminotransferase, neither under tumor growth, nor under treatment with applied chemotherapies. Thus, no signs of the hepatotoxicity of Dx were observed in B16 melanoma-bearing animals suggesting that SeMet and D-Pt do not possess visible protective effects here.
Previously, we found that SeMet and D-Pt possessed strong myeloprotective activities toward Dx action in mice with NK/Ly lymphoma (11). Here, the in-depth studies of blood profile of animals with B16 melanoma treated with the same combination of drugs were performed. Blood samples were taken from tumor-bearing animals at the 10th day after chemotherapy start (the 20th day after tumor inoculation), and blood smears were prepared and compared with such smears, prepared from blood of healthy (control) animals ( Figure 8 and Figure 9). As one can see (Figure 8), growth of B16 melanoma is accompanied by a severe erythropenia and leukocytosis, while treatment of animals with Dx leads to further decrease in the number of erythrocytes, although it partially reverses leukocytosis (P < 0.001). D-Pt reversed erythropenia in tumor-bearing animals, and also partially increased their number in blood under Dx action, while SeMet had no impact here ( Figure 8). Both antioxidants revealed a strong inhibitive impact on the leukocytosis, since combined treatment of mice with SeMet and Dx lowered the number of leukocytes almost to the level found in healthy animals, while a combination of Dx+D-Pt diminished this index even further -up to 60% of the control level ( Figure 8).
Tumor-derived leukocytosis was characterized by two more important changes -2-fold decrease in number of small lymphocytes with a simultaneous 3-fold increase in the level of segmented neutrophils and young neutrophils with ring-shaped nuclei (Figure 9). Treatment of animals with Dx led to partial normalization (150% of control level) of all above mentioned indices, while a combination of Dx and SeMet or, to lower impact, with D-Pt completely reversed the number of neutrophils and small lymphocytes in blood of mice to the appropriate indices found in control group of healthy animals.
Finally, Dx treatment led to a significant monocytosis in B16-melanoma bearing animals ( Figure 9). Such phenomenon was observed when another experimental model, NK/ Ly lymphoma, was studied (11). Co-treatment of animals with SeMet or D-Pt reversed this parameter to sub-control levels, thus, indicating their myeloprotective properties.
Summarizing, the observed reversal of leukocytosis, neutrophilia, lymphopenia and monocytosis in B16 melanoma-bearing mice under their co-treatment with Dx and antioxidants might decrease the intensity of inflammatory processes switched on by tumor growth that could provide better quality of animals' life. This, in turn, might lead to lowering of cachexia effects in tumor-bearing animals, stabilization of their muscle mass, and normalization of creatinine levels.
diSCuSSioN
Use of antioxidants, especially vitamin C, as a supportive therapy for treatment of nearly all diseases -starting from flu and finishing with cancer, gained an extreme popularity in the last decades. It is known that cancer patients often use without doctors control the dietary antioxidant supplements during the conventional cancer treatment in hope to palliate side effects of the chemotherapeutic drugs and, thus, to improve their health conditions (7). General idea of such a massive use of dietary supplements at cancer treatment is based on the opinion that antioxidants help to protect and repair healthy cells that are damaged by the chemotherapy via quenching free radicals whose production is induced by the anti-cancer drugs. However, there is still no high-level evidence of the benefits of the combined use of antioxidants with conventional anticancer therapies for safety of cancer patients (21,22). Moreover, it is known that cancer cells are characterized by an increased ROS level (23), and application of antioxidants might actually decrease the efficiency of chemotherapy and worsen the FiGuRe 9. Changes in leukogram in B16 melanoma-bearing animals, treated with dx and antioxidant compounds, at 20th day after tumor inoculation.*P < 0.05 related to control, unpaired t-test. **P < 0.01 related to control, unpaired t-test. ***P < 0.001 related to control, unpaired t-test.
prognosis in cancer patient (24). Thus, more studies of molecular mechanisms of tissue-protecting action of the antioxidants have to be performed in order to reveal positive effects of their application in clinical medicine.
We have shown (10) that the organic selenium derivative -selenomethionine (SeMet) and vitamin B 5 precursor -Dpantethine (D-Pt) are capable of diminishing several side effects of Dx action in healthy rats, namely, they abolished the oxidative stress in blood cells and protected from a decrease of CoA level in liver. We conducted studies using mice with NK/Ly lymphoma and showed that these dietary supplements, besides lowering the myelotoxicity of Dx, also lead to a boost of survival time of tumor-bearing animals (11). One of the aims of present study was to verify those data using more aggressive solid tumor model -murine B16 melanoma. We also evaluated the potential molecular mechanisms of tissue-protective action of SeMet and D-Pt.
The dissection of ROS production at a systemic cancer therapy is a critical issue, since ROS might not only be mode-ofaction for the anticancer drugs, but they also might be the main cause of dose-limiting adverse effects. In that respect, the use of anthracyclines (eg, Dx) is particularly problematic. While the role of ROS in their in vitro and in vivo anticancer effects is questionable (25,26), the contribution of superoxide in cardiotoxicity is the major side effect (27).
It is believed that antioxidants protect normal cells from ROS-producing drugs by using a direct scavenging of the reactive oxygen species (28). Here we demonstrated that neither SeMet, nor D-Pt possessed such activity toward the action of Dx -a typical ROS inducing drug (3). In vitro studies conducted on B16 melanoma cells have revealed these cells are internally resistant to Dx action, with LC 50 = 5 µM (drug concentration that leads to killing 50% of tumor cells), and LC 75 dose 10 µM. In such doses, Dx caused a significant (4-5fold) and time-dependent increase in the level of hydrogen peroxide and superoxide anions that were measured by the DCFDA and DHE assays, correspondingly (Figures 4 and 5). However, SeMet and D-Pt failed to modulate these effects of Dx at most of studied time points (3-12 h). A statistically significant decrease (30%, P < 0.001) of superoxide anions under SeMet addition was observed only in 24 h after the start of B16 melanoma cell treatment with high dose of Dx (10 µM). D-Pt was capable of partial decreasing hydrogen peroxide levels, but only at 24 hour cell treatment with a lower dose of Dx (5 µM). These results allow one to suggest that in the case of B16 melanoma ROS scavenging capacity of SeMet and D-Pt do not play a decisive role in modu-lating Dx toxicity. As mitochondria are considered to be one of the main cellular sources of ROS (19), we evaluated the impact of SeMet and D-Pt on Dx-induced mitochondrial damage using Rhodamine 123 assay. It was revealed that both dietary supplements lacked any mitochondria-protective activity toward B16 melanoma cells, and, thus, failed to protect them from Dx-induced oxidative stress.
It should be stressed that in vitro studies of ROS-scavenging activities of SeMet and D-Pt were done only on tumor cell line, while these compounds might differentially act toward normal cell lines, and this question needs to be further investigated. Moreover, it was known that melanoma cells are usually characterized by the elevated basal ROS content compared to normal cells (29). Thus, absence of cyto-and ROS-protective effects of SeMet and D-Pt toward cultured B16 melanoma cells under Dx action might suggest that these compounds do not interfere with Dx therapeutic action in vivo, which was in fact observed by us. This might be a huge benefit for using SeMet and D-Pt in clinics. Both SeMet and D-Pt gradually increased quality of life of tumor-bearing animals by lowering the nephrotoxicity and monocytosis caused by Dx, as well as by a significant boost of the immune status of tumor-bearing animals, as revealed by a decreased neutrophilia and increased level of small lymphocytes in blood. It should be stressed that most of the observed positive effects of SeMet and D-Pt were found only at their usage in a combination with Dx. Normalization of creatinine level in blood of B16 melanoma-bearing animals that was decreased in control (nontreated) group comparing to healthy animals, was the only detected positive impact of SeMet and D-Pt applied alone. Such a decrease in creatinine level in blood of tumor-bearing mice might be explained by a severe cachexia (30) that was observed as a weak increase of animal body weight, in contrast to an intensive growth of tumor volume.
A systemic inflammation and disorders of lipid metabolism are considered to be the main triggering factors at cancer cachexia (31). Therefore, one might hypothesize that a reduced manifestation of cancer cachexia in animals treated with SeMet or D-Pt is associated with their immunomodulatory action aimed at decreasing of the inflammation processes. This, in turn, could diminish muscle mass loss that is the main consequence of cachexia in tumor-bearing organism.
However, the immunomodulatory action of SeMet and D-Pt cannot explain their nephroprotective properties under Dx therapy, since it is known that Dx-induced neph-rotoxicity is caused mainly by ROS produced by this drug (5). Our findings demonstrated that the molecular mechanisms of cell protection by SeMet and D-Pt against Dx action seem to be more complicated than a simple scavenging of ROS whose production is induced by Dx in damaged mitochondria. The observed phenomenon could be explained by a potential involvement of cellular glutathione system, as shown by us earlier (10,11), however, it needs further elucidation.
It should be stressed that in most animal tumor model studies, measuring of only the acute toxicity of applied drugs was possible, and the experiments were usually terminated in 30-60 days, while Dx-induced cardiotoxicity is usually observed much later, up to a year after chemotherapy start in human cancer patients (32). Thus, the performed experiments using murine B16 melanoma have limitations, since they cannot reveal the long-term outcomes of the proposed poly-chemotherapy scheme based on a combination of Dx, SeMet and/or D-Pt. This was the reason why we did not include the cardiotoxicity tests in our studies, as the analyzed periods of time -22 days for control group and 60 days for drug-treated groups -were too short for the development of visible manifestations of heart failure in the experimental animals. For such studies, less aggressive and slowly growing solid tumor models should be used, since they could allow animal observation during a longer period of 90-120 days.
The absence of cumulative therapeutic effects of Dx+SeMet/D-Pt co-treatment, comparing to single Dx injections, might suggest a preferable use of Dx in a combination with the studied antioxidants. Normalization of blood formula and the level of red blood cells, as well as stabilization of protein metabolism (according to the creatinine level), are important factors increasing chances for a long-term survival of cancer patient due to an abolishing of negative impact of chemotherapy on the organism.
In conclusion, current study demonstrated a distinct tissueprotective activity of SeMet and D-Pt toward acute toxicity of Dx on B16 murine melanoma. As revealed by the results of our in vitro assays on B16 melanoma cells, such effects of SeMet and D-Pt are not connected with a direct ROS scavenging and protection of mitochondria from damage, they rather suggest other mechanisms underlying the cytoprotective action of these antioxidants toward normal cells and tissues. Further in vivo studies addressed on revealing of the molecular mechanisms of tissue-protecting activity of SeMet and D-Pt are in progress. | v3-fos-license |
2020-11-26T09:04:42.419Z | 2020-11-16T00:00:00.000 | 229511022 | {
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} | pes2o/s2orc | MICROBIAL BIOMASS CARBON AND ENZYMATIC DEGRADATION OF CARBOHYDRATES BY APPLICATION OF VERMICULITE TO RECLAIMED SUBSTRATES
Abstract: A vegetation experiment was carried out with different amounts of vermiculite mixed with humus depot substrates, tailings pond and mine, in different proportions, and with an application of mineral fertilization and liming. Biomass carbon of microbial origin has the highest values after the addition of 10% vermiculite, simultaneous application of fertilization and liming, and alone fertilization, compared to controls, without ameliorants. The activity of the studied enzymes cellulase, amylase, invertase and catalase increased with increasing concentration of vermiculite, as well as in the combined application of fertilization and liming. The values of microbial biomass carbon and enzymes are highest in the variants with substrates from the mine.
Introduction
Soil formation at mining sites is essential for restoring ecosystem functions and depends on soil organic matter accumulation and development of an active microbial community. According to there are soil formation processes in reclaimed terrains whose speed and qualities depend on the soil-forming materials and the type of vegetation. Creating a future sustainable ecosystem by biological re-cultivation of mine embankments and incorporating them into the surrounding landscape is essential (Petrov, 2019). Technical and biological reclamation measures should be identified to limit the occurrence of erosion processes, soil pollution and create conditions for vegetation development (Petrova, 2009). The monitoring of microbiological and enzyme activity is an important part of the methods for assessing the effectiveness of reclamation treatments applied to degraded soil. The microbial community and enzyme activity are important biological indicators of soil quality and net ecosystem productivity in natural and regenerated areas (Dimitriu et al., 2010;Burns et al., 2013). Easily degradable soil organic matter is rapidly consumed by microorganisms and then degradation is dominated by the accumulation of microbial biomass (Ladd et al., 1995;Veen and Kuikman, 1990). Chodak and Niklińska (2012) established that mining soils contain significantly less organic C and total N. However, in some of them, microbial biomass and basal respiration reach values typical of natural forest soils. Microbiological and enzymatic (dehydrogenase, urease and phosphomonoesterase) activity are positively related to microbial biomass. In a study of enzyme activity and microbial biomass in the upper soil layer after brown coal mining, Baldrian et al. (2008) found that bacterial content, cellobiohydrolase and β-xylosidase were more affected by increasing the age of the site, while fungal biomass, chitinase and phosphatases were more affected by season. According to Wang et al. (2008) analyzes of microbial quantity (mainly bacteria followed by actinomycetes and micromycetes) and enzyme activity in tails contaminated with iron show positive correlations. The soil microorganisms and enzymatic activities of urease, invertase, cellulase, and acid phosphatase are reduced in tailings with high concentrations of Pb, Zn, Cd, and Cu (Qiang et al., 2018). The accumulation of microbial biomass in the initial soil-forming process in reclaimed sites is mainly due to the active development of non-spore-forming bacteria . Kuimei et al. (2012) found that the inoculation of mycorrhizal fungi into mini substrates leads to an increase in organic carbon and enzyme activity after 12 months of ryegrass cultivation and improves soil fertility during mine reclamation (Bi et al., 2007;Wang et al., 2009). The addition of coarse wood fragments increases the functional diversity of the microbial community, but not enzymatic activities of highland reclamation areas after oil production in Northern Alberta, Canada (Kwak et al., 2015). The activity of β-glucosidase enzyme in soil is considered to be a good indicator for efficiency of recultivation processes (Doni et al., 2012;Cele and Maboeta, 2016). The addition of bio-crust significantly improves soil microbial biomass and enzyme activities (alkaline phosphatase, dehydrogenase and urease) in the upper layer of copper-contaminated tailings ponds (Chen et al., 2009). A study of microbial and biochemical properties is necessary to evaluate the recovery and quality of soil after disturbance (Harris, 2003;Gil-Sotres, 2005). The purpose of the present study is to determine the biomass carbon accumulation and the degree of enzymatic degradation of carbohydrates when applying vermiculite to reclaimed substrates.
Materials and methods
A vegetation experiment was conducted in March 2019 with different amounts of vermiculite (5%, 10% and 20%) mixed with substrates in different ratios, with mineral fertilization (N150P150K150 kg/ha) and liming (substrate neutralization) (table 1). Substrates from the mine are contaminated with heavy metals, mainly copper and cadmium above the maximum permissible concentrations (MPC). Whereas in the soil and tailings substrates, the concentrations of these elements are below the MPC. The arsenic content follows the same distribution -higher concentrations in the mine samples and lower in the other variants. Source: Author Studies were conducted on the 30th and 95th days from the start of experimentation -before and after liming and fertilizing, respectively, using the following methods: ▪ Microbial biomass carbon (MBC) -fumigation spectrophotometric method (Cai et al., 2011); ▪ Cellulase, amylase and invertase activity -spectrophotometric method (Gradova et al., 2004); ▪ Catalase activity -manganometric titration method (Khaziev, 1976).
Results and discussion
Starting materials used for the vegetation experience are poor in total organic carbon. MBC on the 30th day is presented in the following figure 1. Figure 1: Microbial biomass carbon on the 30 th day, mg C/g soil Source: Author Microbial carbon biomass on the 30th day of the vegetation experiment was higher in variants with the addition of vermiculite compared to controls. In the mine substrate samples, the microbial organic carbon values were highest and up to 1.13 times against control (M-0), decreasing with the addition of 20% vermiculite. For the variants with vermiculite from soil depot, biomass C values increased to 1.2fold relative to the control (SD-0), being highest when 10% vermiculite was added. A similar tendency is observed in the variants with tailings substrates -the highest values of MBC are found in the variant with the addition of 10% vermiculite -1.4 times above SD-0. Biomass carbon on the 95th day of reporting follows its distribution trend on the 30th day, increasing by about 1.3-fold (Fig. 2). Author Microbial carbon biomass on the 95th day of the vegetation experiment was higher in the variants where activities were conducted compared to the control samples, such as S-0F˃S-0. The combined application of lime and fertilizer maximizes microbial biomass. Self-lime application gives better results on this indicator than fertilization alone. The highest MBC values are again established in substrates from the mine. In general, fertilization has a more favorable effect on microbial biomass in tailings samples, with higher values being obtained than in soil depot variants. Overall, the addition of 10% vermiculite maintains the best results for MBC accumulation. Cellulase activity is highest in the mine variants (up to 1 time with the addition of 5% vermiculite and with similar values in the other two samples relative to the control), followed by variants with substrate from the soil depot (up to 1 time relative to the control) and the lowest for tail variants (lower than controls) (Fig. 3). Figure 3: Cellulase activity on the 30 th day, mg glucose/g soil Source: Author On the 30th day after starting the experiment (before liming and fertilizing), a decrease in cellulase activity was observed with increasing vermiculite concentration. While on the 95th day of starting the experiment (after liming, fertilizing and mowing), this trend reverses -cellulase enzyme activity increases with increasing vermiculite concentration -up to 1.4 times with the addition of 20% vermiculite, fertilization and liming compared to the control. The highest values of cellulase activity for the variants from mine are found in conducted liming and fertilization, with concentrations growing with increasing amount of vermiculite. Variants "with fertilizing, without liming" show better results than "with liming, without fertilizing". Fertilization increases the activity of cellulase in the other variants also -with substrates from the soil depot and tailing pond, the dependence being again proportional to the increase in the concentration of vermiculite. Higher values of cellulase activity are found compared to day 30 (up to 1.85 times for mine variants, 1.64 times for soil depot variants and 1.85 times for tail variants), which may to be explained, on the one hand, by the fertilization and liming carried out, as well as by an increase in the amount of organic matter in the samples of dying plant residues over time and, accordingly, stimulating the development and activity of the soil microorganisms (Fig. 4). In general, on the 30th day of the experiment (before liming and fertilizing), a decrease in amylase activity was observed with an increase in the amount of vermiculite. It is highest for the vermiculite mine variants (up to 1.4 times against control), followed by the soil depot variants (up to 1.1 times against control soil) with the lowest enzyme activity seen in the tail variants (up to 1.1 times against controls) (Fig. 5). The opposite trend was observed in the results of day 95 -a promotion in amylase activity with an increase in the amount of vermiculite. The combined application of fertilization and liming in variants from the mine increases activity of this enzyme to a greater extent -1.7 times for variant M-IV (20%) compared to the control. Variants "with fertilizing, without liming" show in better results than "with liming, without fertilizing". Similar to the break down of cellulose, the degradation of starch in the variants increases after fertilization and liming (Fig. 6). Author Invertase activity is highest in the mine variants (up to 1.2 times the control), followed by the soil depot (up to 1.1 times the control) and lowest (even lower than the control samples) is the enzyme activity in the tail variants. On the 30th day after starting the experiment a decrease in the activity of invertase enzyme was observed with an increase in the amount of vermiculite. This tendency is clearer for variants of the mine, while for the variants of soil depot the values are close, and for variants with tail the same regardless of the vermiculite concentrations (Fig. 7). On the 95th day from starting the experiment (after liming, fertilizing and mowing), invertase activity increased with increasing amount of vermiculite in all variants as well as on the 30th day (Fig. 8). Figure 8: Invertase activity on the 95 th day, mg glucose/g soil Source: Author The combined application of fertilization and liming in variants from the mine increases the activity of enzyme to a greater extent -up to 1.7 times in variant M-IV (20%). Variants "with fertilizing, without liming" show better results than "with liming, without fertilizing". Fertilization and the addition of vermiculite increase invertase activity by up to 1.3 times in soil depot variants, while in tail variants the values of invertase are below control, but in fertilizer variants the results are better than in those without fertilization. In general, activities of the three enzymes follows in descending order: amylase> cellulase> invertase. This trend depends on the type of vegetation and development of certain groups of microorganisms capable of producing the enzymes studied. Cellulase, amylase and invertase activity break down cellulose, starch and sucrose to glucose, respectively. Degradation of soil glucose to hydrogen peroxide and water is catalyzed by βglucosidase enzyme. The toxic hydrogen peroxide is degraded to water and oxygen by the catalase enzyme (final stage of carbohydrate degradation in the soil) ( Fig. 9 and Fig. 10). Just like the 30th day, and the 95th day from the start of the experiment, the catalase activity was highest in the variants from the mine, followed by the variants from the soil depot and tailings pond. Activity of this enzyme increases with increasing amount of vermiculite and is highest with the addition of 20% vermiculite in both study periods -up to 1.3 (30th day) -1.4 (95th day) times compared to the control sample M-0. Catalase activity in the tailings variants has values above the control ones, i. catalase activity is less inhibited than amylase, cellulase and invertase activity. The highest values of catalase enzyme are after liming and fertilizing. Variants "with fertilizing, without liming" show better results than "with liming, without fertilizing". The lowest and closest to the control samples are the values of catalase without liming and fertilizing. Fertilization raises catalase activity as values increase with increasing amount of vermiculite in the samples. As noted, the accumulation of microbial biomass C and enzyme activities have the highest values in the most highly contaminated with toxic elements variants -those of the mine. In recent years vermiculite has been widely used for the adsorption of heavy metals (Malandrino et al., 2011;Sis and Uysal, 2014;De Freitas et al., 2017). Furthermore, vermiculite significantly improves the drainage properties of substrates, increases porosity and looseness, prevents sealing and soil compaction, and protects roots from sharp temperature fluctuations.
Conclusion
Microbial biomass has the highest values in the mine variants, followed by the variants with tail and lowest values has MBC in the variants of soil depot. MBC had higher values after the addition of vermiculite compared to controls, with best results in all variants being obtained at 10% vermiculitis. The combined application of liming and fertilizing in the mine variants, as well as fertilization in the variants from the soil depot and tailing, increases МBC and enzyme activities to a greater extent.
The activity of all enzymes tested is highest for the mine variants, followed soil depot variants and lowest for the tail variants. On the 30th day from starting the experiment (before liming and fertilizing), a decrease in activity of cellulase, amylase and invertase was observed with increasing vermiculite concentration. Probably irrespective of the accumulated MBC, initially the activity of the microorganisms (respectively the enzyme activity) is suppressed. This conclusion is not borne out by the results of day 95 -where a raising in the enzyme activities with an increase in the amount of vermiculite is seen. While catalase activity increased with increasing amount of vermiculite, it was highest with the addition of 20% vermiculite in both study periods. Variants "with fertilizing, without liming" show better results than "with liming, without fertilizing". | v3-fos-license |
2016-05-12T22:15:10.714Z | 2014-12-05T00:00:00.000 | 18154000 | {
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} | pes2o/s2orc | Polarity Specific Effects of Transcranial Direct Current Stimulation on Interhemispheric Inhibition
Transcranial direct current stimulation (tDCS) has been used as a useful interventional brain stimulation technique to improve unilateral upper-limb motor function in healthy humans, as well as in stroke patients. Although tDCS applications are supposed to modify the interhemispheric balance between the motor cortices, the tDCS after-effects on interhemispheric interactions are still poorly understood. To address this issue, we investigated the tDCS after-effects on interhemispheric inhibition (IHI) between the primary motor cortices (M1) in healthy humans. Three types of tDCS electrode montage were tested on separate days; anodal tDCS over the right M1, cathodal tDCS over the left M1, bilateral tDCS with anode over the right M1 and cathode over the left M1. Single-pulse and paired-pulse transcranial magnetic stimulations were given to the left M1 and right M1 before and after tDCS to assess the bilateral corticospinal excitabilities and mutual direction of IHI. Regardless of the electrode montages, corticospinal excitability was increased on the same side of anodal stimulation and decreased on the same side of cathodal stimulation. However, neither unilateral tDCS changed the corticospinal excitability at the unstimulated side. Unilateral anodal tDCS increased IHI from the facilitated side M1 to the unchanged side M1, but it did not change IHI in the other direction. Unilateral cathodal tDCS suppressed IHI both from the inhibited side M1 to the unchanged side M1 and from the unchanged side M1 to the inhibited side M1. Bilateral tDCS increased IHI from the facilitated side M1 to the inhibited side M1 and attenuated IHI in the opposite direction. Sham-tDCS affected neither corticospinal excitability nor IHI. These findings indicate that tDCS produced polarity-specific after-effects on the interhemispheric interactions between M1 and that those after-effects on interhemispheric interactions were mainly dependent on whether tDCS resulted in the facilitation or inhibition of the M1 sending interhemispheric volleys.
Abstract
Transcranial direct current stimulation (tDCS) has been used as a useful interventional brain stimulation technique to improve unilateral upper-limb motor function in healthy humans, as well as in stroke patients. Although tDCS applications are supposed to modify the interhemispheric balance between the motor cortices, the tDCS after-effects on interhemispheric interactions are still poorly understood. To address this issue, we investigated the tDCS after-effects on interhemispheric inhibition (IHI) between the primary motor cortices (M1) in healthy humans. Three types of tDCS electrode montage were tested on separate days; anodal tDCS over the right M1, cathodal tDCS over the left M1, bilateral tDCS with anode over the right M1 and cathode over the left M1. Single-pulse and pairedpulse transcranial magnetic stimulations were given to the left M1 and right M1 before and after tDCS to assess the bilateral corticospinal excitabilities and mutual direction of IHI. Regardless of the electrode montages, corticospinal excitability was increased on the same side of anodal stimulation and decreased on the same side of cathodal stimulation. However, neither unilateral tDCS changed the corticospinal excitability at the unstimulated side. Unilateral anodal tDCS increased IHI from the facilitated side M1 to the unchanged side M1, but it did not change IHI in the other direction. Unilateral cathodal tDCS suppressed IHI both from the inhibited side M1 to the unchanged side M1 and from the unchanged side M1 to the inhibited side M1. Bilateral tDCS increased IHI from the facilitated side M1 to the inhibited side M1 and attenuated IHI in the opposite direction. Sham-tDCS affected neither corticospinal excitability nor IHI. These findings indicate that tDCS produced
Studies using transcranial magnetic stimulation (TMS) have demonstrated that transcallosal inhibition is affected by the modulation of intracortical motor circuits in both M1 that project and receive callosal volleys [21][22][23][24]. Hence, it is possible that tDCS-induced neuromodulation in the M1 neural circuits affects transcallosal inhibition. Indeed, Lang et al. [25] demonstrated that transcallosal inhibition measured by the duration of ipsilateral silent period (iSP) was increased and decreased by anodal and cathodal tDCS, respectively, that were unilaterally delivered to the motor cortex receiving transcallosal inhibition. However, the robust effects on iSP were not observed after unilateral tDCS given to the motor cortex projecting callosal volleys [25]. These findings may not be in line with the idea that tDCS given to a motor cortex influences the contralateral motor cortex through the modulation of transcallosal pathways. Subsequently, Williams et al. [5] investigated short-interval interhemispheric inhibition (IHI) elicited by paired-pulse TMS and found that IHI was suppressed after the application of bilateral tDCS combined with unimanual motor training. Although the reduction of IHI was accompanied by the decrease of corticospinal excitability in the side of motor cortex projecting callosal volleys, their causal association was not fully elucidated [5].
IHI and iSP are thought to be mediated by different neuronal populations in the transcallosal pathways [26], suggesting the possibility that tDCS does not affect their different neuronal populations in a similar way. Indeed, Gilio et al. [27] demonstrated that 1 Hz repetitive TMS (rTMS) given to the left M1 suppressed IHI from the left M1 to right M1 with minor effects on iSP. Given these physiological backgrounds, we hypothesized that tDCS given at rest would induce polarity-specific after-effects on IHI from the stimulated M1 in which the corticospinal excitability was changed. To examine this hypothesis, we investigated the after-effects of tDCS applied at rest with three different electrode montages (i.e., unilateral anodal, unilateral cathodal, and bilateral). Each montage was intended to elicit either facilitation of right corticospinal excitability, inhibition of left corticospinal excitability, or both. It should be noted that the intended relative change between the left and right corticospinal excitabilities was the same across the three electrode montages, with right greater than left. Before and after each tDCS, single-pulse TMS and paired-pulse TMS were given to the left M1 and right M1 in order to assess the corticospinal excitability and mutual direction of IHI.
Participants
Participants were sixteen healthy right-handed volunteers (22-34 years old, 3 females). All participants gave their written informed consent to participate in this study. The experimental and consent procedures were approved by the ethical review board of the National Rehabilitation Center for Persons with Disabilities and which was in accordance with the guidelines established in the Declaration of Helsinki. All participants were naïve to the purpose of the experiments.
Recordings
Electromyography (EMG) was recorded from the bilateral first dorsal interosseous (FDI) muscles. Self-adhesive Ag/AgCl electrodes were placed over the muscle belly and the metacarpophalangeal joint. The EMG signals were amplified and filtered (bandwidth, 20-3000 Hz) with a conventional bioamplifier (BIOTOP 6R12, NEC San-ei, Tokyo, Japan). Their digital data were acquired with a sampling rate of 5 kHz with a CED 1401 A/D converter (Cambridge Electronic Design, Cambridge, UK) and stored on a computer for off-line analysis.
TMS
Corticospinal excitability and IHI were investigated by single-pulse and pairedpulse TMS, respectively. TMS was delivered to the left M1 and the right M1 with a figure 8-shaped coil (70-mm diameter) connected to a Magstim 200 (Magstim, Whitland, UK). The stimulus location was determined to be the hot spot where weak stimulation could elicit the largest motor evoked potential (MEP) in the FDI muscle. The coil was held tangentially over the scalp with the handle pointing backward and 45˚lateral away from the midline. The resting motor threshold (RMT) was defined as the minimum stimulus intensity that produced MEPs that were greater than 50 mV in at least 5 out of 10 consecutive trials. For the singlepulse TMS, the intensity of test stimulation (TS) was set at 120% of the RMT. Stimuli were consecutively delivered about every 10 s. Both the left and right hemispheres were examined sequentially with a randomized order across the participants. Fifteen MEPs were obtained at each hemisphere. Paired-pulse TMS was used to elicit IHI both from the left M1 to the right M1 and from the right M1 to the left M1. A suprathreshold conditioning stimulation (CS) with an intensity at 120% of RMT was delivered to M1 on one side 10 ms before a TS was delivered to M1 on the other side. For a few participants, it was impossible to place both coils at the optimal direction due to the size of the coil. Thus, the handle of the coil for the CS was pointed backward and more than 45˚away from the midline until both coils did not contact each other. The TS intensity was adjusted so that the peak-to-peak amplitude of the MEP was about 1 mV. The paired-pulse stimulation and TS alone were randomly given every 10 s. Fifteen control MEPs and 15 conditioned MEPs were obtained at each side of tested FDI. For both single-pulse and paired-pulse TMS, if trials showed more than 20 mV of EMG activity in the window of 100 ms before TMS, additional stimuli were given instead of those trials.
tDCS
Direct current stimulation was delivered by a battery-driven constant-current stimulator (Eldith DC-Stimulator, NeuroConn, Ilmenau, Germany) through a pair of rubber electrodes (565 cm) covered with saline-soaked sponges (566 cm). We examined three kinds of electrode montages; anodal tDCS over the right M1, cathodal tDCS over the left M1, and bilateral tDCS over the right M1 and left M1. For anodal tDCS, the anode and cathode were positioned on the right M1 (i.e., the hot spot of the left FDI) and the superior edge of the left orbit, respectively ( Figure 1A). For cathodal tDCS the anode and cathode were positioned on the superior edge of the right orbit and the left M1 (i.e., the hot spot of the right FDI), respectively ( Figure 1B). For bilateral tDCS, the anode and cathode were over the right M1 and left M1, respectively ( Figure 1C). The current polarity at each electrode was masked to the participants. 1.5 mA of direct current stimulation was delivered for 15 min. The current was gradually increased and decreased during the first and last 10 s of the stimulation, respectively. Sham-tDCS was conducted for 15 min with the montages of anodal tDCS and bilateral tDCS ( Figure 1D, E). The 1.5 mA of direct current stimulation was delivered for first 30 s subsequent to 10 s of current increment.
Experimental procedures
The experiments were composed of real-tDCS and sham-tDCS sessions. 12 participants joined the real-tDCS session and 9 participants joined the sham-tDCS session. 5 out of 16 participants were involved in both sessions; three of them participated in the real-tDCS session first and two of them participated in the sham-tDCS session first. Each kind of electrode montage was tested on a different day. At least 3 weeks were interleaved across the experimental days. At each tDCS session, the order of the electrode montages was randomized across participants.
In the experiments, the participants sat comfortably on a reclining chair with their shoulders and elbows semi-flexed. Both of their hands were placed on the table with palms downward. Before the tDCS application, RMT was measured in both M1. Then, the single-pulse and paired-pulse TMS protocols were conducted. After these baseline measurements were made, real-or sham-tDCS with an electrode montage was given for 15 min. After tDCS application, the same measurements were conducted on each side of M1.
Data analysis
For the evaluation of corticospinal excitability, the peak-to-peak amplitudes of the MEPs elicited by single-pulse TMS were measured in the window 18-50 after the TMS trigger. The extent of after-effects was expressed as the ratio of the MEP amplitude obtained after tDCS to the baseline MEP amplitude obtained before tDCS. In order to evaluate IHI, the amplitude of the conditioned MEPs elicited by paired-pulse stimulation were normalized by the amplitude of the control MEPs evoked by TS alone. Trials with more than 20 mV of peak-to-peak amplitude in background EMG activity for 100 ms pre-stimulus period were discarded from the analysis. For the statistical analysis, a three-way analysis of variance (ANOVA) with repeated measures was performed with factors of time (before and after tDCS), tDCS type (real-anodal, real-cathodal, real-bilateral, sham-anodal, shambilateral), and TS side (left and right M1). In case a significant interaction between three factors was obtained, appropriate follow-up two-way ANOVA was conducted to examine the interaction of time and TS side factors at each tDCS type. In order to compare the magnitude of after-effects across conditions, oneway ANOVA with repeated measures was conducted with factor of tDCS type at each TS side. For the comparison of baseline level in each measurement, two-way ANOVA with repeated measures was performed with factors of tDCS type and TS side. Post-hoc comparisons were conducted by Tukey's test.
IHI was expressed as the ratio of the conditioned MEP amplitude normalized by the control MEP amplitude (i.e., larger value indicates less IHI). The sets of the left-and right-sided columns represent IHI from the left M1 to the right one (L to R) and that from the right M1 to the left one (R to L), respectively. The black and gray columns represent before and after tDCS, respectively. Error bas show standard error of means. The asterisks indicate a significant difference; * p,0.05.
According to the findings in the previous studies [12,16], we expected that real-tDCS induced the polarity-specific modulation in the M1 underneath the active electrode. Thus, we anticipated that real-tDCS influenced the excitability of callosal neurons in the same M1. Therefore, to examine the relationship between the after-effects on MEP amplitude and IHI, we also conducted Pearson correlation analysis in the real-tDCS after-effects between MEP amplitude and IHI. P values less than 0.05 were recognized as statistically significant in all analyses. Group data are presented as the mean ¡ standard deviation in the text.
RMT, MEP
RMT was different across TS sides (F 1,49 55.53, p50.02). TMS given to the left M1 showed slightly lower RMT than the right M1 (Table 1) Figure 1A illustrates representative example of MEPs elicited before and after tDCS. Consistent with the findings in the previous studies [12,16], facilitation and inhibition were observed in the MEPs elicited by single-pulse TMS over the M1 under the anode and the cathode, respectively. Three-way ANOVA revealed significant interactions of time and tDCS type and TS side (F 4,49 54.39, p50.004) on MEP amplitude, indicating that the interaction of time and TS side was dependent on the tDCS type. Then, we performed follow-up two-way ANOVA for each tDCS type. Regardless of electrode montage, real-tDCS showed significant interaction of time and TS side (real-anodal, F 1,11 58.32, p50.02; real-cathodal, F 1,11 55.76, p50.04; real-bilateral, F 1,11 523.53, p,0.001), indicating that all electrode montage had tDCS after-effect on MEP amplitude such that their tDCS after-effects were different depending on the TS side. Post-hoc analysis revealed that after real-anodal tDCS over the right M1, the MEP elicited from the right M1 was increased (232.0¡144.7%, p,0.001) and the MEP elicited from the left M1 was unchanged (111.1¡41.7%, p50.54) compared with the baseline ( Figure 1A). After real-cathodal tDCS over the left M1, the MEP elicited from the left M1 was decreased (76.2¡27.6%, p50.01) and the MEP elicited from the right M1 was unchanged (109.0¡36.5%, p50.45, Figure 1C). After real-bilateral tDCS (anode over the right M1, cathode over the left M1), the MEP elicited from the right M1 was increased (157.6¡68.2%, p,0.001) and the MEP elicited from the left M1 was decreased (75.4¡28.3%, p50.01, Figure 1E). In contrast to real-tDCS, nether of sham-tDCS showed significant main effect of time (sham-anodal, To sum up, facilitation and inhibition were observed in the MEPs elicited from the M1 under the anode and the cathode, respectively. With real-anodal and realcathodal tDCS, the MEP elicited from the unstimulated M1 was unchanged. The magnitude of after-effects was not different across the conditions that showed significant facilitation (real-anodal 232.0¡144.7%, real-bilateral 157.6¡68.2%, p50.20) or inhibition (real-cathodal 76.2¡27.6%, real-bilateral 75.4¡28.3%, p50.99).
IHI
Both before and after tDCS, IHI was examined both from the left M1 to the right M1 and from the right M1 to the left M1. By adjusting the TS intensity to elicit a 1 mV MEP, the amplitude of the control MEP was not different across conditions. Three-way ANOVA revealed significance of neither main effect of time ( The three-way repeated measures ANOVA revealed significant interaction of time and tDCS type and TS side (F 4,49 52.64, p50.04) on IHI, indicating that the interaction of time and TS side was dependent on the tDCS type. Then, we performed follow-up two-way ANOVA for each tDCS type. Real-anodal and realbilateral tDCS showed significant interaction of time and TS side (real-anodal, . That is, in the real-tDCS session, all electrode montages had tDCS after-effect on IHI. The tDCS after-effect was different depending on the TS side (i.e., direction of IHI) after real-anodal and real-bilateral tDCS. On the other hand, the after-effect of real-cathodal tDCS was independent of TS side. Post-hoc analysis demonstrated that after real-anodal tDCS over the right M1, IHI from the right M1 to the left M1 was significantly increased compared with baseline (p,0.001). However, IHI from the left M1 to the right M1 was unchanged (p50.16, Figure 1B). After real-cathodal tDCS over the left M1, a reduction in IHI magnitude was observed both from the left M1 to the right M1 and from the right M1 to the left M1 (p50.01, Figure 1D). After real-bilateral tDCS (anode over the right M1, cathode over the left M1), IHI from the left M1 to the right M1 was decreased compared with baseline (p50.001). In contrast, IHI from the right M1 to the left M1 was increased compared with baseline (p50.003; Figure 1F). Again, neither of sham-tDCS affected IHI ( Figure 1H, J) In summary, IHI from the M1 under the anode was increased. In contrast, IHI from the M1 under the cathode was decreased. IHI from the unstimulated M1 showed a decrease after cathodal tDCS, but it was unchanged after anodal tDCS. Finally, we tested the correlation of tDCS after-effects between MEP amplitude and IHI. However, we did not find any significant correlations between the modulations of MEP amplitude and IHI regardless of TS side (Table 2).
Discussion
The present study demonstrated that tDCS produced polarity-specific after-effects on IHI from the stimulated M1 at which the corticospinal excitability was changed. Regardless of unilateral or bilateral tDCS, IHI was generally increased from the M1 at which the corticospinal excitability was increased and decreased from the M1 at which the corticospinal excitability was decreased. Bilateral tDCS simultaneously produced the opposite directional modulation in IHI from the left to the right M1 and in IHI from the right to the left M1 in addition to the bidirectional corticospinal modulation. Although unilateral anodal tDCS did not affect the corticospinal excitability at the side of unstimulated hemisphere or IHI from the M1 on that unstimulated hemisphere, unilateral cathodal tDCS suppressed IHI from the M1 on the unstimulated hemisphere even though the corticospinal excitability was unchanged at the unstimulated side.
In most cases, the modulations of IHI were parallel to the modulations of corticospinal excitability at the side sending callosal volleys. Thus, it is likely that the tDCS after-effects on IHI are relevant with the excitability change in the motor cortex sending callosal volleys. However, we did not observe any significant relationships between the modulations of MEP amplitude and IHI. If IHI is mainly derived from the collateral discharges of corticospinal neurons and the tDCS-induced modulation in IHI resulted from the changes in collateral discharges, the modulations in MEP amplitude and IHI could have been correlated. Therefore, the modulation of transcallosal pathways could be partly independent of the changes in corticospinal descending pathways. Transcallosal inhibition is assumed to be derived from the discharge of callosal neurons that are distinct from corticospinal neurons [24,28,29]. Accordingly, tDCS might have similarly influenced both corticospinal and callosal neurons in the same M1. In some previous studies, IHI has been evaluated by matching the size of CS-induced MEPs in order to normalize the CS effect [24,30,31]. However, the adjusted CS intensity may not be sensitive enough to detect the excitability change in callosal neurons when both corticospinal and callosal neurons are modulated in parallel [24,31,32]. In the present study, we used the same CS intensity across before and after the tDCS sessions according to the RMT. Therefore, the modulation of IHI could be observed by detecting parallel modulation in the excitabilities of corticospinal and callosal neurons.
Our findings of the modulation of transcallosal inhibition are partly inconsistent with a previous study that used iSP [25], although the corticospinal excitability was modulated in a similar way. The previous study did not observe changes in iSP from the modulated M1 underneath the tDCS electrode [25]. One possible explanation for this discrepancy may be the differences in the tDCS parameters. The present experiments used a higher intensity (1.5 mA) and a longer duration (15 min) of tDCS compared to the previous study (1.0 mA intensity, 10 min duration). The tDCS after-effects have been shown to increase up to a certain extent of intensity and duration [12,33,34]. Furthermore, because the threshold for eliciting transcallosal inhibition is known to be higher than the RMT for MEPs [29,[35][36][37], callosal neurons might require a relatively high intensity and long duration of tDCS to be modulated. Another possibility is the different neural populations mediating transcallosal inhibition because partly different sets of callosal neurons and target neurons receiving callosal volleys have been assumed to mediate short-interval IHI and iSP [26]. In addition, iSP appears as the inhibition of static voluntary activity, although IHI is the inhibition of synchronized corticospinal discharges that TMS artificially evokes [29]. Accordingly, such physiological differences might relate to the different susceptibilities to tDCS. Indeed, previous study using rTMS demonstrated the modulation of IHI without robust changes of iSP [27]. We also observed a reduction of IHI from the unchanged M1 after unilateral cathodal tDCS, although unilateral anodal tDCS did not modulate IHI from the unchanged M1. These findings suggested that unilateral tDCS affected interneuronal circuits that presynaptically regulate callosal transmission and/or relay them to the corticospinal neurons [25]. Indeed, tDCS-induced plastic modulation has been shown in some intracortical interneurons that mediate gamma-aminobutyric acid activity [38][39][40]. One potential reason that unilateral anodal tDCS failed to modulate IHI in this direction might be due to the asymmetry in transcallosal inhibition. Generally, transcallosal inhibition is greater from the left M1 to the right M1 than from the right M1 to the left M1 in righthanders [41,42], which was also confirmed in our study. Furthermore, previous study reported asymmetric effects of tDCS [4]; tDCS applied over the left dominant hemisphere was more effective than that over the right non-dominant hemisphere. In our study, anodal and cathodal stimuli were given to the different hemispheres. Hence, the lack of modulation of IHI toward the facilitated rightside M1 might be also attributed to the decreased efficiency of tDCS that is applied over the non-dominant hemisphere.
The effect of interventional brain stimulation on transcallosal inhibition has been tested by several stimulation protocols such as low-frequency rTMS [27,43], theta burst stimulation [44,45], paired associative stimulation [46], tDCS [5,25], and quadripulse TMS [47]. Even though their protocols were able to elicit bidirectional modulation on the corticospinal excitability, the modulation of transcallosal inhibition was not always observed [44,45]. Presumably, the neural elements involving with transcallosal inhibition might have different susceptibilities according to the type of brain stimulation protocol. Although our results show that bilateral tDCS was able to elicit the bidirectional modulation in transcallosal inhibition between left M1 and right M1 in addition to the left and right corticospinal excitabilities, it is worth noting that the extent of MEP modulation by bilateral tDCS was not different compared to that by unilateral tDCS. This finding was also reported in recent studies [16,17]. Additionally, in line with previous studies [16,25,48], neither the polarity of unilateral tDCS affected the corticospinal excitability in the contralateral unstimulated motor cortex even though transcallosal inhibition toward that motor cortex showed short-lasting after-effects ( Figure 1). These findings suggest that transcallosal inhibition modulated by tDCS might have minor static effects on the corticospinal excitability in the contralateral motor cortex. Nevertheless, previous studies demonstrated that bilateral tDCS was more effective for improving hand motor performance compared to unilateral anodal tDCS over the target motor cortex [3,10], and that unilateral cathodal tDCS over a motor cortex results in substantial improvement of ipsilateral hand motor function in healthy [2,4] and stroke individuals [7,9,11,49]. These facts could provide us rationale to suppose that suppressed transcallosal inhibition contributes to the contralateral cortical motor activity. Indeed, Williams et al. [5] demonstrated a functional relationship between the suppression of transcallosal inhibition and improvements in motor performance using bilateral tDCS. Conceivably, it might be that a functional role of the decreased transcallosal inhibition can be observed in a time-specific motor event like movement initiation. Transcallosal inhibition is gradually decreased according to the time course of movement initiation [50,51]. Therefore, a sustained reduction of transcallosal inhibition could contribute to such a situation of motor performance rather than a static enhancement of corticospinal excitability. To support this notion, recent studies using functional magnetic resonance imaging demonstrated that motor task-related M1 activation was greater in bilateral tDCS compared to unilateral anodal tDCS, and that the M1 activation changes in laterality were correlated with microstructural status of transcallosal motor fibers [18] although resting-state interhemispheric functional connectivity between the left M1 and the right M1 did not show after-effects regardless of unilateral anodal or bilateral tDCS [20]. Therefore, it seems conceivable that modulated transcallosal pathways contribute to the motor performances without marked changes in the corticospinal excitability at rest.
From the methodological point of view, we need to consider tDCS parameters as limitations of our study. First, strong intensity and long duration of direct current stimulation has a risk of over stimulating that causes reversing facilitatory effect of cathodal tDCS on the corticospinal excitability. A recent study demonstrated that cathodal tDCS with 2 mA of intensity and 20 min of duration facilitated the corticospinal excitability [52]. Because tDCS with a high intensity (2 mA) and a short duration (5 min) retained the general polarity-specific aftereffects [16], the combination of intensity and duration might be a specific factor for the tDCS after-effects. Second, small number of participants should be considered as another limitation. Though we found significant tDCS after-effects on MEP amplitude and IHI, some insignificant results may be due to small sample size. We should make a point that the participants were not completely identical across real-tDCS and sham-tDCS sessions. Finally, our study cannot completely rule out spinal effects [53,54]. Though IHI was demonstrated to be mediated by cortical circuits through transcallosal pathways [55,56], potential contribution of subcortical circuits to IHI need to be considered [57].
As a therapeutic tool, tDCS has been frequently applied in patients with hemiparetic stroke [58]. Thus, our findings that tDCS modulated transcallosal inhibition with polarity-specific manner could provide a useful perspective on the understanding of the tDCS therapeutic effect on the recovery of motor function after stroke. In terms of interhemispheric neural modulations, the application of cathodal tDCS to contralesional hemisphere appears to be reliable as demonstrated by some clinical studies [7,8,10,11,49]. However, we may also need to take into account the tDCS effect on the uncrossed ipsilateral motor pathway [59,60]. A recent study demonstrated that cathodal tDCS over a motor cortex affected presumed uncrossed cortico-propriospinal pathway [60]. As the severely impaired motor function is potentially compensated by ipsilateral cortical activity [61], it is important to note the potential risk that cathodal stimulation over ipsilesional hemisphere deteriorates motor function [62].
In conclusion, the present study demonstrated that tDCS produced polarityspecific after-effects on transcallosal inhibition between motor cortices. Comprehensively, IHI was increased from the M1 at which the corticospinal excitability was increased and decreased from the M1 at which the corticospinal excitability was decreased, suggest that tDCS is capable of modulating neuronal activities that are involved with sending and receiving callosal discharges. | v3-fos-license |
2020-11-30T14:06:40.981Z | 2020-11-30T00:00:00.000 | 227217961 | {
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} | pes2o/s2orc | Exosomal miRNA-16-5p Derived From M1 Macrophages Enhances T Cell-Dependent Immune Response by Regulating PD-L1 in Gastric Cancer
Macrophages have an affinity to developing tumors and have been shown to play a role in tumor combat and immune surveillance. However, the exact mechanism by which macrophages participate in the anti-tumor immune response remains unclear. Hence, the current study aimed to identify the effect of macrophages on gastric cancer (GC) cells via exosomes. Paired cancerous, tumor-adjacent, and non-cancerous stomach tissues were initially from 68 GC patients. T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from both the GC patients as well as the healthy donors. Next, the exosomes were isolated from LPS and IFN-γ-induced PBMCs (M1 macrophages) and co-cultured with human GC cells. Another co-culture system comprised of CD3+ T cells and exosomes-treated GC cells was then performed. BALB/c mice and NOD/SCID nude mice were prepared for effects of exosomal miR-16-5p on tumor growth and anti-tumor immune response in GC in vivo. A relationship between M1 macrophages and the poor survival of GC patients was identified, while they secreted exosomes to inhibit GC development and activate a T cell-dependent immune response. Our results revealed that miR-16-5p was transferred intercellularly from M1 macrophages to GC cells via exosomes and targeted PD-L1. M1 macrophage-derived exosomes containing miR-16-5p were found to trigger a T cell immune response which inhibited tumor formation both in vitro and in vivo by decreasing the expression of PD-L1. Taken together, the key findings of the current study suggest that M1 macrophage-derived exosomes carrying miR-16-5p exert an inhibitory effect on GC progression through activation of T cell immune response via PD-L1. Our study highlights the promise of M1 macrophages as a potential cell-based therapy for GC treatment by increasing miR-16-5p in exosomes.
INTRODUCTION
Gastric cancer (GC) is a malignancy characterized by the growth of neoplastic tumor cells in the stomach As at 2015, GC was ranked as the second most commonly diagnosed cancer as well as the second leading cause of cancer related death in China (Chen et al., 2016). Helicobacter pylori represents one of the chief causative factors of GC, accounting for approximately 65∼80% of new GC cases on an annual basis (Kusters et al., 2006;de Martel et al., 2012). Other known risk factors include age, cigarette smoking, obesity, and dietary factors (Karimi et al., 2014). Tumor resection at the early stage of GC is often accompanied with high rates of survival while poor patient outcomes and survival are often the result of advanced stage GC, often due to metastatic GC cell migration to distant tissues and lymph nodes (Thrumurthy et al., 2015). Although there are various standard treatments approaches including surgery, endoscopic mucosal resection and chemoradiation, all of which are widely applied, the emergence of novel therapies such as targeted therapy and immunotherapy have been highlighted in literature (Digklia and Wagner, 2016). Tumor cells can evade the immune system, which is mediated by combination of tumor associated antigens (TAA) and immune checkpoints (Yousefi et al., 2017). Immunotherapy employs the use of antibodies that are capable of specifically blocking immune checkpoints which help to enhance T cell surveillance of tumor cells. More recently, immunotherapy approaches targeting PD1, PDL1, and CTLA4 have all been successfully applied in GC, with largely promising outcomes (Bonotto et al., 2017). PD1 and PDL1 are immune checkpoints both of which are located on the cellular membrane and are capable of regulating the T cell receptor (TCR). PDL1 is expressed by a wide variety of cells including that of cancer cells and has been shown to inhibit cellular antigen presentation. PD1 preferentially appears in immune cells such as NK, B, and T cells (Bonotto et al., 2017). The interaction between PDL1 and PD1 has been widely reported to interfere with the TCR signaling transduction of T cells. Existing literature has revealed that monocytes such as macrophages can secrete immune factors that are able to regulate B, T, and NK cells. Recent studies have suggested macrophage-derived exosome carrying non-coding RNAs and immune factors can also control the immune effectors either by immune inhibition or immune activation (Veerman et al., 2019). For instance, exosomes generated by activated macrophages carry polarized M1 or M2 mRNAs and miRNAs (Garzetti et al., 2014). Furthermore, a correlation between miR-16 and cancer progression such as breast cancer and lung cancer has been reported (Sromek et al., 2017;Usmani et al., 2017). In GC, miR-16 has been identified as an antioncogenic factor that acts to inhibit the proliferation and migration of GC cells by targeting SALL4 (Jiang and Wang, 2018). Furthermore, miR-16 targets PDL1 and breaks the interaction between PDL1 and PD1 in prostate cancer, thus improving the radiotherapy via T cell activation (Jiang and Wang, 2018). The regulation of miR-16 on PD1/PDL1 axis in GC is still unknown. Hence, the current study aimed to elucidate the regulation of M1-derived exosomes carrying miR-16-5p on T cell.
Ethics Statement
Written informed consent was obtained from all patients prior to enrollment into the study. Study protocols were approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University, with the protocol performed in strict accordance with the ethical principles for medical research involving human subjects of the Helsinki Declaration. All animal experiments were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Harbin Medical University. Animal experiments were performed based on idea of minimizing animal number and minimizing the pains experienced by the animals.
Study Subjects
A total of 68 GC patients (46 male and 22 female, aged 31-79 years with a mean age of 55 years) who had undergone excision procedures at The First Affiliated Hospital of Harbin Medical University between January 2013 and December 2015 were enrolled into the study. Based on tumor, lymph node metastasis and tumor node metastasis (TNM) grading criteria of the 7th edition of International Union Against Cancer, there were 20 cases at stage I, 15 cases at stage II, and 33 cases at stage III. GC, tumor-adjacent, and non-cancerous stomach tissues (5 cm away from the tumor) and peripheral blood were all collected from patients with GC. All enrolled patients were yet to have received any chemotherapy or radiotherapy prior to the operation. Patients diagnosed with infectious disease, autoimmune disease, or multiple primary cancers were excluded. A follow-up visit was performed in order to determine the overall survival (OS) rate of GC patients by means of phone call or follow-up review until December 2018. Furthermore, the peripheral blood from 40 healthy volunteers (16 male and 24 female, aged 32-78 years with a mean age of 57.35 years) was used as the peripheral blood control.
Isolation of Macrophages and T cells
As previously described , GC and nontumor tissues were made into single cell suspension and stained by anti-human antibodies against CD68, CD11c, and CD206. Fluorescence activated cell sorter FACSAria II (BD Biosciences, Franklin Lakes, NJ, United States) was employed to classify the macrophages from GC and non-tumor tissues.
Next, Ficoll-Paque Plus density gradient centrifugation was utilized to isolate the peripheral blood mononuclear cells (PBMCs) from both the healthy donors and GC patients. Later, anti-CD3 magnetic bead (Miltenyi Biotec, Miltenyi, Germany) was applied to purify CD3 + T cells from PBMCs.
Reverse Transcription Quantitative Polymerase Chain Reaction
Total RNA was extracted in accordance with the instructions of the Trizol kit (Shanghai Haling Biotechnology Co., Ltd., Shanghai, China). Next, miRNA was reversely transcribed into complementary DNA (cDNA) as per the instructions of the TaqMan MicroRNA Assays Reverse Transcription prime kit, while mRNA was reversely transcribed into cDNA based on the method prescribed by the EasyScript First-Strand cDNA Synthesis SuperMix kit (AE301-02, Beijing TransGen Biotech Co., Ltd., Beijing, China). Fluorescent quantitative PCR was conducted based on the instructions of the SYBR R Premix Ex TaqTM II kit (TaKaRa, Tokyo, Japan), while reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using 7500-type fluorescent quantitative PCR instrument (ABI Company, Oyster Bay, NY, United States). U6 was regarded as the loading control of miR-16-5p, while glyceraldehyde-3phosphate dehydrogenase (GAPDH) was considered as the internal reference of PD-L1. All primers were synthesized by Beijing Genomics Institute, Co., Ltd., (Beijing, China) ( Table 1). Quantitative analyses were conducted using the 2 − Ct method.
Western Blot Analysis
Total protein was extracted from the tissues or cells. After isolation by means of polyacrylamide gel electrophoresis, the protein was transferred onto a polyvinylidene fluoride membrane. The membrane was subsequently incubated with primary antibodies (from Abcam) PD-L1 (ab205921, 1:25),
Cell Treatment
The GC cell lines AGS and NCI-N87, purchased from Shanghai Institute of Cellular Research, Chinese Academy of Sciences (Shanghai, China), were incubated with DMEM (Gibco by Life technologies, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin (Gibco by Life technologies, Grand Island, NY, United States) at 37 • C with 5% CO 2 . After detachment using 0.25% trypsin, the cells were passaged (1:3) and inoculated into a 6-well plate (3 × 10 5 cells/well). When cell confluence reached 70−80%, the cells in the logarithmic growth phase were collected for further treatment. PBMCs were isolated using gradient centrifugation, and CD14 + mononuclear cells were purified by immunomagnetic beads. CD14 + mononuclear cells after purification were induced by granulocyte-macrophage colony stimulating factor for 5day. Following medium change, M1 macrophages were induced by LPS (100 ng/ml) and IFN-γ (20 ng/ml). M1 macrophages were later inoculated into a 6-well plate (4 × 10 5 cells/well) and transduced with miR-16-5p mimic and miR-16-5p inhibitor lentivirus (2 mL/well) at 37 • C overnight. After 48 h, RT-qPCR was conducted in order to determine the relevant gene expression.
Isolation and Identification of Exosomes Derived From Macrophages
M1-polarized macrophages were cultured with serum-free medium for 24 h, after which the supernatant was collected, followed by centrifugation at 2,000 × g and 4 • C for 20 min. The supernatant was subjected to further centrifugation at 10,000 × g and 4 • C for 1 h. The pellets were suspended by serum-free DMEM containing 25 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (pH = 7.4). The aforementioned centrifugation process was repeated again, after which the pellets were collected. Later, a transmission electron microscope (TEM) was utilized to identify exosomes (Cheng et al., 2017), after which the flow cytometer Guava easyCyteTM system together with CD63-phycoerythrin (PE) antibody was utilized to ascertain the level of the exosome surface marker CD63. The cells that were not subject to antibody addition were regarded as the blank control, while those added with PE-labeled anti-human IgG were considered to be the negative control (NC).
Co-culture of M1 Macrophage-Secreted Exosomes With GC Cells
Exosome inhibitor GW4869 was employed to treat the macrophages with the objective of inhibiting the release of exosomes. The macrophages were settled on a 6-well plate (1 × 10 6 cells/well). When cell confluence reached 80-90%, the cells were treated with 10% GW4869 (D1692-5MG, Sigma-Aldrich Chemical Company, St. Louis, MO, United States), while those treated with 0.005% dimethyl sulfoxide (DMSO) were regarded as the control. After 24 h, the cells and the supernatant were collected for subsequent use.
M1 macrophages were settled on the basolateral chamber of a 24-well transwell chamber (1 × 10 4 cells/well), while the AGS cells (5 × 10 4 cells/well) were covered on its apical chamber. The insert aperture between the apical and basolateral chambers was 0.4 µm. After 24 h of co-culture of AGS cells with macrophage-derived exosomes, AGS cells were isolated to evaluate the expression of miR-16-5p and PD-L1.
The macrophage-secreted exosomes were then labeled with PKH76 (green) dye liquor (MINI67-1KT, Sigma-Aldrich Chemical Company, St. Louis, MO, United States) and cocultured with the supernatant of the AGS cells inoculated in the 24-well plate after their confluence had reached 50-60%. The co-cultured AGS/NCI-N87 cells were treated with exo-miR-16-5p mimic or exo-miR-16-5p inhibitor (the concentration of these exosomes was determined by BCA and appropriate concentration was selected according to experiments). RT-qPCR and western blot analysis were performed in order to determine the expression of miR-16-5p and PD-L1.
Enzyme-Linked Immunoassay
Isolation of PBMCs from mouse peripheral blood was conducted using the Mouse Peripheral Blood Lymphocyte Separation Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Briefly, the mice were anesthetized with the eyeball obtained for blood collection. The blood was then quickly diluted with an equal volume of PBS, added with the separation solution, and subjected to gradient centrifugation to finally obtain the lymphocytes in peripheral blood. CD3 + T cells were then isolated by flow cytometry and cultured in the supernatant for 24 h. Cytokine expression was determined in accordance with the instructions of the ELISA kits including interleukin (IL)-2 (ab174444, Abcam, Cambridge, MA, United States), tumor necrosis factor (TNF)-α (ab181421, Abcam), and INFγ (DIF50, R&D Systems, Minneapolis, MN, United States). A multifunctional microplate reader (Synergy TM 2, Bio Tek Instruments, Winooski, VT, United States) was applied to measure the optical density (OD) value of each well at a wavelength of 450 nm. With the standard sample concentration as the abscissa, and the OD value as the ordinate, the regression equation of standard curve was calculated. The OD value was substituted into the equation to calculate target protein concentration.
Dual Luciferase Reporter Gene Assay
Dual luciferase reporter gene vector 3 untranslated region (3 UTR) of PD-L1 (CD247) and mutant (MUT) plasmid mutated on its conjugated binding site with miR-16-5p were constructed, namely, PmirGLO-PD-L1-wild type (WT) and PmirGLO-PD-L1-MUT. The plasmids were co-transfected with miR-16-5p mimic or NC mimic into the HEK293T cells. After 24 h, the cells were lysed and centrifuged at 25,764 × g for 1 min. Dual-Luciferase R Reporter Assay System (E1910, Promega Corporation, Madison, WI, United States) was employed to detect the luciferase activity. The ratio of firefly luciferase activity to renilla luciferase activity was regarded as the relative luciferase activity.
Additionally, a total of 30 female NOD/SCID mice (age: 5-7 weeks) were selected. AGS cells were incubated with exosomes and exosomes-containing miR-16-5p inhibitor for 48 h. Next, 100 µL of AGS cells (1 × 10 6 cells per mouse) were subcutaneously injected into the armpit region after which the tumors were permitted to grow over a period of 10 days. Later, 5 × 10 6 polyclone T cells [treated with 2 mg/mL anti-CD3 (ab135372, Abcam, Cambridge, MA, United States) and 1 mg/mL anti-CD28 (ab243228, Abcam, Cambridge, MA, United States)] were co-cultured with the AGS cells (1 × 10 6 cells per mouse) and then with 5 µg/mL PD-L1 or 5 µg/mL control IgG for 24 h. The co-culture system of AGS cells and T cells were injected into the peritoneum of mice bearing 10-day subcutaneous tumors. The tumor size and volume were measured every 2 days. The mice were euthanized with 50 mg/kg pentobarbital sodium (57-33-0, Shanghai Beizhuo Biotechnology Co., Ltd., Shanghai, China), after which their respective tumors were removed, photographed and fixed for immumohistochemical staining, RT-qPCR and ELISA. The spleen was split into single cells for flow cytometry purposes as previously reported method . Additionally, tumor tissue supernatants followed by determination of expression of anti-tumor molecules perforin and granzyme B in the supernatants using ELISA kits according to the manufacturer's instructions.
Statistical Analysis
All data, expressed as mean ± standard deviation, were analyzed using a Statistic Package for the Social Science (SPSS) 21.0 statistical software (IBM Corp., Armonk, NY, United States). If departure from normal distribution and homogeneity of variance was not observed, comparisons between two groups were analyzed by means of a non-paired t test, while comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA) with Tukey's post hoc test, while date comparisons at various time points were analyzed by repeated measures ANOVA with Bonferroni's post hoc test. Kaplan-Meier method was applied in order to calculate the OS of the GC patients, and Log-rank test was applied for single factor analysis. Significant difference was reflected by p < 0.05.
Enrichment of M1 Macrophages Was Associated With GC Progression
To elucidate the role of macrophages in GC, enrichment of the macrophages at various stages was analyzed in different samples. Initially, flow cytometry was performed to evaluate its enrichment in peripheral blood. The ratio of CD68 + macrophages in peripheral blood of the GC patients was observed to be higher than that of the healthy donors. Next, enrichment in the GC tissues was also revealed that the ratio of CD68 + macrophages in the GC tissues was greater than that in the tumoradjacent and non-cancerous stomach tissues. As depicted in Figure 1A, the ratio of macrophages in each sample was elevated with the development of GC. Similarly, immumohistochemical staining further illustrated a large number of macrophages were accumulated in GC tissues (Figure 1B), highlighting the potential involvement of macrophages in the GC microenvironment.
Flow cytometry was further conducted to assess the ratio of CD68 + macrophages in M1 macrophages (CD11c) and M2 macrophages (CD206) in patients at different stages. Our results revealed that as GC deteriorated, the ratio of M1 macrophages decreased from 32 to 8% (Figure 1C), while the ratio to M2 macrophages was elevated from 22 to 44% (Figure 1D). The Kaplan-Meier method demonstrated that poor expression of CD11c or high expression of CD206 was linked to poor OS (Figures 1E,F).
M1 Macrophage-Derived Exosomes Inhibited GC Progression
To further investigate the effects associated with M1 macrophages on GC progression, PBMCs-induced macrophages were induced into M1 macrophages by LPS (100 ng/ml) and IFN-γ (40 ng/mL) in vitro. Membrane protein was extracted by the Membrane Protein Extraction Kit. Western blot analysis was conducted to determine the expression of cell surface marker iNOS with Na/KATPase used as an internal reference (Figures 2A,B). Our results revealed that the expression of iNOS was elevated after LPS (100 ng/ml) and IFN-γ (20 ng/ml) induction. Next, exosomes were isolated from M1 macrophages. TEM observed that the M1 macrophage-derived exosomes had uniformly circular or oval-shaped membranous vesicles ( Figure 2C). Dynamic light scattering revealed that their size ranged from 30 to 120 nm ( Figure 2D). Western blot analysis displayed that the expression of the exosome surface markers (CD63 and HSP70) was higher in the aforementioned exosomes ( Figure 2E). Besides, the level of CD63 was increased as reflected by flow cytometry, suggesting that exosomes had been successfully extracted ( Figure 2F).
Next, to further determine whether the GC cells could absorb M1 macrophage-secreted exosomes, PKH76 (Green)-labeled exosomes were co-cultured with GC cells for 48 h. The uptake of PKH76-labeled exosomes by GC cells was analyzed under a confocal fluorescence microscope ( Figure 2G). After 48 h of coculture, the GC cells were noted to exhibit a green fluorescence, while a distinct uptake of PKH76-labeled exosomes by the GC cells was identified, indicating that exosomes could transfer from donor M1 macrophages to receptor GC cells. Besides, the above assays were repeated in NCI-N87 cells, from which the same results were obtained (Supplementary Figures 1, 2).
After the mice were treated with 10 µg M1 macrophagesecreted exosomes via tail vein injection, the tumor growth, volume and weight of the mice decreased in vivo (Figures 2H-J). The GEPIA database 1 found that PDL1 was upregulated in the GC samples from TCGA ( Figure 2K). Next, the effect of M1 macrophages on PD-L1 in GC microenvironment was explored. As reflected by RT-qPCR and western blot analysis, the expression of PD-L1 was increased in the tumors of the 1 http://gepia.cancer-pku.cn/ GC mice and reduced in those treated with M1 macrophagesecreted exosomes ( Figure 2L). Therefore, we concluded that the M1 macrophage-secreted exosomes could inhibit GC progression and down-regulate PD-L1 expression.
M1 Macrophage-Secreted Exosomes Enhanced Immune Response of T Cells in GC
M1 macrophages can inhibit disease progression by enhancing the T cell immune response (Cheng et al., 2017). Meanwhile, elevated PD-L1 expression in GC can stimulate the immune tolerance of T cells (Lazar et al., 2018). Thus, it could be speculated that M1 macrophage-secreted exosomes could repress the tumor growth in GC by inducing the T cell immune response. To verify this hypothesis, immunohistochemistry was conducted to detect CD3 + T cell inflammation in the tumors of mice, the result of which revealed that CD3 + T cell inflammation was enhanced in those treated with M1 macrophage-secreted exosomes ( Figure 3A). Next, CD3 + T cells were isolated from the tumors. Flow cytometry was conducted to assess the INF-γ + T cells in CD3 + T cells ( Figure 3B). The results obtained provided evidence demonstrating that T cell activation enhanced in mice treated with M1 macrophagesecreted exosomes, suggesting that M1 macrophage-secreted exosomes could activate T cells. In the following, ELISA was performed to determine the expression of IL-2, TNF-α and INF-γ in the supernatant of T cells (Figure 3C). The results demonstrated that the expression of these factors was elevated after the mice treated with M1 macrophage-secreted exosomes, revealing that M1 macrophage-secreted exosomes could enhance the immune response of T cells.
Subsequently, to study the interactive impact of PD-L1 and PD-1 on T cell activation, GC cells treated with M1 macrophagederived exosomes were added with anti-PD-L1 after co-cultured with PD-1 + and PD-1 − T cells. Based on the results from flow cytometry (Figures 3D,E), the treatment of the M1 macrophagederived exosomes were found to have potentiated PD-1 + T cell proliferation. The treatment of M1 macrophage-derived exosomes and anti-PD-L1 promoted PD-1 + T cell proliferation, however, it did not alter PD-1 − T cell proliferation. Based on the aforementioned results, we concluded that M1 macrophagederived exosomes could activate the immune response of T cells by inhibiting PD-L1 expression.
M1 Macrophage-Secreted Exosomes Carrying miR-16-5p Reduced PD-L1 Expression in GC
Existing literature has suggested that miR-16-5p can inhibit PD-L1 expression and induce the polarization of macrophages to its M1 phenotype (Jia et al., 2016). Hence, we speculated that high levels of miR-16-5p expression in M1 macrophagesecreted exosomes may inhibit PD-L1 expression in GC. First, RT-qPCR confirmed that compared with unpolarized M0-macrophages, miR-16-5p expression increased in M1 macrophage and its secreted exosomes ( Figure 4A). Next, protein expression in model mice with GC normalized to GAPDH after protein quantitation by Image J (n = 5). *p < 0.05 compared with macrophages without the induction of IFN-γ or M1 exosome or mouse GC models without any treatment; #p < 0.05 compared with mouse GC models treated with PBS. Measurement data was representative of three independently conducted experiments and expressed as the mean ± standard deviation. Comparisons between two groups are analyzed by non-paired t test, and comparisons among multiple groups are analyzed by one-way ANOVA. Post hoc test is conducted using Tukey's test.
to evaluate the regulatory mechanism associated with miR-16-5p and PDL1 in exosomes, Starbase 2 provided data predicting the existence of a binding site between miR-16-5p and PDL1 (Figure 4B), which was subsequently verified by dual luciferase reporter gene assay that in the presence of miR-16-5p mimic, the luciferase activity of PD-L1-WT 2 http://starbase.sysu.edu.cn decreased (p < 0.05), while no significant difference was detected in relation to PD-L1-MUT (p > 0.05) (Figure 4C), suggesting that miR-16-5p could specifically bind to PD-L1. Moreover, as reflected by RT-qPCR and western blot analysis, PD-L1 expression was weakened in M1 macrophages by miR-16-5p mimic and strengthened by miR-16-5p inhibitor (Figures 4D-F). Therefore, PD-L1 expression was negatively regulated by miR-16-5p. macrophage-derived exosomes and anti-PD-L1 after co-cultured with PD-1 -T cells or PD-1 -T cells. *p < 0.05 compared to mice treated with PBS as well as in comparison to the GC cells without any treatment; #p < 0.05 compared with GC cells treated with M1 macrophage-secreted exosomes. Measurement data representative of three independently conducted experiments expressed as mean ± standard deviation. Comparisons between two groups analyzed by non-paired t test as well as comparisons among multiple groups are analyzed by one-way ANOVA. Post hoc test was conducted using Tukey's test.
M1 Macrophage-Derived Exosomes
Carrying miR-16-5p Promoted the Activation of T Cells in the Co-culture System With GC Cells In the following, GC cells treated with M1 macrophage-derived exosomes were co-cultured with T cells. After GC cells were treated with exo-miR-16-5p mimic and exo-miR-16-5p inhibitor, flow cytometry was conducted to determine PD-L1 expression ( Figure 5A). Key observations were made indicating that PD-L1 expression was decreased following treatment with exo-miR-16-5p mimic and increased by the treatment of exo-miR-16-5p inhibitor. After the co-culture of GC cells and CD3 + T cells, flow cytometry was employed to assess CD3 + T cell proliferation and the number of INF-γ + T cells. As depicted in Figures 5B,C, in the cells treated with exo-miR-16-5p mimic, enhanced CD3 + T cell proliferation as well as an increased number of INF-γ + T cells were detected, while contrasting results were identified in cells treated with exo-miR-16-5p inhibitor. ELISA found that the introduction of exo-miR-16-5p mimic facilitated the expression of IL-2, TNF-α and INF-γ, while the introduction of exo-miR-16-5p inhibitor blocked their expression ( Figure 5D). Next, mouse models of GC were treated with exo-mimic-NC, exo-miR-16-5p mimic, exo-inhibitor-NC, and exo-miR-16-5p inhibitor by tail injection, respectively, to detect the effect of miR-16-5p in the exosomes on GC progression. The results displayed that up-regulation of miR-16-5p reduced the tumor growth rate, volume, and weight, while silencing miR-16-5p led to the opposite trends ( Figure 5E). Thus, M1 macrophage-derived exosomes carrying miR-16-5p were confirmed to inhibit PD-L1 expression and trigger T cell activation, thereby inhibiting GC development.
M1 Macrophage-Secreted Exosomes Inhibited Anti-tumor Immune Response and Tumor Formation in vivo by Repressing PD-L1 Expression
To further investigate the effect of M1 macrophage-secreted exosomes carrying miR-16-5p on GC progression in vivo, the mice were initially injected with GC cells treated with M1 macrophage-secreted exosomes and subsequently with T cells (2 mg/mL anti-CD3 and 1 mg/mL anti-CD28) co-cultured with GC cells. After assessment, we found that relative to the treatment of medium, the tumor growth, volume and weight were impeded by the treatment of M1 macrophagesecreted exosomes and T cells. When compared with matched controls, the tumor growth, volume and weight were increased by the treatment of exo-miR-16-5p inhibitor and T cells and decreased by the treatment of exo-miR-16-5p inhibitor, T cells and anti-PD-L1 (Figures 6A-C), suggesting that M1 macrophage-secreted exosomes carrying miR-16-5p inhibited tumor formation by repressing PD-L1 expression in vivo. The above assays were also conducted in NCI-N87 cells and the same results were obtained.
Next, to detect T cell activation in the tumors of the mice, flow cytometry was conducted to evaluate cell suspension of the spleen with the results indicated that the INF-γ + T cells were reduced following treatment with the exo-miR-16-5p inhibitor and T cells while an increase was detected following the treatment of exo-miR-16-5p inhibitor, T cells and anti-PD-L1 ( Figure 6D). Meanwhile, EILSA was utilized to examine the expression of anti-tumor molecules perforin and granzyme B in lysis buffer of the spleen cells. The results demonstrated that the treatment of exo-miR-16-5p inhibitor and T cells decreased the expression of perforin and granzyme B, while the treatment of exo-miR-16-5p inhibitor, T cells and anti-PD-L1 led to a contrasting trend of results (Figures 6E,F). Therefore, M1 macrophage-secreted exosomes carrying miR-16-5p inhibited PD-L1 expression to activate the immune response of T cells, thus suppressing tumor formation in vivo.
DISCUSSION
The reported survival rates in the advanced stages of GC remain poor. In a previous pembrolizumab clinical trial targeting PDL1, the tumors in more than 50% of patients with advanced stage GC shrunk from baseline, highlighting the promising outcomes associated with GC immunotherapy (Matsueda and Graham, 2014). During the current study, we investigated another form of immunotherapy mediated by exosome-derived miRNA targeting a key finding of our study revealed that M1 polarized macrophages were able to generate exosomes carrying miR-16-5p which subsequently targeted and downregulated the expression of PDL1 of GC cells. The downregulation of PDL1 was found to be capable of diminishing GC immune evasion from T cells monitor and activate the immune effects of T cells.
Initially, we identified diminished M1 macrophage numbers in relation to GC tumor growth with exosomes secreted by M1 macrophages found to suppress the proliferation of GC cells. There are two types of macrophages including M1 and M2. M1 macrophages represent the classically activated macrophages, which are key effector cells capable of eliminating infectious pathogens and cancer cells. Alternatively, M2 macrophages when activated are responsible for wound-healing and immune FIGURE 5 | Exosomes secreted from M1 macrophages harboring miR-16-5p promote the activation of T cells in the co-culture system with GC cells. (A) Flow cytometry of PD-L1 expression in GC cells treated with exo-miR-16-5p mimic and exo-miR-16-5p inhibitor. (B) Flow cytometry of CD3 + T cell proliferation in GC cells co-cultured with T cells for 24 h after treated with exo-miR-16-5p mimic or exo-miR-16-5p inhibitor. (C) Flow cytometry of the number of INF-γ + T cells in GC cells co-cultured with T cells for 24 h after treated with exo-miR-16-5p inhibitor or exo-miR-16-5p inhibitor. (D) ELISA was performed to determine levels of IL-2, TNF-α and INF-γ in the supernatant of T cells after treated with exosomes. (E) Representative images of tumors in mice after tail injection of GC cells stably transfected with exo-miR-16-5p mimic or exo-miR-16-5p inhibitor as well as quantification of tumor volume and weight (n = 5). *p < 0.05 cells treated with exo-mimic-NC; #p < 0.05 compared with cells treated with exo-inhibitor-NC. Measurement data representative of three independently conducted experiments expressed as the mean ± standard deviation. Comparisons between two groups are analyzed by non-paired t test.
regulation (Italiani and Boraschi, 2014). However, existing literature has suggested that ornithine produced by M2 macrophages can promote cellular proliferation and repair collagen synthesis as well as remodel regional tissue, suggesting that M2 macrophages play a stimulatory role in tumor progression (Italiani and Boraschi, 2014). This was consistent with the findings of our study whereby M1 macrophages were reduced while M2 macrophages exhibited elevated numbers in line with GC progression. Similarly, one study shows that M2 macrophages are enriched in gastric and breast cancer tissues and promote metastasis of cancer cells mediated by CHI3L1 protein .
Secondly, the exosomes secreted by M1 macrophages were demonstrated to inhibit GC cells proliferation. Exosomes represent bio membrane vesicles capable of loading macro biomolecules that participate in a functional manner in cellular communications. In the midst of inflammation and immune responses, exosomes have been reported to present antigens and act as a shuttle for RNA and immune factors that regulate the surrounding immune cells (Schorey and Bhatnagar, 2008). A previous study reported that M1-derived nanovesicles could repolarize M2 tumor-associated macrophages to M1 type which ultimately enhances immune checkpoint inhibitor therapy against tumor growth (Choo et al., 2018). Likewise, in melanoma therapy, M1 as opposed to that of M2-derived exosomes represent an excellent vaccine adjuvant capable of potentiating vaccine effects by increasing proinflammatory responses (Cheng et al., 2017). The results of the present study illustrated that miR-16-5p plays a role as a functional molecule regulating PDL1 in tumor cells. The regulation between macrophages and tumor cells through exosomal miRNAs is a common way. For example, miR-501-3p loaded by M2derived exosomes has been shown to downregulate TGFBR3, thus activating the TGF-β signaling pathway and promoting the progression of pancreatic ductal adenocarcinoma (Yin et al., 2019). Furthermore, cancer-derived exosomes have been demonstrated to influence macrophages in a reverse manner. Hypoxic epithelial ovarian cancer cells have also been shown to generate exosomes carrying miR-940 to promote M1 to M2 polarization . The aforementioned data highlights the effectiveness of exosomal miRNAs as regulators between immune and cancer cells.
Finally, the miR-16-5p carried by exosomes was found to be sufficient to activate T cells responses to GC cells. Although the target relation between miR-16 and PDL1 has been reported in prostate cancer (Tao et al., 2018), we first identified the miR-16 in tumor microenvironment was loaded on M1-derived exosomes. The PD1/PDL1 checkpoint is generally utilized by cancer cells as a means of escaping T cell surveillance. In clinical therapy, dozens of antibodies have been designed to target PD1 or PDL1, and have shown an encouraging capacity to block the interaction between the two molecules. However, the side effects associated with artificial antibodies are still significant enough to limit their clinical application in humans (Sood et al., 2018). In contrast, exosomes structure owning double lipid membrane can reduce immunogenicity, which can also easily transfer molecules to target cells, highlighting the promise associated with developing exosome-based drugs that function as inhibitors of checkpoints to activate T cell immune responses and achieve successful immunotherapy.
CONCLUSION
Taken together, the key findings of the present study indicate that M1 macrophages are negatively associated with GC progression, highlighting the anti-tumor role of M1 macrophages. M1 macrophages are able to generate exosomal miR-16-5p that specifically targeted and downregulated PDL1 on GC cells. Blockade of PD1/PDL1 checkpoints could lead to T cells activation and inhibit GC proliferation. However, the downstream signaling pathway after miR-16-5p blocking PD1/PDL1 checkpoints still requires further exploration in order to fully elucidate the mechanism.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Harbin Medical University.
AUTHOR CONTRIBUTIONS
ZL, BS, DW, JS, MZ, and BF designed the study. ZL, BS, and DW collated the data, carried out data analyses, and produced the initial draft of the manuscript. JS, MZ, and BF contributed to drafting the manuscript. GL, YG, and CS contributed substantially to its revision. All authors have read and approved the final submitted manuscript. | v3-fos-license |
2020-01-30T09:04:34.339Z | 2019-07-15T00:00:00.000 | 214429228 | {
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} | pes2o/s2orc | Chemical Composition and Rheological Parametrs of Helianthus Tuberosus Flour Used as a Sources of Bioactive Compounds in Bakery
LIVIA APOSTOL1, NASTASIA BELC1*, LIVIU GACEU2*, VALENTIN VLADUT 3, OANA BIANCA OPREA2 1National Research & Development Institute for Food Bioresources IBA Bucharest, 6 Dinu Vintila Str., 0211202, Bucharest, Romania 2Transilvania University of Brasov, 29 Eroilor Blvd., 500036, Brasov, Romania 3National Research and Development Institute for Machines and Installations Designed to Agriculture and Food Industry INMA Bucharest, 6 Ion Ionescu de la Brad, 013813, Bucharest, Romania
Known for over 2000 years, Jerusalem artichoke (Helianthus tuberosus L.) from the Asteraceae family is a perennial plant found in the Northeast of USA. It is cultivated in temperate areas for its edible tuber. As a source of inulin, which has aperient, cholagogue, diuretic, spermatogenic, stomachic and tonic effects, its tuber has been used as a traditional remedy in the treatment of diabetes and rheumatism [1].
More recently, research done by multiple teams has proven that the chemical composition of H. tuberosus has a positive influence on gastrointestinal system mechanisms, as the plant contains a high concentration of minerals [2][3][4] and inulin [5,7,8].
Literature surveys reveal a massive consumer interest in bakery products enriched with functional ingredients; thus, technological developments have been made towards developing more products of this kind [9]. Many research studies have been carried out, with the purpose to improve the nutritional values and functional properties of wheat flour that involved the addition of numerous other ingredients, such as dietary fibre from coconut flour [10], mango peels as an antioxidant source [9,11], soy protein [12], apple pomace [13], olives, onion, garlic [14], potato peel [15] and guar gum [16]. In addition, Bajiae et al. [17] have used plant extracts (rosemary, thyme, sage) in the production of bakery products. Inulin mixed with water creates a gel network, which gives a smooth and creamy texture [18][19]. The aim of this study shows how H. tuberosus also can be utilized to increase the dietary fibre and mineral content of wheat flour.
Experimental part Materials and methods
Jerusalem artichoke (Helianthus tuberosus L.) tuber flour was supplied by SC Hofigal Export Import SA, (Bucharest, Romania) and obtained by finely grinding the tubers. The wheat flour used in the study was type 550 (0.55% dry matter (DM) ash content) and was provided by Titan SA (Bucharest, Romania).
Chemical analysis
Moisture was determined at 103°C (±2°C) using test samples weighing 2 g, until constant weight was achieved between measurements, as described in the ICC Standard No. 110/1. The ash content was determined by incineration at 525 ± 25°C (ICC No. 104/1). Total fat was determined by extracting 10 g of sample with petroleum ether 40-65°C, using a semiautomatic Soxhlet Foss Extraction System 2055 (Foss, Sweden). Total nitrogen (N) and crude protein content (N 6.50, conversion factor) was estimated using the Macro Kjeldahl method (Kjeltec System, FOSS, Sweden). Total fibre was measured using the enzymatic gravimetric method, Mes-Tris buffer, AOAC (1995) method 991.43. The determination was performed using the Fibertec 1023 system (FOSS Sweden). Each sample was analysed in triplicate.
Inulin analysis
Inulin was determined according to the Determination of inulin in dough products method, Petkovaet al. [20]: the inulin extraction from the samples was carried out in an Ultrawave ultrasonic bath operating at a 60 Hz ultrasonic frequency and at 240 V. Then the samples were centrifuged in an Eppendorf 5804 R centrifuge. The spectrophotometric determination of fructans was carried out by the resorcinol assay. The absorbance of pink colored compound was read at 480 nm against distilled water.
The concentration of inulin in the sample extracts was calculated using the calibration curve of fructose [20][21]. Measurements were performed using a Jasco V 550 UV-Vis spectrophotometer.
The content of inulin was calculated by the method described in [20], using the formula: Y = 0.1174 X + 0.0087; where: Y is absorbance at 480 nm; X -concentration of fructose, µg mL -1 .
Mineral analysis
The mineral contents were determined using the plasma-mass spectrometer ICP-MS (Perkin Elmer NexION 300Q). Total ash was determined by incineration of the samples at 550°C, in an oven. Analysis was performed using an external standard (Merck, multi element standard solution) and all calibration curves were obtained for six different concentrations. The total mineral content was measured using their most abundant isotopes. The dried samples were digested in a mixture of concentrated HCl. All measurements were made in triplicate.
Testing of rheological properties of doughs
The rheological behaviour of doughs was analysed using the predefined Chopin + protocol on Mixolab (www.chopin.fr), a piece of equipment created by CHOPIN Technologies, which uses the international standard ICC-Standard Method No. 173 protocol for a complete characterization of flours.
The procedure parameters used for the analysis with Mixolab are as follows: tank temperature 30°C, mixing speed 80 min -1 , heating rate 2°Cmin -1 , total analysis time 45 min.
The Mixolab curves are characterised by their torque in five defined points (C1-C5, N•m), as described in table 2, their temperatures and corresponding processing time. Mixolab tests the correlation between the parameters in table 3 during the mixing and heating of the dough [22].
Dough preparation and baking procedure
The bread formula is made from wheat flour, dried yeast (3.0 g), sodium chloride (1.5 g), H. tuberosus tuber flour, and water according to the Mixolab water absorption. Samples were coded according to table 1.
The mixtures of flours were sieved twice (sieve Nos. 70 and 212 mm). All ingredients were added into a mixer (Diosna, Germany). The program consisted of a kneading step of 10 min, then resting for 5 min prior to rounding and fermentation for 30 min at 28-30°C. Two dough pieces of 600 g were formed by re-shaping, placed in tins and then fermented at 30°C, RH 90%, 60 min. The fermented samples were baked at 200 ± 5°C in a baking oven (Mondial Forni-Verona). After baking, the bread was cooled down at room temperature for 2 h before measurements.
Specific volume measurement
Data is reported as the mean of three measurements, each loaf was weighed and its volume was determined by the rapeseed displacement method (AACC, 2000).
Porosity measurement
Porosity was determined by measuring the total volume of the holes in a known volume of crumb while mass and density are known. Porosity is expressed in % volume. Elasticity was measured by applying a pressing force on a piece of bread crumb to bring it to half of its initial height and then removing the pressing force, and measuring the height recovery of the test sample one minute after removal of the load. Crumb elasticity is the ratio between the height expressed in % by pressing and return, and the initial height of the bread crumb.
Determination of sensory characteristics
The bread score is determined based on the quantification of a set of sensory characteristics, reported Table 3 MIXOLAB PARAMETERS to a standard volume of 400 cm 3 100g -1 and 85% porosity, validated by Institute for Food Bioresources, Bucharest.
Moisture content measurement
Moisture content was determined by drying the bread crumb at 103°C (±2°C) to constant weight. For determination, approximately 5 g of crumb was taken from central slice of the loaf. Data are reported as the mean of three measurements, each one performed on a freshly made loaf. In table 4, is presented a summar y of organoleptic evaluation scores.
Acidity measurement
Acidity, expressed in degrees (SR 91/2007), was determined by titration of an aqueous extract of bread with 0.1 N NaOH solution, in the presence of phenolphthalein as indicator.
Statistical analysis
All analyses were performed in triplicate and the mean values with the standard deviations were reported. Microsoft Excel 2007 was employed for statistical analysis of the data with the level of significance set at 95%. Analysis of variance (ANOVA) followed by Tukey's test was used to assess statistical differences between samples. Differences were considered significant for a value of P < 0.05.
Results and discussions
Chemical analysis of flour mixtures. H. tuberosus tuber flour was chemically analysed to determine its contents of: proteins, ash, lipids and dietary fibre (soluble and insoluble) (table 5). These data confirm that H. tuberosus tuber flour is a good source of nutrients, especially inulin, which is the major component (63.01% DM) of total fibres (79.46% DM).
H. tuberosus tuber flour (P6) presents a high mineral content, being particularly rich in potassium (1.3%), calcium (0.29%), and magnesium (0.6%) (table 6). It can be noticed that compared to the low mineral content of wheat flour (P1), mixtures of wheat flour and H. tuberosus tuber flour have higher contents of minerals as the percentage of the latter component in the flour mixture increases. (fig. 2). A low resistance of dough to mixing was noted. A decrease in consistency (C2), showing a higher softening under the effect of temperature, reveals some negative qualitative changes in flour protein composition, i.e. changes or dilution of gluten content.
Dough stability times ranged from 5.05 to 8.97 min, except for sample P5 (4.28 min,) ( fig. 3). The lowest C3 was determined for pure wheat dough (P1) (table 5). The difference in C3 results between P1 and P2 samples was 0.04 N•m; thus, the influence of flour mixture used for dough was insignificant; the same applies for C2. As mentioned above, the C4 parameter corresponds to the stability of the starch gel formed ( fig. 4). This parameter was strongly influenced by the amount of H. tuberosus flour used in the flour mix. For P1 and P2, the difference between C4 parameter values (1.35 N•m and, respectively, 1.38) is insignificant.
For the other samples, a gradual increase of the C4 parameter was noticed (1.8 for P3 and 1.94 for P4), except in the case of P5 where it decreased to 1.84.
The retrogradation stage of starch (C5) in the analyzed wheat flour and wheat-tuber flour mixtures, demonstrated similar differences as the measurement for starch gel stability. It can be observed that differences in C5 between consecutive samples are not notable, but that the difference between P1 and P5 is considerable (2.05 and 2.80 N•m, respectively).
Bread properties
The final moisture content of bread depends on the absorption of water during dough formation and water loss during baking. From table 8, it is apparent that the addition of H. tuberosus flour has a significant effect on the moisture of the samples: P5 had the lowest moisture content (37.61) compared with the control (P1). The final bread volume depends on dough expansion during fermentation and baking, and on the ability of the matrix to stabilize the retained gas. The sample made from the mixture of wheat flour with 20% H. tuberosus, (P5), had a volume of 154 cm 3 , compared to the P1 sample volume which was 384 3 . This significant decrease is due to the dilution effect: soluble fibre affects gas retention as it interacts with the gluten network, but does not increase the gas production, resulting in a disrupted structure [24,25].
These results seem to be correlated with the rheological data presented in table 7, as the specific volume decrease coincides with decreased consistency of the dough bread (C2). That means that the protein flour composition undergoes some negative qualitative changes, i.e. dilution of gluten content and changes in gluten structures. As it can be observed in table 8 In terms of acidity of the bread samples, the increase in percentage of H. tuberosus flour results in the increase of acidity up to 2.2 degrees, which is the typical value obtained for bread made from whole wheat.
According to the results obtained in porosity measurement experiments, it was noted that the high percentage of H. tuberosus flour did not allow gas formation and retention during baking, with visible consequences on bread porosity. However, the sample with the lowest content of H. tuberosus flour showed an acceptable porosity compared to that obtained from whole wheat bread. This is true for elasticity as well.
These studies indicated that wheat bread can be enriched with inulin up to 2.5 g /100 g flour and still retain the quality attributes of conventional bread. Of note, in our work, the wheat flour mixture with 5% H. tuberosus contains about 3 g inulin per 100 g sample.
It can be seen in table 9 that wheat flour with 5% H. tuberosus flour is acceptable for making bread of similar organoleptic quality to whole-wheat flour bread. However, to improve the quality of the bread obtained from the flour mixture, it is necessary to choose a technology that improves both the formation and retention of gases and bread elasticity.
Conclusions
The chemical characterization performed in this study proved that Jerusalem artichoke (H. tuberosus L.) flour is a valuable source of nutritional components, mainly inulin and minerals.
The main conclusion in our study with respect to rheological properties of dough made from wheat flour and H. tuberosus flour was that the P2 sample (5 g H. tuberosus tuber flour added to 95 g wheat flour) retained suitable rheological parameters for obtaining bakery products of a good quality. After performing the baking test, it was observed that the best sensory and physicochemical values were obtained using an addition of 5% H. tuberosus tuber flour, and these were comparable to those of whole wheat bread.
This study provides useful information toward using H. tuberosus flour as source of functional ingredients in the bakery industry; in particular, this flour can be regarded as a valuable source of fibre (more than 3 g100 g -1 ), according to Regulation 1924/2006. | v3-fos-license |
2017-03-30T21:59:47.984Z | 2014-05-13T00:00:00.000 | 17075388 | {
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} | pes2o/s2orc | Palladium-catalysed cross-coupling reaction of ultra-stabilised 2-aryl-1,3-dihydro-1H-benzo[d]1,3,2-diazaborole compounds with aryl bromides: A direct protocol for the preparation of unsymmetrical biaryls
Summary There has been a significant interest in organoboron compounds such as arylboronic acids, arylboronate esters and potassium aryltrifluoroborate salts because they are versatile coupling partners in metal-catalysed cross-coupling reactions. On the other hand, their nitrogen analogues, namely, 1,3,2-benzodiazaborole-type compounds have been studied extensively for their intriguing absorption and fluorescence characteristics. Here we describe the first palladium-catalysed Suzuki–Miyaura cross-coupling reaction of easily accessible and ultra-stabilised 2-aryl-1,3-dihydro-1H-benzo[d]1,3,2-diazaborole derivatives with various aryl bromides. Aryl bromides bearing electron-withdrawing, electron-neutral and electron-donating substituents are reacted under the catalytic system furnishing unsymmetrical biaryl products in isolated yields of up to 96% in only 10 minutes.
Introduction
Arylboronic acids 1, arylboronate esters 2 and potassium aryltrifluoroborate salts 3 (Figure 1) have received considerable attention and have found a special place as mild and versatile nucleophilic coupling partners for carbon-carbon bond-forming cross-coupling reactions [1][2][3][4][5].Amongst them, the Suzuki-Miyaura cross-coupling reaction of aryl halides/triflates and organoboron compounds is one of the most documented and versatile cross-coupling reaction in the literature [6].The use of organoboron compounds 1, 2 and 3 (Figure 1) as nucleophilic coupling partners in the Suzuki-Miyaura cross-coupling reaction is particularly attractive due to the non-toxicity of the byproducts, the ease with which they are transmetalated and their high stability towards air and moisture, which are the key features for coupling reactions [7].On one hand, structural diverse π-conjugated organic molecules containing a three-coordinate boron moiety such as trimesitylborane (4), arylalkynyldimesitylborane 5 and 2-aryl-1,3diethyl-1H-benzo[d]1,3,2-diazaborole 6 (Figure 2) are well known and have received considerable attention due to their interesting luminescence characteristics, fluoride ion sensing abilities, emissive as well as electron-transporting properties [8][9][10][11].Three-coordinate boron compounds are electron-poor and strong π-electron acceptors owing to the empty boron p z -orbital, which is capable of significant delocalisation when attached to an organic π-system [11].These compounds exhibit an unusual stability because of the bulky aryl groups, such as mesityl (2,4,6-trimethylphenyl) groups, which provide steric conjunction around the empty boron p z -orbital thereby blocking the incoming nucleophile (Figure 2, compounds 4 and 5) [12].Alternatively, three-coordinate boron compounds functionalized with 1,3,2-benzodiazaborole units are greatly stabilised by electron back-donation from the two nitrogen atoms to the empty boron p z -orbital (Figure 2, compound 6) [13,14].
Despite their popularity in the organic community, their profound stability towards air and moisture, their ease with which they are accessible and their non-toxicity, these compounds have, to the best of our knowledge, never been used in any transition metal-catalysed C-C bond formation reaction as coupling partners except for their 2-alkyl/alkenyl-substituted analogues [14].We are aware of reports describing the Suzuki-Miyaura cross-coupling reaction aryl diaminoborole containing compounds {ArB(dan)} which are structurally similar to our compounds, however, these compounds {ArB(dan)} are used as protecting groups not as coupling partners [15,16].During the course of our studies on the syntheses, crystal structures, fluorescence and theoretical characteristics of 1,3,2-diazaborolane functionalised organic molecules, which is reported in details elsewhere [17], we were encouraged by the high yields of 2-aryl-1,3-dihydro-1H-benzo[d]1,3,2-diazaborole compounds (Scheme 1), their solubility in various organic solvents and their high stability towards air and moisture to investigate their reactivity in transition metal-catalysed cross-coupling reaction.These compounds could be left on a bench top in a basic media for weeks without any noticeable degradation [17].Herein, we report the first palladiumcatalyzed cross-coupling reaction of 2-aryl-1,3-dihydro-1Hbenzo[d]1,3,2-diazaborole compounds with aryl bromides in only 10 minutes.
Suzuki-Miyaura cross-coupling reaction
To find optimal reaction conditions, we initially studied the reaction of bromobenzene (13a) with compound 9 in a toluene/ water mixture under different conditions as a model reaction (Table 1).Attempted cross-coupling reaction of compound 9 with bromobenzene (13a), in the absence of both the ligand and a base, gave, as expected, zero conversion of the starting materials (Table 1, entry 1).
The addition of PPh 3 as a ligand and K 3 PO 4 as a base failed to afford the desired coupled product in any substantive yield (Table 1, entry 2).Poor conversion of the starting material and low assay yield of the desired product were observed when more bulky Pd(PPh 3 ) 4 as a catalyst was used ( the most effective combination and was thus chosen as optimal reaction conditions for the purpose of this study.With the optimized reaction conditions in hand, the scope and the limitations of the cross-coupling reaction was investigated using bromobenzene (13a) and 4-bromoanisole (13b) as elelctrophilic coupling partners and boronates 9-12 (Scheme 1) as the corresponding nucleophilic coupling partners (Table 2).The crosscoupling reaction of bromobenzene (13a) with boronates 9-12 went smooth affording the coupled products in yields ranging from 68% to 88% (Table 2, entries 1, 3, 5 and 7).The sterically hindered ortho-substituted boronate 11 generally afforded lower yields when compared to other boronate derivatives (Table 2, entries 5 and 6).This was attributed to incomplete conversion of the starting materials possibly due to a steric effect around the boron atom which is consistent with the literature [6,18,19].The cross-coupling of an electron-rich aromatic system (4-bromoanisole) was generally less efficient compared to bromobenzene (Table 2, entries 2 and 6).This effect was attributed to the deactivation of the carbon-bromine bond as a result of electron-donating substituent (OMe) [20].Encouraged by our results (Table 2), we then turned our attention to investigate the reactivity of activated as well as conjugated electrophiles in our catalytic system (Table 3).Unlike bromobenzene and 4-bromoanisole, the cross-coupling reaction of electron-deficient electrophiles (4-bromoacetophenone (13d) and 4-bromonitrobenzene (13c)) furnished the desired coupled products in excellent yields ranging from 85 to 96% (Table 3) [6,21].These observations are consistent with the literature and are attributed to the activation of the carbon-bromine bonds due to the electron-withdrawing functional groups.The presence of an electron-withdrawing group induces oxidative addition of the carbon-bromine bond to the metal centre (catalyst) compared to the corresponding electron-neutral and electron-rich functionalities [22].The cross-coupling reaction of boronate 9 with both substrates 13c and 13d afforded the desired products, as expected, in high yields (Table 3, entries 1 and 2).We noticed that steric hinderance on the boronate 10 did not have any negative impact on the cross-coupling reaction investigated herein.
Conclusion
Although arylboronic acids, arylboronate esters and potassium aryltrifluoroborate salts are powerful coupling partners in the Suzuki-Miyaura cross-coupling realm, extending the scope of organoboron compounds that can participate effectively as coupling partners in the cross-coupling reaction is still necessary.We have synthesised a range of 2-aryl-1,3-dihydro-1Hbenzo[d]1,3,2-diazaborole compounds and developed their first Pd-catalysed Suzuki-Miyaura cross-coupling reaction with a range of aryl bromides bearing electron-rich, electron-neutral and electron-deficient functionalities using cost-effective and commercially available combination of Pd(OAc) 2 /PCy 3 as a catalyst and K 3 PO 4 •H 2 O as a base.The catalytic system appeared versatile and general, tolerating a large range of functional groups such as NO 2 , OMe, COMe and diazaborolyl whilst furnishing the coupled product with isolated yields of up to 96% in only 10 minutes. | v3-fos-license |
2021-03-24T13:24:29.375Z | 2021-03-18T00:00:00.000 | 232327301 | {
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} | pes2o/s2orc | Myosin turnover controls actomyosin contractile instability
Significance Contractile force produced by myosin II that binds and pulls constrained filamentous actin is harnessed by cells for diverse processes such as cell division. However, contractile actomyosin systems are vulnerable to an intrinsic aggregation instability that destroys actomyosin architecture if unchecked. Punctate myosin distributions are widely observed, but how cells prevent more advanced aggregation remains unclear. Here, we studied cytokinetic contractile rings in fission yeast cell ghosts lacking component turnover, when myosin aggregated hierarchically. Simulations reproduced the severe organizational disruption and a dead-end state with isolated aggregates and ring tension loss. We conclude that in normal cells, myosin turnover regulates actomyosin contractile instability by continuous injection of homogeneously distributed myosin, permitting functional aggregates to develop but intercepting catastrophic runaway aggregation.
Actomyosin contractile force produced by myosin II molecules that bind and pull actin filaments is harnessed for diverse functions, from cell division by the cytokinetic contractile ring to morphogenesis driven by supracellular actomyosin networks during development. However, actomyosin contractility is intrinsically unstable to self-reinforcing spatial variations that may destroy the actomyosin architecture if unopposed. How cells control this threat is not established, and while large myosin fluctuations and punctateness are widely reported, the full course of the instability in cells has not been observed. Here, we observed the instability run its full course in isolated cytokinetic contractile rings in cell ghosts where component turnover processes are absent. Unprotected by turnover, myosin II merged hierarchically into aggregates with increasing amounts of myosin and increasing separation, up to a maximum separation. Molecularly explicit simulations reproduced the hierarchical aggregation which precipitated tension loss and ring fracture and identified the maximum separation as the length of actin filaments mediating mechanical communication between aggregates. In the final simulated dead-end state, aggregates were morphologically quiescent, including asters with polarity-sorted actin, similar to the dead-end state observed in actomyosin systems in vitro. Our results suggest the myosin II turnover time controls actomyosin contractile instability in normal cells, long enough for aggregation to build robust aggregates but sufficiently short to intercept catastrophic hierarchical aggregation and fracture. myosin j actomyosin j aggregation j cytokinesis j turnover Many fundamental single-cell and tissue-level processes rely on force production by actomyosin assemblies. During cytokinesis the actomyosin contractile ring develops tension that guides or drives furrow ingression and physical division of the cell (1)(2)(3). Tension gradients in the actomyosin cortex produce cortical flows that establish cell polarity (4) and cortical actomyosin forces are harnessed for cell migration (5). Contraction of supracellular actomyosin networks drives early tissue morphogenetic events such as gastrulation (6,7) and neurulation in vertebrates (8).
In these systems, actomyosin contractility finds use as a powerful and highly adaptable tool, in which contractile stress generated by constrained myosin II molecules that bind and exert force on oriented filamentous actin is harnessed for diverse functions. However, the mechanism has an intrinsic instability. A chance fluctuation in the density of myosin II and other components may enhance local contractile stress and draw in more actomyosin material at the expense of weaker neighboring regions, further amplifying the stress difference in a potentially runaway aggregation process (Fig. 1A). Unchecked, catastrophic fracture and tension loss may result.
How the cytokinetic contractile ring and other actomyosin machineries deal with this threat is not established. Some examples of the instability have been documented. In cells, actomyosin stress fibers spontaneously sever or undergo repair in regions of high strain (9,10), and in vitro myosin II spontaneously aggregated into puncta in reconstituted actomyosin bundles (11). Theoretical evidence for contractile instability is abundant, as in molecularly explicit (12) and continuum (13,14) mathematical models of the fission yeast contractile ring and continuum models of the animal cell cortex (15,16).
Observing actomyosin contractile instability run its full course in cells has proved challenging, presumably because mechanisms intervene before its full maturation. Nevertheless, numerous observations are suggestive of incipient instability. Large fluctuations and punctateness characterize myosin II distributions in fission yeast and animal cell contractile rings (17,18) and animal cell cortices (19,20). Punctate cortical myosin in some cases has pulsatile time dependence over seconds to minutes (21), which may be associated with pulsatile dynamics of the GTPase RhoA that promotes myosin II activity and minifilament assembly (4,16,22). Many morphogenetic events feature Significance Contractile force produced by myosin II that binds and pulls constrained filamentous actin is harnessed by cells for diverse processes such as cell division. However, contractile actomyosin systems are vulnerable to an intrinsic aggregation instability that destroys actomyosin architecture if unchecked. Punctate myosin distributions are widely observed, but how cells prevent more advanced aggregation remains unclear. Here, we studied cytokinetic contractile rings in fission yeast cell ghosts lacking component turnover, when myosin aggregated hierarchically. Simulations reproduced the severe organizational disruption and a dead-end state with isolated aggregates and ring tension loss. We conclude that in normal cells, myosin turnover regulates actomyosin contractile instability by continuous injection of homogeneously distributed myosin, permitting functional aggregates to develop but intercepting catastrophic runaway aggregation.
Several candidate mechanisms that could control actomyosin contractile instability have been proposed. One possibility is turnover of myosin and actin (24,25), which presumably tends to homogenize spatial variations (12). Another suggestion is myosin II diffusivity, which could serve this purpose by smoothing smallscale density variations characteristic of the instability (15,26). Another proposal, suggested for C. elegans zygotes, is that the instabilities are controlled by pulsatile RhoA dynamics (16).
Here we examined the role of component turnover as a possible regulator of actomyosin contractile instability by studying cytokinetic contractile rings in cell ghosts under conditions where turnover is absent (27,28). We find runaway aggregation of myosin II, in which the amount of myosin per aggregate and the separation between aggregates increases with time, up to a certain maximum separation. The aggregation is hierarchical, with repeated rounds of aggregation yielding aggregates with yet more myosin. In molecularly explicit simulations the maximum separation was identified as the length of actin filaments which mediate mechanical communication between aggregates, and small-scale instability was controlled not by myosin diffusivity but by local excluded volume and actin filament polarity sorting effects. Our results suggest that in normal cells myosin II turnover controls contractile instability in actomyosin assemblies, setting the size of myosin II aggregates and preventing catastrophic hierarchical aggregation and fracture.
Component Turnover Is Absent in Contractile Rings of Cell
Ghosts of the Fission Yeast Schizosaccharomyces japonicus. To investigate whether component turnover plays a role in maintaining organization in the cytokinetic contractile ring we studied cell ghosts of the fission yeast S. japonicus. In normal S. japonicus cells many of the molecular components of the ring turn over, including the two isoforms of myosin II, Myo2 and Myp2 (28). In the fission yeast Schizosaccharomyces pombe, Myo2 has a turnover time of ∼19 s, while Myp2 undergoes little turnover (29,30). Cell ghosts are prepared by enzymatic digestion of the cell walls to generate protoplasts and then permeabilizing the plasma membranes of mitotic protoplast cells to release the cytoplasm and organelles, leaving isolated membrane-bound cytokinetic rings (27,28) (Fig. 1B and Methods). Since cytoplasm is absent there is no component association, while dissociation rates are much reduced relative to normal cells (28). Thus, cell ghosts allow the organization of isolated contractile rings to be studied in the absence of component turnover.
In Contractile Rings without Turnover, Myosin II Aggregates into Puncta with Increasing Amounts of Myosin. We imaged contractile rings in ghost cells expressing myosin II light chain Rlc1-GFP and we analyzed images from this set and from our earlier study (28). Following membrane permeabilization the myosin II distribution was mildly punctate. On incubation with A fluctuation in the local density of contractile actomyosin material (Left) generates contractile stress gradients and flows (arrows) that pull material inward and amplify the fluctuation. Unopposed, the result is runaway aggregation and fracture (Right). Yellow/orange/brown background and black lines symbolically represent myosin density and actin filaments, respectively. Density profiles are shown above schematics of the actomyosin material. (B) Preparation of cell ghosts. Following enzymatic digestion of cell walls of normal cells of the fission yeast S. japonicus, the plasma membrane is permeabilized to release cytoplasm. In cells which have assembled cytokinetic rings, the rings remain anchored to the membrane. On addition of ATP, myosin II in rings aggregates progressively. (C) Time-lapse fluorescence micrographs of S. japonicus cell ghosts labeled with Rlc1-GFP following addition of 0.5 mM ATP. Images are sum-intensity projections of 3D reconstructions of rings from z-series data. Imaging commenced at least 1 to 2 min after ATP addition (see Methods). Contractile rings remain fully anchored to the membrane (Top), or ring segments appear to detach from the membrane and shorten (Middle), or segments detach, shorten, and sever (Bottom). Arrows indicate detached segments. We observed n = 6, 5, and 16 rings in each category, respectively. (Scale bars, 2 μm.) (D) Rlc1-GFP intensity along the ring at the indicated times for the intact ring of C. Intensity normalized by the total intensity around the ring at each time. (See SI Appendix, Fig. S1A for the intensity profiles around the partially unanchored and the partially unanchored and severed ring, respectively.) ATP, the distribution became progressively more punctate over ∼30 min ( Fig. 1C and SI Appendix, Fig. S6). As the punctateness increased, contractile rings suffered three distinct fates (Fig. 1C). Some rings appeared to remain robustly anchored to the membrane, with myosin II aggregates located around the contour defined by the initial ring. In other rings, segments appeared to detach from the membrane and straighten, presumably due to compromised anchoring to the plasma membrane that is weakened by permeabilization (31). In a third category, segments detached from the membrane and severed, following which myosin puncta in the severed segment merged with the apparently anchored segment. For each ring we identified the longest segment attached to the membrane that neither severed nor underwent large length changes, and we measured the Rlc1-GFP intensity along the segment. Following ATP addition, peaks of myosin fluorescence intensity appeared which became increasingly prominent with time ( Fig. 1D and SI Appendix, Fig. S1A). The relative peak amplitudes increased with time while intensity decreased in regions neighboring the peaks. The peak intensities significantly exceeded the initial intensity, indicating that the puncta were at least in part due to movement of myosin around the ring.
Thus, myosin progressively merges into aggregates of growing size in contractile rings lacking component turnover. This behavior would be expected of unopposed actomyosin contractile instability, suggesting that turnover may control the instability in normal cells.
Myosin II Aggregates Hierarchically in the Absence of Turnover.
Kymographs of myosin fluorescence intensity profiles around contractile rings showed that, in regions where the myosin density was initially above the mean, the density increased in time and the region of higher density became smaller ( Fig. 2A). In regions with an initial deficit the density decreased and the region expanded. Thus, excess density fluctuations grew and sharpened into distinct aggregates, while deficit density fluctuations diminished and became empty regions separating aggregates.
Myosin aggregated hierarchically. Neighboring aggregates moved toward one another and merged, and in some cases we observed merged aggregates themselves merging into higher-order aggregates containing yet more myosin ( Fig. 2A). Occasionally aggregates split into two parts. The progressive, hierarchical aggregation steadily increased the mean distance between neighboring aggregates from 1.00 ± 0.28 μm to 1.77 ± 0.56 μm over 60 min ( To quantify the punctateness we measured the fluctuation in myosin fluorescence intensity relative to the mean, which increased approximately twofold over 60 min (averaged over n = 10 rings; Fig. 2C). A measure of aggregate size is the halfwidth of the spatial correlation function of the intensity around the ring ( Fig. 2D and SI Appendix, Fig. S1B), which decreased from 1.15 ± 0.55 μm to 0.46 ± 0.16 μm over 60 min, with most of the decrease in the first 20 min ( Fig. 2E; values are mean ± SD).
Molecularly Explicit Mathematical Model of the Contractile
Ring in Cell Ghosts. To explore the mechanisms underlying the observed myosin aggregation (Figs. 1 and 2), we developed a three-dimensional (3D) molecularly explicit mathematical model of the cytokinetic ring in S. japonicus cell ghosts, closely related to our previous model of the ring of the fission yeast S. pombe (12, 31) ( Fig. 3A and SI Appendix, Fig. S2). For details, see Methods. Membrane-anchored formin Cdc12 dimers anchor actin filament barbed ends to the membrane. Actin filaments dynamically cross-linked by α-actinin are bound and pulled by myosin II according to a force-velocity relation set by the measured myosin II gliding velocity (32). Myosin II is anchored to the plasma membrane in coarse-grained clusters of 8 Myo2 dimers (33), with anchor drag coefficient in the membrane chosen to be consistent with the observed aggregation times.
Throughout, we refer to these groups of eight Myo2 dimers as clusters, while the term "aggregates" will denote assemblies of several myosin clusters that merge over time (see below). For simplicity we do not attempt to model the second myosin II isoform, Myp2, which is likely unanchored from the membrane (18,30).
To model the turnover-free situation in S. japonicus cell ghosts, component association is entirely absent and the ring of radius 3.7 μm does not constrict (Fig. 3A). Myosin, formin, and actin are present in cell ghost rings after ATP addition (28). The dissociation rate of α-actinin was taken as the value measured for S. pombe (12,34), while that of myosin II was chosen to reproduce the slow decrease in total myosin II fluorescence over time measured here (Methods and SI Appendix, Fig. S3A). Since rings in S. japonicus ghosts with phallacidinstabilized actin filaments showed negligible actin loss (28), we assumed formins do not dissociate. Rates of cofilin-mediated severing of actin filaments were set to reproduce the previously measured actin loss over ∼40 min (28). The initial ring at the instant of ATP addition was assumed normal ( Fig. 1 C and D) and was generated by preequilibration of the ring for 11 min with normal component turnover, using component densities, actin filament lengths, and association rates determined by previous measurements in S. pombe (33,35). In the following, images of simulated rings were convolved with a Gaussian of width 600 nm to mimic the experimental microscope point spread function (see Methods).
In Simulated Contractile Rings, Myosin II Aggregates Hierarchically in the Absence of Turnover. In simulations of the model, the myosin distribution evolved in very similar fashion to that seen experimentally (Figs. 1 and 2). Qualitatively similar behavior was seen in 10 simulations, as follows. Within 5 to 10 min, isolated myosin II puncta had developed (Fig. 3B), presumably due to the lack of component turnover. Kymographs revealed that small initial aggregates emerged from the relatively homogenous initial distribution, some of which then merged into aggregates containing more myosin (Fig. 3C). Some of these aggregates underwent further rounds of merging, yielding aggregates with yet more myosin. As hierarchical aggregation proceeded, peaks in the myosin density profile sharpened ( Fig. 3D and SI Appendix, Fig. S4) similar to the experimental fluorescence intensity (Fig. 1D). As a result, relative myosin density fluctuations increased ∼2.5-fold over 60 min (Fig. 3E), compared with the approximately twofold experimental increase in relative myosin fluorescence intensity (Fig. 2C).
The distinct myosin II aggregates that emerged after 5 to 10 min had a characteristic organization, with myosin II at the center and polarity-sorted outward-pointing actin filaments (filament barbed ends colocalizing with the myosin, and distal pointed ends) (Fig. 4A). In a typical aggregation event, two such aggregates were pulled toward one another when the myosin in each aggregate engaged with the actin filaments hosted by the other aggregate and stepped toward the barbed ends. This occurred provided the aggregates were within a range of order the mean actin filament length of each other (Fig. 4A), taken as ∼1.4 μm in our simulations from measurements on S. pombe rings (36). Aggregates separated by more than this distance no longer affected each other, since filaments mediated the mechanical interaction. The end result of an aggregation was a similarly organized larger aggregate, with more myosin II and more polarity-sorted actin filaments.
These kinetics led to increasingly compact aggregates ( Fig. 4B) separated by increasingly large distances (Fig. 4C). The size of a compact aggregate depended on the amount of myosin II it contained and the excluded volume interactions, reflecting steric interactions among myosin II molecules, which imposed a physically meaningful maximum density. The evolution of the correlation length and mean aggregate separation were both quantitatively consistent with experiment ( Fig. 4 B and C).
Runaway Myosin Aggregation Leads to Ring Fracture and
Tension Loss. Our experiments revealed growing myosin aggregates (Fig. 1C), and simulations showed progressive polarity sorting of actin filaments, with increasing colocalization of myosin and actin filament barbed ends (Fig. 4A). Thus, we anticipated the contractile ring tension would decrease over time, since myosin contributes less to tension when it binds and pulls a filament close to the anchored barbed end, compared with a location near the pointed end (12). We expected the tension would ultimately drop to a very low value, since the separation of the growing aggregates increased and eventually exceeded the typical actin filament length, presumably signaling ring fracture.
Measurements of simulated ring tensions confirmed these expectations. With time, the unopposed myosin aggregation destroyed the structural integrity of simulated rings and virtually abolished the ring tension. After ∼20 min rings lost mechanical connectivity (Movie S1) and ring tension decreased from ∼500 to 9 pN over 40 min (Fig. 4D), compared with the ∼400 pN measured in contractile rings of S. pombe protoplasts (12).
Runaway Aggregation Is Rescued by Myosin II Turnover but
Not by Actin Turnover. Our results suggest that in normal cytokinetic rings turnover of myosin and actin protect the organization against contractile instability. We next asked if prevention of runaway aggregation required turnover of one or both of these components. In 10 cell ghost simulations with both myosin II and actin turnover restored to the values in normal intact cells, runaway aggregation was prevented and the organization was normal (Fig. 5A). Myosin II turnover alone [with the turnover rate measured in S. pombe, 0.026 s À1 (34)] prevented aggregation and rescued the ring organization in five other simulations (Fig. 5 B and D). However, restoring actin turnover (with turnover rates consistent with experimental measurements in S. pombe; see Methods) failed to rescue rings from catastrophic aggregation in another five simulations (Fig. 5C). In the latter case, compared with cell ghosts late-stage aggregates had a larger separation (Fig. 5E), since actin turnover maintained longer filaments that allowed myosin aggregates to communicate over greater distances, and the rate of tension loss was lower (SI Appendix, Fig. S3C). Thus, myosin II turnover but not actin turnover is sufficient to prevent runaway aggregation. In ring simulations using a range of turnover times interpolating between the extremely small values in cell ghosts and the values in normal cells (SI Appendix, Table S1), myosin aggregation effects were apparent for myosin turnover times approximately fivefold the normal value or greater, while ring morphology was insensitive to the actin turnover time (SI Appendix, Fig. S5 and Methods).
Hierarchical Aggregation Is Progressively Slowed Down by
Increasing Degrees of Myosin II Inhibition. These simulation results suggest that myosin II contractile forces drive the aggregation observed in the absence of turnover. Thus, we would expect aggregation to be progressively curtailed by increasing doses of the myosin II inhibitor blebbistatin. We simulated blebbistatin effects by turning off forces exerted on actin filaments by a randomly selected subset of myosin clusters whose identity was refreshed every second, since experiments show blebbistatin biases myosin to a state interacting weakly with actin filaments (37,38). The fraction of deactivated myosin was varied, to mimic increasing blebbistatin levels. To quantify the degree of aggregation we counted the number of myosin gaps in contractile rings, defined as regions of width 600 nm or greater lacking myosin. After 9 min of simulation, all rings developed at least one gap for myosin deactivation levels 10% or lower (SI Appendix, Fig. S6B). For deactivation levels 30%, 50%, 70%, and 90%, gaps had appeared after 9 min in four, three, one, and zero rings, respectively, out of five rings for each deactivation level. Thus, simulations show slower aggregation when myosin is partially deactivated.
To compare these model results with experiment we imaged rings in cell ghosts treated with blebbistatin at concentrations 0 to 40 μM prior to ATP-mediated activation (see Methods). After 9 min, two of three rings with no blebbistatin had already developed gaps; one out of four rings had developed a gap for 10 μM blebbistatin, and no gaps had developed for 25 μM and 40 μM blebbistatin (in seven and four rings, respectively) (SI Appendix, Fig. S6A). Thus, consistent with simulations, increasing levels of blebbistatin-mediated myosin deactivation progressively slowed down aggregation of myosin.
Myosin Diffusion Is Too Weak to Prevent Runaway Aggregation
Due to Contractile Instability. The present work implicates myosin II turnover as the mechanism that controls contractile instability in cells. It has been suggested that myosin II diffusivity serves this purpose, by smoothing small-scale density variations that characterize the instability (15,26). To test this proposal we ran simulations of contractile rings without turnover as previously, but now the membrane-anchored myosin II clusters could diffuse laterally in the membrane with diffusivity D.
We tested diffusivities in the range 10 À5 ≤ D ≤ 10 À2 μm 2 Á s À1 (Fig. 6). In five simulations per diffusivity value, myosin aggregation was not prevented by diffusion, except for the highest values tested D ∼ 10 À2 μm 2 Á s À1 . This value is almost three orders of magnitude greater than measured diffusivities of membrane-bound myosin II in fission yeast contractile rings, D ∼ 2 × 10 À5 μm 2 Á s À1 (39). We conclude that physiologically relevant myosin diffusion is far too weak to oppose contractility-induced aggregation.
Discussion
The cytokinetic contractile ring and other actomyosin machineries require mechanisms to intercept contractile instability (Fig. 1A), but these have been difficult to identify since the full course of the instability is rarely observed. Here, we observed the instability run its full course in isolated contractile rings in cell ghosts, from initially normal organization to a final catastrophically disrupted dead-end state. In a hierarchical process, myosin II aggregated into puncta that merged with one another, some of these larger aggregates merging into yet larger aggregates, and so on ( Fig. 2A). Aggregation progressively increased the myosin per aggregate and the aggregate separation, up to ∼1 to 2 μm (Fig. 2B). We observed no greater separations, suggesting that this separation effectively terminated the process, consistent with simulations which identified the maximum separation as the mean actin filament length (Fig. 5). Here, hierarchical aggregation is driven by active myosinmediated contractile forces. It is of interest to compare this with analogous systems where the driving forces are passive particle-particle adhesive interactions, well known in contexts such as aggregation of atmospheric pollutant particles or colloidal suspensions (40). In these cases larger aggregates are effectively more adhesive due to their greater surface area, which can lead to hierarchical kinetics dominated by ever-larger aggregates (41).
We reasoned that the uncontrolled aggregation was caused by the absence of component turnover. Since cell ghosts lack cytoplasm, association of myosin II, actin and other components is absent and component dissociation rates are severely reduced. Turnover would tend to restore homogeneity to the ring, so its absence could explain the runaway aggregation. Molecularly explicit simulations corroborated this interpretation (Figs. 3-6). Simulations of cell ghost rings lacking turnover quantitatively reproduced the experimental hierarchical myosin aggregation. Aggregates emerged from local stochastic myosin density peaks that drew in myosin and actin filaments (Fig. 1A). Late-stage aggregates had a myosin II core and outward pointing polaritysorted actin filaments. Interestingly, some late aggregates had an aster structure, with a relatively compact myosin core and actin filaments pointing radially outward in multiple directions (Fig. 4A), similar to in vitro actomyosin systems which evolved to a final state consisting of morphologically quiescent asters with polarity sorted actin filaments (42). Other late aggregates had a more extended myosin II core, with longitudinally oriented actin filaments emerging at the edges (Fig. 4A).
Hierarchical aggregation, in which aggregates repeatedly merged into yet larger aggregates, continued until the aggregate separation exceeded the typical actin filament connector length when aggregates could no longer communicate mechanically. As aggregates became mechanically isolated, the ring tension decayed to zero and the ring fractured (Fig. 4). In this deadend state the contractile instability had run its full course, with morphologically quiescent aggregates similar to those observed in vitro (42).
By contrast, in simulations of rings in normal cells with turnover, myosin reached a steady-state punctateness with moderately sized aggregates (Fig. 5A). Myosin II turnover was the key process averting runaway aggregation, by replacing large myosin aggregates with more homogenously distributed moderately sized aggregates, before hierarchical aggregation could run its catastrophic course. When actin turnover was switched off, turnover of myosin II alone was sufficient to prevent aggregation (Fig. 5 B and D). Actin turnover alone was insufficient to prevent aggregation (Fig. 5 C and E). Simulations assumed myosin and actin turn over independently, consistent with experiments showing myosin Myo2 is anchored to the plasma membrane in the fission yeast ring (33) and remains in the ring following actin disassembly by latrunculin A treatment (18).
We conclude that rapid myosin turnover in contractile rings prevents runaway aggregation and tension loss. While not the emphasis of this study, normal cytokinesis also requires actin turnover (28), and contractile ring homeostasis in normal cells likely relies on turnover of both myosin II and actin in complimentary ways. In fission yeast, contractile rings are assembled from a ∼2-μm-broad band of precursor protein complexes called nodes containing myosin and other components. Nodes grow actin filaments to assemble the nodes into a narrow ring, and experimental and computational observations suggest sufficiently rapid actin turnover is required to avoid unproductive node aggregation (39,43). In mutants of the actin-severing protein cofilin that contributes to actin turnover, contractile rings suffer structural instabilities in which actin bundles peel away from the plasma membrane into straight bridges (43-45) containing Myp2 but not Myo2 (44). We recently modeled these mutants and concluded that bridging instability originates in ring tension being higher due to the longer actin filaments, generating higher centripetal forces that pull Myo2-anchored actin filaments away from the membrane (46). Our previous experimental work emphasized the role of actin turnover for normal organization and function of contractile rings (28). Myosin II aggregation in S. japonicus cell ghosts was abolished by treatment with the myosin II inhibitor blebbistatin, and stabilization of actin with jasplakinolide suppressed formation of small myosin puncta and increased the fraction of rings that shortened. These rings appeared to fully constrict, but broad myosin II aggregates were apparent in these shortening rings, similar to our simulations with restored actin turnover (compare Figs. 3B and 5C). The shortening segments in these rings likely have zero tension, since a mathematical model (31) of the analogous phenomenon seen experimentally (27) in S. pombe fission yeast ghosts accurately reproduced the observed shortening rates and showed that rings shortened due to zero tension detached segments being reeled in by myosin II against zero load. We have found that myosin turnover protects contractile rings from large-scale contractile instabilities that would provoke aggregation. Contractile instability also affects the smallest scales since, if unopposed, it would aggregate myosin II into clumps of arbitrarily high density (see Fig. 1A). In simulations without turnover, aggregate sizes were set by the amount of myosin II in the aggregates, excluded volume interactions and local polarity sorting (Fig. 4A). Our results suggest that in the cytokinetic ring and other actomyosin machineries, small-scale contractile instability is controlled by several small-scale effects. These include excluded volume interactions due to steric intermolecular interactions that impose packing limits, small-scale actomyosin polarity sorting that suppresses contractility (42), and other local interactions such as myosin II minifilament stacking in animal cells (47).
It is often assumed that myosin diffusion provides this smallscale control. Active gel models of actomyosin cortices commonly assume a diffusive contribution to the myosin motion, which helps suppress actomyosin instability that would otherwise cause uncontrolled short scale density blow up (15,26). To be effective, diffusivities of order D ∼ 0:01 μm 2 Á s À1 or larger are invoked. However, measured myosin II diffusivities in contractile rings in fission yeast (39) are ∼ 2 × 10 À5 μm 2 Á s À1 , ∼3 orders of magnitude smaller, and were reported unmeasurably small in the C. elegans embryo cortex (16). Thus, myosin diffusivity is presumably not responsible for controlling actomyosin contractile instability, and its inclusion in models is a numerical convenience that misrepresents at least the small-scale behavior. We confirmed this in simulations with added myosin cluster diffusivity. A diffusivity ∼500 times larger than the experimental value was required to prevent uncontrolled aggregation (Fig. 6).
Overall, our results suggest that in actomyosin assemblies a balance of turnover and small-scale effects regulates contractile instability on large and small scales, respectively (Fig. 7). We 6. Myosin II diffusion does not prevent runaway aggregation due to contractile instability. (A-C) Simulated confocal fluorescence micrographs of myosin II in cell ghost contractile rings that lack turnover, with lateral diffusion of myosin II aggregates in the plasma membrane implemented. Three diffusivities were simulated, D myo = 10 À5 , 10 À3 , and 10 À2 μm 2 Á s À1 . Myosin diffusion rescues the normal myosin II distribution only for the largest diffusivity, a value ∼500-fold larger than physiological values measured in fission yeast S. pombe (39). (Scale bar, 2 μm.) (D and E) Kymographs of the rings of B and C, respectively. Arrows indicate instants at which turnover is switched off.
suggest contractile instability serves the useful function of assembling myosin II into force-generating aggregates, regulated by turnover which imposes a time limit on the contractile instability-driven aggregation. The steady-state myosin aggregate size is set by the turnover time, together with local effects that dictate a maximum density of actomyosin components. The turnover time allows aggregation to proceed for long enough that powerful myosin aggregates are built, but short enough to prevent runaway aggregation and disastrous organizational disruption, tension loss and ring fracture (Fig. 7).
S. japonicus Cell Ghost Preparation and ATP-Dependent Ring Activation.
S. japonicus cell ghosts were prepared using protocols published previously (48,49). A brief summary is given below. For strains, see ref. 28. S. japonicus cells were cultured until midlog phase using a rich YEA medium, and cell walls were digested using lytic enzymes Lallzyme MMX (Lallemand; ref. 50) for protoplast preparation. Protoplasts were then washed and recovered in sorbitolcontaining medium. Following ring assembly, protoplasts were washed once with wash buffer (20 mM Pipes-NaOH, pH 7.0, 0.8 M sorbitol, 2 mM EGTA, and 5 mM MgCl 2 ) and permeabilized with isolation buffer (50 mM Pipes-NaOH, pH 7.0, 0.16 M sucrose, 50 mM EGTA, 5 mM MgCl 2 , and 50 mM potassium acetate) containing 0.5% Nonidet P-40 to obtain cell ghosts. Ghosts were washed twice with reactivation buffer (0.16 M sucrose, 5 mM MgCl 2 , 50 mM potassium acetate, and 20 mM MOPS-NaOH, pH 7.0; pH adjusted to 7.5) after permeabilization. Both the isolation and reactivation buffers were cooled to 4°C. The isolation and washing steps were performed on ice. Equal volumes of cell ghosts and reactivation buffer containing 1 mM ATP (A6559; Sigma-Aldrich) were then mixed to induce ATP-dependent actomyosin contractile forces in the ring. It takes at least 1 to 2 min after the addition of ATP to mount the ghosts and to find those appropriate for imaging. Thus, time 0 in figures represents the instant at which imaging commences. Cytokinetic ring constriction in the presence of blebbistatin. The cytokinetic rings were isolated from S. japonicus ghost cells using the protocol mentioned in Huang et al. (48,49). The isolated rings were mixed with different concentrations (0, 10, 25, and 40 mM) of blebbistatin (B0560; Sigma). The rings were then activated with 0.5 mM ATP (A6559; Sigma) and imaged using the Andor Revolution XD spinning disk confocal system, equipped with an Andor iXon Ultra EMCCD (electron-multiplying charge-coupled device) camera. The images were captured with a Z-step size of 0.5 μm for 25 min. The images were projected along the z axis by Fiji.
Sample Preparation for Microscopy
Imaging. An equal volume of reactivation buffer containing 1 mM ATP was added to cell ghosts and imaging was performed in an Ibidi μ-Slide eight-well glass-bottom dish (80827). To prevent water evaporation during imaging, an adhesive film membrane was used to seal all imaging dishes.
3. Spinning-Disk Confocal Microscopy. Image acquisition was performed using Andor Revolution XD spinning-disk confocal microscopy. The microscope was equipped with a Nikon Eclipse Ti inverted microscope, Nikon Plan Apo Lambda 100×/1.45 numerical aperture oil-immersion objective lens, a spinning-disk system (CSU-X1; Yokogawa Electric Corporation), and an Andor iXon Ultra EMCCD camera. Andor IQ3 software was used to acquire images at 80 nm/pixel. Laser lines at wavelength of 488 nm were used to excite the fluorophores. Most images were acquired with z-step sizes of 0.5 μm, and at an interval time of 1 min.
Three-Dimensional Molecularly Explicit Model of the Cytokinetic
Ring in S. japonicus. We developed a fully 3D and molecularly explicit mathematical model of the S. japonicus cytokinetic ring. Formin Cdc12 dimers anchored to the membrane nucleate and grow actin filaments that bind myosin II, assumed membrane-anchored from previous studies. In this section, we describe the model for a normal ring with normal turnover processes, as realized in normal intact cells or protoplasts. Later sections describe the model's adaptation to the situation in cell ghosts where turnover processes are switched off. 4.1. Ring geometry. The ring lies on the inner surface of the plasma membrane. Ring components bind with uniform probability in a binding zone of width 0.2 μm (12, 35) on the plasma membrane, thus determining the width of the ring. The thickness of the ring in the direction perpendicular to the plasma membrane is an output of the simulation dynamics. We used a ring radius of 3.7 μm as the rings we observe here have a mean length up to ∼22 to 25 μm. 4.2. Components in the ring. We describe actin filaments as inextensible but flexible polymers with bending modulus κ = k B Tl p where the persistence length l p = 10 μm (51, 52). In the simulation, we keep track of every 37th actin subunit on a filament, with spacing 0.1 μm from each other, as in our earlier work (12,31). The subunits at the barbed end of each filament represent formins, which are anchored to the plasma membrane and subject to anchoring drag.
Myosin II in the ring is described as clusters containing 16 myosin heavy chains (31) and are anchored to the plasma membrane and subject to anchoring drag (12). A cluster binds and pulls on actin filaments within its capture radius r myo = 80 nm measured from the center of the cluster (53).
Alpha-actinin cross-linkers are modeled as springs with rest length r 0 x = 30 nm and spring constant k x = 25 pN=μm, connecting pairs of actin subunits (only those that we keep track of) on different filaments that are within distance r bind x = 50 nm from each other.
Forces in the ring.
Binding of actin filaments to myosin clusters. A myosin II cluster α binds and exerts a capture force f cap i,α on every actin subunit i within its capture radius r myo , implemented as a spring with spring constant k cap = 400 pN=μm and zero rest length that connects the center of the cluster r α to the actin subunit located at r i . In order not to interfere with the pulling force of the myosin, we turnover myosin aggregate size hierarchical aggregation turn Fig. 7. Turnover regulates contractile instability to set myosin II aggregate size and prevent runaway aggregation. Model of turnover-regulated contractile instability. Contractile instability progressively increases punctateness in the contractile ring and other actomyosin machineries. Given a homogenous initial distribution (t = 0), in normal cells component turnover tends to restore homogeneity by intercepting the instability on the turnover timescale τ turn , setting a functional steady state myosin II aggregate size. Without turnover contractile instability progresses unopposed, separating the aggregates and hierarchically merging them into yet bigger aggregates with increased separation. Late-stage aggregates have a myosin II core and outward pointing polarity-sorted actin filaments, with a size governed by local excluded volume interactions that cap the density and polarity sorting that effectively switches off contractility. Once the aggregate separation exceeds the actin filament length aggregates can no longer communicate mechanically, and contractile instability has run its full course (Bottom Left). Schematic depicts myosin density (yellow/orange/brown shading), actin filaments (black), and membrane-anchored actin filament barbed ends (blue).
decompose this force along and perpendicular to the actin filament, and only the latter is actually applied: whereT i is the unit tangent vector of the actin filament at subunit i. Force-velocity relation of myosin. Myosin II cluster α pulls on bound actin subunits i with a force tangent to the filament: wheret i is the unit vector from the (i À 1) th subunit to the i th subunit. This force decreases linearly with the velocity that the myosin II cluster moves toward the barbed end ðv i À v α Þ and is equal to the stall force f s = 4 pN or zero when the velocity is equal to zero or the load-free velocity v 0 myo , respectively. When one myosin II cluster pulls on n fil > 10 actin filaments, the stall force is lowered to a value f s = 4 pN ð Þ× ð10=n fil Þ. Excluded volume interactions. Myosin II clusters repel other clusters located within a distance d myo = 45 nm, with an elastic force that increases linearly as the distance decreases. The elastic constant k excl myo = 4 pN=nm. The value of the distance was chosen so that the aggregates from the simulation and experiment had a similar morphology, which is a mix of broad and pointlike aggregates.
Tension in actin filaments and ring tension. Tension is implemented as pairwise attractive force of magnitude f tension i,i+1 between adjacent actin subunits labeled by i and i + 1. The constraint force ensures that the subunit separation is maintained at 0.1 μm (see subsection 4.2). The force is implicitly set by the dynamics, and in practice is calculated together with component velocities. See Numerical scheme. The sum of filament tension forces exerted across each ring cross-section were obtained and averaged over all cross-sections to obtain ring tension at every time instant.
Bending forces of actin filaments. Following the scheme in ref. 54, first the bending energy of one filament is calculated as The filament has N subunits, each separated by l 0 = 0.1 μm, and a bending modulus κ. The bending force is given by Àð∂H B Þ=ð∂r i Þ, the negative derivative of H B with respect to the coordinates of the subunits.
Cross-linking. Cross-linkers exert a spring force f x i between actin subunits. See subsection 4.2 for details.
Ring component confinement. This constraint is implemented as an elastic restoring force with elastic constant k mb = 20 pN=μm pointing toward the origin that is activated when any ring component is greater than R away from the origin, where R is the radius of the cell.
Normal anchoring forces. Formin Cdc12 dimers and myosin II clusters are anchored to the plasma membrane (33), and an anchoring force normal to the plasma membrane maintains their radial coordinate at R À d for and R À d myo respectively, where R, d for , d myo are the radius of the cell, and distances of formin and myosin from the membrane, respectively. The magnitude of this force is implicitly set by the dynamics, and in practice is calculated together with component velocities. See Numerical scheme. The values of d for and d myo are 30 nm and 80 nm, respectively, based on superresolution measurements (33). Tangential anchoring forces due to membrane drag. As all formin Cdc12 dimers and myosin II clusters are membrane anchored, they experience drag forces f drag, mb for = Àγ for v and f drag,mb myo = Àγ myo v, respectively.
Cytosolic drag forces. Drag forces for actin subunits in the cytosol are set by their velocities and the drag coefficient γ act,proto = 0:2 pN Á s Á μm À1 (12). Cytosolic drag forces for myosin II clusters and formin Cdc12p dimers are neglected as they are much smaller than the membrane drag force.
Stochastic forces. A myosin II cluster α of the simulations in Fig. 6 has a diffusivity D due to a newly added stochastic, zero-mean force f diff α . The correlation of the force is given by hf diff α ðtÞf diff α 0 ðt 0 Þi = D myo γ 2 myo δðt À t 0 Þδ αα 0 I where γ myo and I are, respectively, the drag coefficient parallel to the membrane and the 3 × 3 matrix whose only nonzero entries are I xx = I yy = 1. 4.4. Turnover of ring components. Formin Cdc12p dimers, myosin II clusters, and α-actinin cross-links dissociate from the ring at rates k for off,proto , k myo off,proto , and k x off , respectively. In addition, α-actinin unbinds the ring if the two actin subunits that it cross-links are separated by > r bind x = 50 nm. Formin Cdc12 dimers and myosin II clusters bind the plasma membrane in a binding zone of width 0.2 μm. Alpha-actinin cross-links bind the ring with equal probability between any pair of actin subunits within r bind x unless this pair has already been crosslinked. Component binding rates are chosen to maintain steady state of component densities (SI Appendix, Table S1). Cross-linker binding rates are tuned to achieve a steady-state cross-linker density of 25 μm À1 (35).
Actin is polymerized by formin at v pol = 70 nm=s and subject to severing by ADF-cofilin at rate r sev,proto = 1:8 μm À1 Á min À1 , at a random location along the filament with uniform probability (12). Once severed, the portion from the severing point to the pointed end is removed from the simulation.
Mean actin filament length in simulations is ∼1.4 μm, and is set by formin density, filament growth rate, and filament severing rate. (1) Previously measured formin densities are ∼15 per micrometer (SI Appendix, Table S1). (2) Electron micrographs showed ∼20 filaments in the cross section of the S. pombe ring (55).
(3) Upon addition of the actin monomer sequestering drug latrunculin A to intact cells, in 60 s, ∼90% of cells lost their rings (56). The growth and severing rates v pol and r sev were obtained as best-fit parameters to reproduce (2) and (3) above.
The initial experimentally measured Rlc1-GFP fluorescence distribution had a correlation length ∼1.0 μm (Fig. 2E). In our simulations we used a simple measure to reproduce these statistics. We divided the perimeter of the simulated ring into four equal sectors and chose the association kinetics such that incoming myosin clusters had a preference to bind to two nonneighboring sectors. This generated steady-state rings with a correlation length of myosin density at t = 0 similar to that seen experimentally before turnover was abolished (Fig. 4B). Irrespective of whether or not we used such a spatial turnover bias to set up the initial state of the ring, we observed the same hierarchically aggregating myosin behavior once turnover was switched off. 4.5. Simulation of the model. Initial configuration of the ring. The ring is initially a 0.2-μm-wide bundle composed of myosin II clusters and actin filaments that have a clockwise or anticlockwise orientation with equal probability. Their barbed ends (formin Cdc12p dimers) and myosin II clusters are randomly distributed in a band-like zone 0.2 μm wide on the plasma membrane, with uniform probability per area. Eleven minutes of ring dynamics is simulated in order for the ring to reach steady state.
Numerical scheme. Given the ring configuration at any time step, we numerically solve the linear system of force balance equations for actin subunit i not at the pointed end represent the anchoring of formins and myosins to the membrane, and the maintenance of distance between neighboring actin subunits (see subsection 1.3). The Euler method with a time step Δt = 0:01 s and τ = 10Δt is used to evolve the system. The scheme was adapted from Witkin et al. (57). At each time step, components are added to and removed from the simulation according to turnover dynamics.
To optimize simulation running time and enable larger timesteps, we added a pairwise drag force f = Àγ a Δv between every pair of neighboring actin subunits and between an actin subunit and its bound myosin cluster, in order to suppress spurious oscillations. Here, Δv is the relative velocity in each pair and the artificial drag coefficient γ a = 4 pN Á s=μm. No net force was added to the simulation as this is a pairwise interaction.
Three-Dimensional Molecularly Explicit Model of the Cytokinetic
Ring in Permeabilized S. japonicus Protoplasts (Ghosts). We adapted the model of the normal contractile ring to describe the ring in ghost cells, permeabilized S. japonicus protoplasts. Initially, following ATP addition, formin and actin filaments are present in these ghost cell rings, and both components remain present as time progresses (28). We assume the actin cross-linker α-actinin is also initially present in these rings (however, α-actinin rapidly dissociates, so its initial presence had essentially no influence on our simulation results.) In ghost cells, ring components no longer bind to the ring, and unbinding dynamics are slowed down. The fraction of actin in the ring in permeabilized protoplasts does not decrease with time upon treatment with ATP and phallacidin (28). This implies that formin (bound to the barbed end of actin filaments) unbinds the ring with negligible rate, k for off,ghost = 0. We tuned the cofilin-mediated severing rate r sev,ghost in permeabilized protoplasts to reproduce the fraction of actin remaining in the ring at 40 min after treatment with ATP alone (SI Appendix, Table S1). Actin polymerization is absent, v pol = 0. We tuned the myosin II cluster off rate k myo off to be consistent with our measurements of the time course of Rlc1-GFP intensity (SI Appendix, Fig. S3A). Here, we have interpreted the decrease in myosin II intensity over time as due to myosin II loss from the ring. However, we found that myosin II aggregation kinetics were qualitatively unchanged even when we assumed there was no myosin II loss whatsoever, by setting k myo off to zero in five simulated runs, snapshots from one of which are shown in SI Appendix, Fig. S3B. (In this interpretation, the decrease in Rlc1-GFP intensity of SI Appendix, Fig. S3A would be attributed to photobleaching.) Cross-linker binding rates are set to zero to reflect an absence of cross-linker association to the ring in cell ghosts. For the rings of Fig. 5, along with crosslinker turnover, either actin or myosin turnover processes were abolished as indicated. To simulate cell ghost rings with blebbistatin, SI Appendix, Fig. S6, in addition to abolishing turnover we switched off forces exerted on actin filaments by a subset of myosin clusters whose identity was refreshed every second. 6. Image Analysis. We analyzed 3D confocal micrographs of rings in cell ghosts in Fiji (58). After isolating a 3D volume containing the ring, we identified a best-fit plane where the maximum amount of Rlc1-GFP fluorescence was present. We then rotated the image such that the z axis of the rotated 3D image is parallel to the normal to the best-fit plane and performed a sum intensity projection to get a two-dimensional image. We performed background subtraction using the rolling ball technique with a radius of 50 pixels. We measured the total fluorescence in a rectangular region containing the ring (SI Appendix, Fig. S3A). We used contours of finite thickness coincident with the ring to generate kymographs using the "max" option in the Kymograph plugin developed by Seitz and Surrey (59). Kymographs were analyzed in MATLAB. Aggregates were identified as peaks in the intensity at each time instant in kymographs. We only indicated aggregation events in Figs. 2A and 3C which were flagged by the algorithm, with the exception of the third red asterisk from the left in Fig. 3C which we judged to be a valid aggregation event despite not being flagged by the algorithm. Brightness and contrast of each entire image was adjusted for presentation purposes in Figs. 1C and 2A and SI Appendix, Fig. S6A. In SI Appendix, Fig. S6A we performed maximum intensity projection along the z axis without 3D rotation and additionally corrected for drift in the X-Y plane. We detected gaps by eye in SI Appendix, Fig. S6 and then measured gap size in Fiji.
Images of simulations were generated by convolving myosin positions with a Gaussian with a full-width half-maximum of 600 nm to mimic the experimental microscope point spread function (PSF). Rings in our experimental images are oriented arbitrarily in 3D space and thus are affected by the small PSF width in the X-Y plane and the comparatively larger PSF widths in the X-Z and Y-Z planes. 7. Variation of Parameters in the Turnover Scans of SI Appendix, Fig. S5.
We varied myosin and actin turnover times in the runs of SI Appendix, Fig. S5. For myosin turnover, we varied the off-rate constant k myo off so that the lifetime of myosin clusters is variable. At each ring location we chose the on-rate constant such that the ratio of on-and off-rate constants had the value we used in simulations of normal rings, where the ratio was chosen to generate the nonuniform myosin density profile observed experimentally, with its ∼ 1-μm correlation length (see subsection 4.4). We varied the off-rate constant k for off of formin dimers while maintaining the ratio of on-and off-rate constants to preserve mean formin density and thus the total number of filaments. To preserve filament length statistics, we also simultaneously varied the rate of filament growth v pol and severing r sev such that the ratios k for off =v pol and r sev =v pol remain fixed. Steady-state filament length distributions depend only on these ratios (see next paragraph). We set the load-free velocity of myosin v 0 myo to five times the normal value to enable comparison of simulations with different actin turnover times with minimal complications caused by the different values of v pol (a change in v pol changes the forces that myosin exerts on growing actin filaments, according to the myosin force-velocity relation). Initial conditions were randomly generated rings, as described in subsection 4.5. 7.1 Derivation of the steady-state filament length distribution. Let pðlÞ denote the steady-state probability distribution of filament lengths l. During a small time interval such that a filament grows a small amount Δl, the processes that alter pðlÞ are growth of filaments whose length was l À Δl, growth of filaments whose length was l, and severing of filaments with lengths larger than l. In steady state, there is no net change in the distribution. To order Δl, the change of pðlÞ in this small time interval is Δp l ð Þ = p l À Δl ð Þ 1 À k for off Δl v pol À r sev lΔl v pol ! À p l ð Þ + ∑ ∞ k=1 p l + kΔl ð Þ r sev Δl ð Þ 2 v pol = 0: Subtracting a similar equation for Δp l À Δl ð Þfrom Δp l ð Þ and using coefficients of the lowest-order term, in the continuous limit we get d 2 p dl 2 + k for off v pol + r sev l v pol ! dp dl + 2r sev p v pol = 0: It follows that the length distribution pðlÞ depends only on the ratios k for off =v pol and r sev =v pol .
Data, Materials, and Software Availability. Experimental data and simulation results supporting the findings of this paper and codes to perform the simulations, to analyze the data, and to generate the technical figures are available in the Zenodo repositories https://zenodo.org/record/6989129 (60) and https:// zenodo.org/record/6639126 (61). The codes are also available in a GitHub repository (https://github.com/sathish-t/myoagg/releases/tag/v1.0.0). | v3-fos-license |
2020-08-06T09:04:06.163Z | 2020-08-01T00:00:00.000 | 221036979 | {
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} | pes2o/s2orc | Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study
Simple Summary There are few publications on the subject of canine platelet concentrates and further studies are required to characterize these for clinical applications. The aim of this study determined platelet, erythrocyte, and leukocyte counts in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy). Whole blood (WB) from 30 healthy dogs was used. In 10 L-PRP bovine thrombin activated samples platelet-derived growth factor isoform BB (PDGF-BB) concentration was also measured. The CPUNT 20 produced clinically useful quantities of sterile canine L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Activated L-PRP has an average of 3442 ± 2061 pg/mL of PDGF-BB. Abstract Background: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. Methods: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) according to the manufacturer’s instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. Results: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Conclusion: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.
Introduction
Platelet-rich plasma (PRP) is a product derived from whole blood, characterized by platelet (PLT) concentrations higher than baseline in a small volume of plasma [1]. Growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta, vascular endothelial growth factor, basic fibroblastic growth factor, and epidermal growth factor usually contained in platelet α granules are released when platelets are activated or destroyed [2,3]. The release of these growth factors, that can act either individually or synergistically, into damaged tissue has the potential to facilitate cell proliferation, angiogenesis, tendon and wound healing, production of fibroblasts, collagen, osteoblasts, decreases the inflammatory reaction and accelerates the healing process [2,3]. The use of PRP for delivery of growth factors has emerged as a convenient method for promoting wound healing and tissue regeneration. The autologous nature of PRP preparations makes them safer and less expensive than allogenic cell-based regenerative therapies. As a result, there is widespread use of PRP therapy in human [4][5][6][7][8][9] and in veterinary medicine [10][11][12][13][14][15][16][17][18][19][20][21] prompting the need for development of simple and reproducible methods for preparation of high-quality PRP for clinical use.
In human medicine various authors have tried to define simple parameters for assessing the quality of platelet concentrates, a difficult target to achieve. Marx [22] has reported that the ideal PRP should have a platelet count with a 3-to 5-fold increase over whole blood or an ideal concentration of one million platelet/µL; similarly to Marx, Mazzucco [23] defines one million platelet/µL as a "reasonable compromise" for a quality platelet concentrate for non-transfusion use, while 300,000 platelets/µL is described by Anitua [24] as the minimum platelet concentration needed in a quality PRP. Platelet concentration is not the only important component of a PRP product; inclusion or exclusion of leukocytes not only defines a platelet concentrate [25,26] but has also been reported to affect the efficacy of the product and to have a significant effect on the inflammatory responses after clinical use of PRP [27][28][29][30][31][32].
The collection and processing of PRP should preserve the platelets in a quiescent state so that activation and subsequent growth factor release will be delayed until the moment of clinical use [33]. An important question is whether exogenous activation of PRP with thrombin or calcium should be performed before clinical application: the use of non-activated PRP would result in a greater and longer in time release of growth factors when the platelets are exposed to collagen and therefore activated in the injured site, but this is difficult to confirm in vivo and activation at the lesion site may not occur consistently [34][35][36]. The amount of growth factors available at the end of PRP production process depends on the techniques used (from blood collection to centrifugation) [37][38][39][40], on the biological variability of growth factor concentration among individuals [41,42] and on method of exogenous activation [36], but the relationship between platelet and growth factors concentration is far from clear [2,12,43].
Despite the popularity and advantages of PRP, there are conflicting reports in the literature regarding its clinical efficacy [44][45][46][47][48][49]. These differences may, in part, be attributable to variations in the composition of PRP preparations, therefore considerable research effort has been focused on optimizing human [28,38,[50][51][52][53] and equine [54][55][56][57] protocols with regard to reproducible recovery, yield and cellular composition. However, there have been relatively fewer studies addressing optimization of PRP protocols in dogs. Many different manual or semi-automated methods for PRP production in dogs are described [58][59][60][61] achieving wide ranges of PLT concentration, but many of these studies have been performed on a small number of subjects [58][59][60] and few studies provide information about concentrations of platelet-derived growth factor or p-selectin externalization when PRP is activated [43,60,[62][63][64][65]. Analysis of the previous studies indicates that it cannot be assumed that a system will produce the same PRP from canine blood as that obtained from human blood. Furthermore, the data highlight that the PRPs obtained by use of various commercially available systems can potentially differ in their characteristics [63]. Characterization of PRP products with specific focus on canine patients is needed to understand results of past, current, and future clinical applications or in vivo experiments [59] and more detailed evaluations including the reproducibility in cell content, growth factor concentration, and platelet activation status in the PRP have yet to be performed [63].
The aim of this study was to characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine the platelet-derived growth factor-BB (PDGF-BB) concentration after activation in the final product from a commercial semi-automated platelet-rich plasma closed system using canine blood.
Subjects
After approval by the Ethics Committee of the University of Milan (protocol number 13-01-15) and with owner-informed consent, citrated blood samples were collected from the cephalic vein of 30 healthy un-sedated adult canine blood donors (PLT count within the reference range), 17 males and 13 females, weighing between 20 and 45 kg and between 2 and 8 years of age (mean ± standard deviation: 4.2 ± 1.9 years). A variety of breeds were represented (e.g., Labrador retriever, Rhodesian ridgeback, Corso dog) and all dogs were admitted to the Veterinary Transfusion Research Laboratory (REVLab) of the Department of Veterinary Medicine for routine blood donor check-ups. All dogs were fasted for 12 h before blood sample collection.
Samples Collection and PRP Production
A closed semi-automatic platelet-rich plasma (PRP) collection and producing system for veterinary use (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) was used, according to the manufacturer's instructions. Briefly, the system consists of a sterile, single use, disposable collection kit for blood sampling, a dedicated centrifuge and an automatic instrument for the separation of the PRP ( Figure 1). Animals 2020, 10, x FOR PEER REVIEW 3 of 16 that a system will produce the same PRP from canine blood as that obtained from human blood. Furthermore, the data highlight that the PRPs obtained by use of various commercially available systems can potentially differ in their characteristics [63]. Characterization of PRP products with specific focus on canine patients is needed to understand results of past, current, and future clinical applications or in vivo experiments [59] and more detailed evaluations including the reproducibility in cell content, growth factor concentration, and platelet activation status in the PRP have yet to be performed [63] The aim of this study was to characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine the platelet-derived growth factor-BB (PDGF-BB) concentration after activation in the final product from a commercial semi-automated platelet-rich plasma closed system using canine blood.
Subjects
After approval by the Ethics Committee of the University of Milan (protocol number 13-01-15) and with owner-informed consent, citrated blood samples were collected from the cephalic vein of 30 healthy un-sedated adult canine blood donors (PLT count within the reference range), 17 males and 13 females, weighing between 20 and 45 kg and between 2 and 8 years of age (mean ± standard deviation: 4.2 ± 1.9 years). A variety of breeds were represented (e.g., Labrador retriever, Rhodesian ridgeback, Corso dog) and all dogs were admitted to the Veterinary Transfusion Research Laboratory (REVLab) of the Department of Veterinary Medicine for routine blood donor check-ups. All dogs were fasted for 12 h before blood sample collection.
Samples Collection and PRP Production
A closed semi-automatic platelet-rich plasma (PRP) collection and producing system for veterinary use (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) was used, according to the manufacturer's instructions. Briefly, the system consists of a sterile, single use, disposable collection kit for blood sampling, a dedicated centrifuge and an automatic instrument for the separation of the PRP (Figure 1). The collection kit comprises a butterfly needle (19G) connected to a 20 mL syringe for blood aspiration, with a port for anticoagulant addition and an antibacterial filter to preserve kit sterility, and a 10 mL bag for the storage of the PRP ( Figure 2). All three connections are equipped with plastic clips that can be opened or closed as needed during the collection process. Two 10 mL syringes are also supplied in the sterile package together with the collection kit. Before starting the collection procedure, the three plastic clips (red, blue and green) must be closed, being careful to position the clip of the PRP storage bag close to the bag itself. The collection kit comprises a butterfly needle (19G) connected to a 20 mL syringe for blood aspiration, with a port for anticoagulant addition and an antibacterial filter to preserve kit sterility, and a 10 mL bag for the storage of the PRP ( Figure 2). All three connections are equipped with plastic clips that can be opened or closed as needed during the collection process. Two 10 mL syringes are also supplied in the sterile package together with the collection kit. Before starting the collection procedure, the three plastic clips (red, blue and green) must be closed, being careful to position the clip of the PRP storage bag close to the bag itself. To prevent blood clotting, before blood sampling the plastic clip of the anticoagulant port was opened, 3 mL of 3.8% sodium citrate (not provided by the manufacturer) was inserted with the provided 10 mL sterile luer-lock syringe through the antibacterial filter and transferred to the aspiration syringe. The connection between the 2 tubes was then temporarily clipped.
The plastic clip of the blood aspiration port was opened and whole blood (WB) was collected from the cephalic vein of each subject up to the 20 mL mark on the aspiration syringe. The collection route was then clipped, the anticoagulant port was opened and another 1 mL of 3.8% sodium citrate was inserted.
After sample collection, the part of the kit with the butterfly and the anticoagulant port and a portion of the aspiration syringe piston were removed retaining only the part of the kit dedicated to the production of the PRP, i.e., the aspiration syringe connected to the 10 mL storage bag ( Figure 3). These were centrifuged at 1200× g for 15 min, using the dedicated centrifuge equipped with special adapters. Immediately before centrifugation, the aspiration syringe was temporarily disconnected from the storage bag to withdraw 500 microliters of citrate WB for subsequent laboratory analysis. This step was performed only for the purposes of the study. To prevent blood clotting, before blood sampling the plastic clip of the anticoagulant port was opened, 3 mL of 3.8% sodium citrate (not provided by the manufacturer) was inserted with the provided 10 mL sterile luer-lock syringe through the antibacterial filter and transferred to the aspiration syringe. The connection between the 2 tubes was then temporarily clipped.
The plastic clip of the blood aspiration port was opened and whole blood (WB) was collected from the cephalic vein of each subject up to the 20 mL mark on the aspiration syringe. The collection route was then clipped, the anticoagulant port was opened and another 1 mL of 3.8% sodium citrate was inserted.
After sample collection, the part of the kit with the butterfly and the anticoagulant port and a portion of the aspiration syringe piston were removed retaining only the part of the kit dedicated to the production of the PRP, i.e., the aspiration syringe connected to the 10 mL storage bag ( Figure 3). These were centrifuged at 1200× g for 15 min, using the dedicated centrifuge equipped with special adapters. Immediately before centrifugation, the aspiration syringe was temporarily disconnected from the storage bag to withdraw 500 microliters of citrate WB for subsequent laboratory analysis. This step was performed only for the purposes of the study. The storage bag was then centrifuged again at × g for 5 min, using the appropriate slots of the same dedicated centrifuge. At the end of this second centrifugation, a pellet suspended in the platelet poor plasma (PPP) was formed inside the storage bag ( Figure 5). At the end of the centrifugation the syringe, in which erythrocytes, buffy coat and supernatant plasma layers are clearly visible, was gently removed from the adapter and the separating plastic clip between syringe and storage bag was opened. At this point, the kit was positioned in the automatic separation instrument. The movement of a plunger positioned under the syringe piston of the syringe under control of an optical reader isolated the supernatant plasma, the buffy coat and the surface of the erythrocytes layer into the storage bag ( Figure 4). The aspiration syringe, now containing only the erythrocyte layer, was then separated from the bag. The storage bag was then centrifuged again at × g for 5 min, using the appropriate slots of the same dedicated centrifuge. At the end of this second centrifugation, a pellet suspended in the platelet poor plasma (PPP) was formed inside the storage bag ( Figure 5). The storage bag was then centrifuged again at × g for 5 min, using the appropriate slots of the same dedicated centrifuge. At the end of this second centrifugation, a pellet suspended in the platelet poor plasma (PPP) was formed inside the storage bag ( Figure 5). Finally, 75% of the supernatant PPP was removed using the provided 10 mL syringe through the appropriate perforable membrane, and the pellet was resuspended in 25% of the remaining PPP by gentle manual mixing.
The leukocyte-and platelet-rich plasma (L-PRP) thus obtained ( Figure 6) was collected through the latex perforable membrane with a sterile syringe and transferred to an empty tube for subsequent laboratory analyses.
Platelet and Leukocyte Counts
For each test subject (30/30), platelet count (PLT/μL) leucocyte count (WBC/μL) and erythrocyte count (RBC/μL), were calculated on WB and L-PRP by an automatic analyzer using optical and volumetric impedance measurements (Cell-Dyn 3500 analyzer, Abbott Diagnostics Europe), while neutrophils (cells/μL), monocytes (cells/μL) and lymphocytes (cells/μL) counts were calculated with the same instrument and checked with a manual differential count of blood smears in only 19/30 subjects, for technical reasons. In the same 19 subjects the smears were also used for assessment of possible platelet clumping. All sample were stored at room temperature on a laboratory blood rocker for a minimum of 5 min before counts were performed. Finally, 75% of the supernatant PPP was removed using the provided 10 mL syringe through the appropriate perforable membrane, and the pellet was resuspended in 25% of the remaining PPP by gentle manual mixing.
The leukocyte-and platelet-rich plasma (L-PRP) thus obtained ( Figure 6) was collected through the latex perforable membrane with a sterile syringe and transferred to an empty tube for subsequent laboratory analyses. Finally, 75% of the supernatant PPP was removed using the provided 10 mL syringe through the appropriate perforable membrane, and the pellet was resuspended in 25% of the remaining PPP by gentle manual mixing.
The leukocyte-and platelet-rich plasma (L-PRP) thus obtained ( Figure 6) was collected through the latex perforable membrane with a sterile syringe and transferred to an empty tube for subsequent laboratory analyses.
Platelet and Leukocyte Counts
For each test subject (30/30), platelet count (PLT/μL) leucocyte count (WBC/μL) and erythrocyte count (RBC/μL), were calculated on WB and L-PRP by an automatic analyzer using optical and volumetric impedance measurements (Cell-Dyn 3500 analyzer, Abbott Diagnostics Europe), while neutrophils (cells/μL), monocytes (cells/μL) and lymphocytes (cells/μL) counts were calculated with the same instrument and checked with a manual differential count of blood smears in only 19/30 subjects, for technical reasons. In the same 19 subjects the smears were also used for assessment of possible platelet clumping. All sample were stored at room temperature on a laboratory blood rocker for a minimum of 5 min before counts were performed.
Platelet and Leukocyte Counts
For each test subject (30/30), platelet count (PLT/µL) leucocyte count (WBC/µL) and erythrocyte count (RBC/µL), were calculated on WB and L-PRP by an automatic analyzer using optical and volumetric impedance measurements (Cell-Dyn 3500 analyzer, Abbott Diagnostics Europe), while neutrophils (cells/µL), monocytes (cells/µL) and lymphocytes (cells/µL) counts were calculated with the same instrument and checked with a manual differential count of blood smears in only 19/30 subjects, for technical reasons. In the same 19 subjects the smears were also used for assessment of possible platelet clumping. All sample were stored at room temperature on a laboratory blood rocker for a minimum of 5 min before counts were performed.
Microbiological Evaluation
This study was performed using a closed system designed to guarantee the sterility of the finished product. However, having temporally disconnected the aspiration syringe from the storage bag to withdraw 500 microliters of citrated WB for subsequent laboratory analysis (step not required by the method) and still wanting to test the validity of the method for further possible clinical uses, we performed bacteriological tests on seven, randomly chosen, L-PRP samples. One hundred microliters of L-PRP was plated by streaking onto blood agar plates (Microbiol, Cagliari, Italy) both for aerobic and anaerobic bacteria and then incubated at 35 ± 2 • C for at least 48 h [66].
Platelet Derived Growth Factor Evaluation
The PDGF-BB levels in 10 L-PRP samples were assessed after L-PRP activation of samples with. bovine thrombin. To each 1 mL of L-PRP, 50 µL of a bovine thrombin solution (BioPharm Laboratories LLC, Bluffdale, UT, USA) containing 500 IU/mL was added. After activation, the samples were kept at 37 • C in an incubator. The supernatant (after spontaneous clot retraction) of each activated L-PRP was collected at 3 h after activation and the concentration of PDGF-BB (pg/mL) was determined using a human ELISA kit (Human PDGF-BB Duoset DY220E, R&D Systems, Minneapolis, MN, USA) previously validated in dog [67] and used in others veterinary studies [41,68]. The mean detection sensitivity was <15 pg/mL. Measurements of the concentrations of PDGF-BB were performed in duplicate at 450 nm according to the manufacturer's instructions.
Statistical Analysis
The normal distribution of parametric data was calculated using the D'Agostino-Pearson test and only RBC and lymphocytes values were found to be normally distributed. Results are presented as mean ± standard deviation. The statistical differences between mean values of PLT, WBC, neutrophils, lymphocytes, monocytes and RBC on WB and L-PRP were compared using Wilcoxon rank sum test or paired t-test depending on data distribution. The statistical differences between mean male and female PLT values were compared using Mann-Whitney test. The increase in platelet concentration in L-PRP over whole blood baseline values was calculated using the following equation: platelet count L-PRP/platelet count WB. Spearman's coefficient of rank correlation (rho) was used to evaluate the relationship between cellular counts (PLT, WBC, neutrophils, lymphocytes, monocytes and RBC) in WB and in L-PRP. Correlation between cellular (PLT, WBC, neutrophils, lymphocytes, monocytes and RBC) and PDGF-BB concentrations in L-PRP were determined using Pearson test. For all tests significance was set at p < 0.05. Statistical analyses were performed using commercial software (MedCalc Software v.11.5.1 Mariakerke, Belgium).
Results
The mean volume of L-PRP obtained was 2.3 ± 0.7 mL. The average PLT, neutrophils, lymphocytes, monocytes and RBC values comparing WB and L-PRP were significantly different (Table 1). There was a 4.4-fold increase in the mean platelet concentration in L-PRP compared to baseline concentrations in WB: the highest value of PLT concentration was 1,898,000/µL (9-fold increase), the lower value was 304,000/µL (2-fold increased). There was no difference in L-PRP PLT concentration between males and females (p = 0.44). The mean PDGF-BB concentration in L-PRP samples after thrombin activation was 3442 ± 2061 pg/mL. Pearson correlation coefficient between cellular (PLT, WBC, neutrophils, lymphocytes, monocytes and RBC) and PDGF concentrations in L-PRP were 0.62 (p = 0.05), 0.60 (p = 0.06), 0.43 (p = 0.22), 0.50 (p = 0.14), 0.88 (p = 0.0008) and 0.23 (p = 0.52), respectively. All samples of L-PRP tested for bacteriological analysis were sterile. Table 1. Mean and standard deviation of whole blood (WB) and leukocyte-platelet rich plasma (L-PRP) cell counts.
Discussion
In this study canine L-PRP was prepared using a commercial semi-automated closed system. All the steps of the production method were easy to perform and the volume of platelet concentrate produced was more than sufficient (on average > 2 mL per subject were obtained) for therapeutic use: PRP has been used in quantities ranging from 0.6 to 3 mL in a variety of canine pathologies [69][70][71][72][73][74].
The fact that the system is closed means the final product is free from bacterial contamination, as confirmed by our microbiological evaluation This cannot be guaranteed in other double centrifugation methods with the exception of a laminar flow chamber under sterile conditions and with trained personnel. This is particularly important when canine platelet concentrates will be used therapeutically [58,60]. The proven sterility makes CPUNT system ideal for using the platelet concentrate for therapeutic regenerative purposes both in regions, such as joints or bone, that require a perfect asepsis and in regions, such as the oral cavity or skin, where asepsis is less critical.
Interestingly, it is also possible to withdraw an aliquot of the L-PRP obtained from the collection bag in a sterile manner, so as to be able to keep the remainder for dermatological or oculistic periodic therapeutic treatments in the same subject. Shelf life of the remaining product may be prolonged by freezing, as carried is out in human medicine [75,76], though studies demonstrate that freezing is detrimental to platelet morphology and function and continuous synthesis of growth factors [36]. In veterinary medicine, there are few studies considering extension of the shelf life by freezing of platelet concentrates [54,[77][78][79][80] and in the dog there are no literature reports that confirm the best preservation method. In fact, there is only one research study focused on growth factors evaluation in which a small number of canine PRP samples was frozen at −80 • C and subsequently analyzed, demonstrating an increase in the growth factors following freezing similar to that obtained with calcium chloride activation [36] and one clinical study reporting PRP preservation (at −76 • C) before application to skin wounds in the dog, with a subsequent excellent clinical response [15].
The system utilized demonstrated a consistent ability to enrich platelets in autologous plasma (about fourfold) producing a significantly higher platelet count in L-PRP than in WB (p < 0.0001). Traditionally, a three-to five-fold increase in platelets has been considered an appropriate concentration for medical applications [22,51,57]. In this study, however, in 7 out of 30 samples the three times target platelet increase was not reached, with increases in platelet numbers of between 2-and 2.8-fold. No correlation was found between sex, age of the subjects, number of platelets in WB and L-PRP samples that did not reach the platelet target concentration. It is not uncommon in studies focused on preparation of PRP in dog, for not all samples to reach the predetermined enrichment value, with great variability between individual samples or very large reported standard deviations [13,58,81], as happened also in our study in which the standard deviation for platelet concentration was large. In a study on the assessment of a PRP produced with a commercial centrifugation and platelet recovery kit the minimum platelet enrichment was still considered to be within an appropriate range for medical application with 90% of samples falling within the range of 4.7-to 8.1-fold enrichment [63]. According to previous literature [24], PRP products must achieve a platelet count of at least 300 × 10 3 platelets/µL to be in the therapeutically effective range and this was reached by the seven sampled in this study that failed to concentrate 4-fold. This proposed minimum platelet number for PRP has been extrapolated from the human literature and key species differences in average platelet counts and platelet physiology preclude an accurate evaluation of literature data [82]. The ideal enrichment of platelets in veterinary PRP remains unknown and may depend on the species, the specific disease and the site of application for the regenerative therapy. In man it has been shown that too many platelets may be detrimental to tissue repair [25,30,31,57,[83][84][85], but to date no studies have been conducted to investigate this in dogs [59].
There was no statistical difference in L-PRP PLT concentration between the two sexes, but, as already found in human patients and horses [41,42] as well as the dog the PLT count in P-PRP was on average higher in females than males.
In our study, the L-PRP system utilized significantly decreased the RBC concentration compared with WB (p < 0.001). Reducing RBC concentration is thought to be important when developing the ideal PRP product [86]. A recent study revealed that an increased RBC concentration in PRP increases the concentrations of unwanted inflammatory mediators, specifically IL-1 and TGF-α. This study also showed that when synoviocytes are treated with RBC concentrate there were significantly more synoviocyte deaths when compared with leukocyte-rich PRP, leukocyte-poor PRP, and phosphate-buffered saline [86].
The CPUNT 20 separation instrument has two modes of use: the production mode of the PRP with the inclusion of leukocytes, which allows L-PRP (leukocyte-and platelet-rich plasma) collection and the production mode without the inclusion of leukocytes, which results in production of P-PRP (pure platelet-rich plasma), according to Dohan Ehrenfest's classification. In this study we used the PRP production modality with the inclusion of leukocytes, obtaining an L-PRP with an average WBC not statistically different from that in WB, with a moderate positive correlation between WBC concentration in WB and L -PRP (rho = 0.387, p = 0.03). The average WBC in our L-PRP did not reach the limit of twice baseline value defined by some authors [20,58] although the high standard deviation underlines a great variability between the samples. Leukocyte concentrations may influence the effects of PRP therapy, but their role is still a matter of debate. Leukocytes-and platelet-rich plasma has been associated with increased pro-inflammatory mediators and a catabolic effect [29,[87][88][89]. P-PRP has been thought to be more beneficial than L-PRP in maintaining tendon homeostasis and counteracting inflammation associated with osteoarthritis [27,31,86,89]. Interestingly, however, a human study found both groups improved with no differences in pain or functional scores between P-PRP and L-PRP for intra-articular knee injections [90]. Other authors have instead shown how the presence of leukocytes in an injectable preparation of PRP can be useful for increasing the in situ production of growth factors [28,91], for the potential analgesic effect through the release of different chemokines, anti-inflammatory cytokines (IL-4, IL-10 and IL-13) and opioid peptides (β-endorphins, encephalins and dinorphine-A), to promote the inhibition of pain in a clinically relevant way [26,92] and to increase the important antimicrobial role in PRP [93,94].
In our study, in L-PRP we achieved a decrease in neutrophils and an increase in lymphocytes and monocytes statistically significant compared to WB, with a positive correlation between lymphocytes concentration in WB and L-PRP (rho = 0.558, p = 0.016). The potentially deleterious effects of leukocytes are largely attributed to the presence of neutrophils [25,27,29,31], while the effect of monocytes and lymphocytes remains largely unknown. Further investigation is needed regarding various leukocyte lineages, their concentrations, and their effects on PRP utilization evaluated according to the purpose of use (e.g., regenerative, antimicrobial, analgesic). Based on the sparse canine literature available it is already apparent that leukocyte profiles differ depending on the PRP processing method; however further comparison of the current canine literature remains difficult until a standardized methodology for assessing cellular enrichment or growth factor concentration is adopted for kit analysis [63]. High WBC counts are generally accepted in platelet concentrates used for autologous topical application [58,87,95]. Thus, the desirable WBC count is still a matter of speculation [25] and to date there are no studies that clarify the significance of leukocytes in canine PRP and the optimal ratio of platelet to leukocytes. Finally, the integrity of leukocytes seems to be of considerable importance: centrifugation can activate or destroy white blood cells and/or stimulate the inflammatory state [26].
In our samples there was no any correlation in PLT count between WB and L-PRP when the specific values for each L-PRP and WB samples were analyzed in line with previous studies in man [96], but in contrast to what has been reported in the horse [54]. This conflicting result could depend on the PRP production process used and/or on the different species evaluated and other studies are needed in the dog to understand the validity of this lack of correlation.
The main objective for delivery of platelets is to increase growth factor concentrations to the affected tissue; however platelet enrichment does not necessarily result in increased delivery of growth factors since degranulation can occur during the centrifugation, leading to a plateau in growth factors that depend on platelet activation [97]. The amount of growth factors available at the end of PRP production process therefore depends on the particular technique used to obtain the PRP [37,38,54,98,99]. However, the platelet growth factor concentrations in PRP could be influenced by other methodological aspects, such as the type of anticoagulant used during blood collection, the ratio of blood to anticoagulant, the type of activating agent and the activation protocol [32,34,36,43,53,68]. Among the numerous growth factors present in platelet α granules, we selected PDGF-BB as an index of activated platelets because it is only secreted by activated platelets and is not found in plasma. Thus, its concentration should represent platelets activation and alpha granules release [60]. In our study the PDGF concentration in activated L-PRP is similar to that found in previous studies in dog [60,65], demonstrating a correct release of growth factors following platelet activation with bovine thrombin. The relevance of this to clinical application remains tenuous as in vivo activation depends on the site of injection, and bovine thrombin cannot be used in vivo due to the potential for adverse immunological reactions seen in human medicine [22,100]. Unfortunately, in our study the PDGF-BB dosage was measured in only 10 samples and this may have affected our results on correlations with sex or breed, which have previously been studied in man, horses and rabbits, with different results [41,96,101].
This study shows a positive and significative correlation between PDGF-BB concentration and PLT concentration in L-PRP (Pearson index 0.62 with p = 0.005). The relationship between platelet and PDGF concentrations is far from clear. Some authors report correlation between platelet and PDGF concentrations [43,60] and others do not [12,37,42,64]. Franklin et al. [36] suggest that activation had a greater effect on growth factor concentration than did cellular composition. Several factors might contribute to these discordant results, for example: manipulation-induced platelet stress and variable susceptibility of platelets to stress. Furthermore the biological variability of growth factor and platelet concentration in PRP among individuals must be taken into account [41,42,96,102]. The presence of a positive correlation between WBC and PDGF-BB and monocytes and PDGF in L-PRP (Pearson index 0.6 with p = 0.06 and 0.88 with p = 0.0008, respectively) in our results seems to corroborate the theory that the quantity of white blood cells in the PRP also influences the concentration of growth factors [32,63,91].
A limitation of our study was that we did not measure PDGF-BB concentrations in whole blood. We could speculate that the concentration of PDGF-BB might increase in L-PRP, after activation, in our samples by comparing our values with those found in WB in the literature [65], but we cannot be sure, also considering that the blood sampling site in the dog seems to affect the activation status of the platelets. [63] Further studies will be needed to investigate this. Another limitation was that we did not measure whether platelets were fully activated in the L-PRP, perhaps with microscopical morphological evaluation of blood smears or with p-selectin dosage [63,64]. Among the PRPs with the same number of platelets, the concentrations of growth factors will depend on the number of activated platelets [60] and there is a correlation between activated platelets and quantitative growth factors, as reported in previous studies in canine PRP obtained with other methods [64]. To activate as many platelets as possible, various agonists (collagen, ADP and thrombin, etc.) and the ratio between PRP and agonist should be further studied in dogs, as suggested by previous authors [60].
Another limitation of our study was that we did not evaluate some key inflammatory markers, such as interleukin-1β or TNF-α, and their correlation with WBC and PLT content in L-PRP. The concentration of these catabolic cytokines, that seems differ in various PRPs, may be clinically relevant. A final limitation was that we didn't evaluate the production mode without the inclusion of leukocytes of the CPUNT 20 separation instrument, which results in production of P-PRP, according to the manufacturer's instructions; it would have been interesting to compare the characteristics of the two options provided by the kit with canine samples.
Conclusions
The closed semi-automated system utilized in this study produced a fourfold mean increase in platelets, but with a large standard deviation and reduced the relative percentage recovery of erythrocytes, with a similar concentration of leukocytes but with a lower concentration of neutrophils as in baseline WB. The autologous L-PRP obtained had PDGF-BB concentration comparable to previous data in the literature after activation with bovine thrombin, was sterile and was produced in a sufficient volume to allow clinical use in various fields of canine medicine. Further studies are recommended to evaluate inflammatory markers and clinical application of this L-PRP product in dogs. | v3-fos-license |
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} | pes2o/s2orc | The first ruthenium-silsesquioxyl complexes – synthesis, structure and mechanistic implications in silylative coupling†
Polyhedral oligosilsesquioxanes (POSS) of the general formula (RSiO3/2)n and particularly those (n = 8) containing the inorganic cubic core of Si–O–Si bonds form a class of versatile building blocks for the production of inorganic–organic materials thanks to the three-dimensional highly symmetrical nature of the POSS core. The properties of the core permit a wide range of technological applications of silsesquioxanes that can be used as nanofillers for the preparation of nanostructured composites, catalysts, and dendrimers, as precursors for optoelectronic materials, as dry resists in microelectronics and as precursors for SiO deposition. Monoand octa-functionalized silsesquioxanes and spherosilicates have been synthesized by TM catalyzed reactions such as hydrosilylation by Si–H substituted POSS and silylative coupling by vinyl-substituted POSS with olefins, which occurs via intermediates containing a TM–silicon bond. The mechanism of silylative coupling of olefins with vinyl-substituted silicon compounds was the subject of our earlier study [see ref. 17; for reviews see ref. 18, 19]. So far only two reports have been published on the isolation and crystal structure of the complexes containing a TM–silicon bond (exactly cobalt– silicon bond). This paper reports the synthesis of the first silsesquioxyl complexes of ruthenium containing Ru–Si bonds and describes the crystal structure of one of them. Moreover, the catalytic activity of this complex in the silylative coupling of vinylheptaalkylsilsesquioxane with styrenes is analysed.
Introduction
Polyhedral oligosilsesquioxanes (POSS) of the general formula (RSiO 3/2 ) n and particularly those (n = 8) containing the inorganic cubic core of Si-O-Si bonds form a class of versatile building blocks for the production of inorganic-organic materials thanks to the three-dimensional highly symmetrical nature of the POSS core. 1 The properties of the core permit a wide range of technological applications of silsesquioxanes that can be used as nanofillers for the preparation of nanostructured composites, catalysts, 2-4 and dendrimers, 5 as precursors for optoelectronic materials, [6][7][8][9] as dry resists in microelectronics 10 and as precursors for SiO deposition. 11 Mono-and octa-functionalized silsesquioxanes and spherosilicates have been synthesized by TM catalyzed reactions such as hydrosilylation by Si-H 12 substituted POSS and silylative coupling [13][14][15][16] by vinyl-substituted POSS with olefins, which occurs via intermediates containing a TM-silicon bond. The mechanism of silylative coupling of olefins with vinyl-substituted silicon compounds was the subject of our earlier study [see ref. 17; for reviews see ref. 18,19]. So far only two reports have been published on the isolation and crystal structure of the complexes containing a TM-silicon bond (exactly cobaltsilicon bond). 20,21 This paper reports the synthesis of the first silsesquioxyl complexes of ruthenium containing Ru-Si bonds and describes the crystal structure of one of them. Moreover, the catalytic activity of this complex in the silylative coupling of vinylheptaalkylsilsesquioxane with styrenes is analysed.
Results and discussion
When a toluene solution of the ruthenium hydride complex [RuHCl(CO)(PPh 3 ) 3 ] 1 was heated in the presence of 1 equiv. of vinylheptaisobutylsilsesquioxane at 110°C the color of the solution gradually turned yellow-brown within 48 h. The 1 H NMR spectrum of the post-reaction mixture revealed disappearance of signals at δ = −6.60 (dt) ppm characteristic of the Ru-H bond, and formation of a new singlet at δ = 5.25 ppm which can be assigned to ethylene. Moreover, the appearance of a new singlet at δ = 37.49 ppm was observed in the 31 P NMR spectrum. On the basis of these observations, a general scheme for the synthesis of ruthenium-silsesquioxyl complexes was proposed (Scheme 1).
Further experiments led to the development of efficient synthetic procedures for the synthesis of silsesquioxyl complexes 3 and 4 (see ESI †). The compounds were isolated in high yields (89% and 86% respectively) as pale yellow powders. The obtained products were fully characterized by 1 H, 13 C, 29 Si, and 31 P NMR spectroscopy and high resolution mass spectrometry (see ESI †). Moreover the structure of complex (3) was confirmed via X-ray analysis (Fig. 1). Single crystals were obtained by slow evaporation of a pentane solution. Ru is 5-coordinated in a square-pyramidal fashion. Four atoms, P1, P2, Cl1 and C1, are almost coplanar (maximum deviation from the least-squares plane is 0.053(3) Å) while the fifth one, Si1, is 2.485 Å out of this plane. The Ru atom is also slightly displaced, by 0.0219 Å, from the basal plane towards the apical Si1. Because of static disorder, in approximately one of six molecules the CO and Cl ligands are interchanged (cf. Experimental part) without a significant influence on the complex geometry. The Si 8 O 12 moiety is quite regular, with deviations from the ideal symmetry caused mainly by the presence of Rucoordination. The Si-O distances involving the coordinated silicon atom are systematically longer than the other Si-O bonds; the mean values are 1.636(4) Å for Si1 and 1.620(5) Å for all other ones. In fact, Si1-O distances are the three longest Si-O distances in the molecule. Also, the majority (8 of 12) of Si-O-Si angles are within quite a narrow range, with the mean value of 145.4(16)°, while the other four (O4,O8, O9, O10) are significantly larger, 154°-161°.
Stoichiometric reactions of the ruthenium-silsesquioxyl complex with styrene
As we previously demonstrated for the silylative coupling mechanism of vinylsilicon compounds and olefins, complexes 3 and 4 can be treated as intermediate ones in the catalytic cycle. In order to better understand the mechanism of silylative coupling of styrenes with vinylsilsesquioxanes an equimolar reaction between ruthenium-silsesquioxyl complex 3 and styrene was performed (Scheme 2).
The tests were monitored by 1 H NMR spectroscopy. Addition of 1 equiv. of styrene and 5 equiv. of CuCl to the solution of complex 3 followed by heating the reacting mixture at 110°C resulted in formation of the cross-coupling product 6.
Although spectroscopic examination does not confirm the formation of a complex 9 containing a Ru-H bond, we observed signals at δ = 6.47 ppm (d, 1H, J = 19.2 Hz, vCHSi) and δ = 7.56 ppm (d, 1H, J = 19.2 Hz, vCHPh) characteristic of product 6. The intermediate complex containing a Ru-H bond was also not observed in the reaction of Ru-Si complexes with olefins, which was previously reported by our group. 18,19 Moreover, GC-MS analysis confirmed the formation of the desired coupling product 6. Formation of compound 6 is evidence for insertion of styrene into the Ru-Si(POSS) bond in complex 3 followed by β-H elimination and evolution of E-phenyl(silyl)ethene.
In the above stoichiometric reaction, the desired product was formed only when the reaction was conducted in the presence of CuCl. Moreover, complex 3 exhibited almost no reactivity when the reaction was performed without Cu(I) salt. To better understand the role of CuCl in the reaction mixture we performed a series of stoichiometric reactions monitored by 1 H and 31 P NMR spectroscopy. When a toluene solution of the ruthenium-silsesquioxyl complex [Ru(POSS)Cl(CO)(PPh 3 ) 2 ] 3 was heated in the presence of 5 equiv. of CuCl at 110°C for 24 h we observed disappearance of the signals at δ = 37.49 ppm characteristic of complex 3 and formation of a new singlet at δ = 25.95 ppm in the 31 P NMR spectrum (Fig. 2).
The formation of this new complex 8 was accompanied by precipitation of an insoluble grey-brown [CuPPh 3 ] complex, whose appearance proves the dissociation of phosphine in the proposed reaction system. The 1 H NMR spectrum of the postreaction mixture revealed changes in the aliphatic region. We observed disappearance of two multiplets at 1.
Isotopic labelling experiments
In order to confirm that the coupling process in the presence of 3 proceeds via the insertion-elimination mechanism which is characteristic of a silylative coupling reaction, [17][18][19] we performed a series of labeling studies. In these experiments monitored by 1 H NMR spectroscopy, a solution of equimolar amounts of styrene-d 8 and complex 3 was heated. The insertion-elimination mechanism should afford the formation of silylstyrene-d 7 and ethylene-d 1 (Scheme 3), at least in the initial stage of the reaction. 1 H NMR spectroscopic analysis of the products formed in the equimolar reaction carried out in the presence of 3 revealed the formation of E-1-phenyl-2-(silyl)ethene-d 7 and ethylene-d 1 .
The results obtained in the experiments with deuterium labelled styrene clearly demonstrate that functionalisation of vinylsilsesquioxanes with styrenes proceeds according to the silylative coupling mechanism involving the activation of vC-H and Si-Cv bonds.
Catalytic examination and mechanistic implications
The ruthenium-silsesquioxyl complex 3 was examined in the silylative coupling of selected styrenes with vinylheptaisobutylsilsesquioxane and its catalytic activity was compared to that of its parent complex [RuHCl(CO)(PPh 3 ) 3 ] 1 and the previously studied more reactive complex [RuHCl(CO)(PCy 3 ) 2 ] 7 (Scheme 4). 15 Selected data for 1 and 3 are presented in Table 1, entry 1. As indicated in this table, the silsesquioxyl catalyst 3 exhibits higher catalytic activity than catalyst 1. For example after five hours in the reaction of vinylsilsesquioxane with styrene catalysed by 3, the conversion of the silsesquioxane reached 33%, while the reaction catalysed by 1 revealed almost no conversion (6%). The activity of catalysts 1 and 3 was examined in a wide temperature range. Our experiments demonstrated that increasing the temperature of the reacting mixture from 40°C to 110°C did not give a higher yield of the coupling product. We compared the catalytic activity of complexes 1 and 3 with that of complex 7 bearing PCy 3 ligands. In all cases, complex 7 was the most active in silylative coupling of vinylheptaisobutylsilsesquioxane with styrenes; however, because of its high reactivity we were unable to isolate ruthenium-silsesquioxyl complex bearing PCy 3 ligands.
Mechanistic implications
On the basis of stoichiometric experiments and labelling studies we proposed a mechanism for the coupling of styrenes with vinylsilsesquioxanes in the presence of [RuHCl(CO)-(PPh 3 ) 3 ] 1 and CuCl as co-catalysts (Fig. 3).
Our studies have shown that the coupling reaction proceeds according to the well-recognized insertion-elimination mechanism 17 in which, in the first step, vinylsilsesquioxane reacts with hydride complex 1 to give β-silylethyl complex. This compound decomposes via β-silsesquioxyl group migration to ruthenium and evolution of ethylene to give Ru-Si[POSS] complex 3. Addition of CuCl to complex 3 causes dissociation of phosphine to produce a less sterically hindered four-coordinate complex 8 which reacts with styrene. The next step of the catalytic cycle involves the migratory insertion of styrene into a Ru-Si bond, followed by β-H elimination to give E-phenyl(silyl)ethene 6.
General methods and chemicals
Unless mentioned otherwise, all operations were performed by using standard Schlenk techniques. 1 H-and 13 C-NMR spectra were recorded on a Varian 400 operating at 402.6 and 101.2 MHz, respectively. 31 P NMR spectra were recorded on a Mercury 300 operating at 121.5 MHz. 29 Si NMR spectra were recorded on a Varian Avance 600 operating at 119.203 MHz. All FT-IR spectra were recorded with IFS 113v FT-IR and VERTEX 70 spectrophotometers (Bruker, Karlsruhe). GC analyses were carried out on a Varian CP-3800 (column: Rtx-5, 30 m, I.D. 0.53 mm) equipped with TCD. Mass spectrometry analyses were performed using a Synapt G2-S mass spectrometer (Waters) equipped with an electrospray ion source and a quadrupole-time-of-flight mass analyzer. Acetonitrile was used as a solvent. The measurements were performed in positive ion mode with the desolvation gas flow 200 L h −1 and capillary voltage set to 5000 V with the flow rate of 20 µl min −1 . The chemicals were obtained from the following sources: vinyltrichlorosilane from ABCR, dichloromethane, acetone, n-pentane, ethanol, dichloromethane-d 2 , benzene-d 6 , toluene-d 8 , styrene-d 8 , decane, dodecane, styrene, 4-chlorostyrene, 4-bromostyrene, 4-methoxystyrene, 2-methoxyethanol, triphenylphosphine, formaldehyde, copper(I) chloride, anthracene, calcium hydride and anhydrous magnesium sulphate from Aldrich, triethylamine and silica gel 60 from Fluka, ruthenium(III) chloride hydrate from Lancaster, trisilanolisobutyl POSS from Hybrid Plastics, 22 and toluene and n-hexane from Chempur.
Synthesis and characterization of ruthenium-silyl complexes
Procedure for stoichiometric reactions of ruthenium-silyl complex 3 with CuCl. The stoichiometric reactions were performed in J-Young valve NMR tubes and controlled by 1 H and 31 P NMR spectroscopy. In a typical procedure, ruthenium complex 3 0.01 g (6.64 × 10 −6 mol) and anthracene 0.0001 g (internal standard) were dissolved in 0.65 mL of toluene-d 8 .
Then the 1 H and 31 P NMR spectra were recorded and 0.0032 g (3.32 × 10 −5 mol) CuCl was added under argon. Then the reaction mixture was heated at 110°C, and after 1 h, 3 h and 24 h, the 1 H and 31 P NMR spectra of the reaction mixture were taken.
General procedure for the catalytic examination
The oven dried 5 mL glass reactor equipped with a condenser and a magnetic stirring bar was charged under argon with 2 mL of CH 2 Cl 2 , monovinylsilsesquioxane (0.1 g, 1.19 × 10 −4 mol), styrene (41 μL, 3.56 × 10 −4 mol) and 20 μL of an internal standard (decane or dodecane). The reaction mixture was placed in an oil bath and preheated to 40°C. Then ruthenium complex 1 or 3 (0.0011 g (complex 1) or 0.0017 g (complex 2), 1.19 × 10 −6 mol) was added under argon. After 5 min of the reaction, copper(I) chloride (0.0006 g, 5.93 × 10 −6 mol) was added. The reaction mixture was heated at 40°C under a gentle flow of argon. Reaction yields were calculated on the basis of the 1 H NMR spectra of the reaction mixture. The corresponding reactions with 4-methoxystyrene, 4-bromostyrene and 4-chlorostyrene were performed using the same procedure.
X-ray analysis (complex 3)
X-ray diffraction data were collected at 100(1) K by the ω-scan technique, on an Agilent Technologies four-circle Xcalibur diffractometer equipped with an Eos detector 24 with an MoK α radiation source (λ = 0.71073 Å). The temperature was controlled with an Oxford Instruments Cryosystem device. The data were corrected for Lorentz-polarization effects as well as for absorption (multiscan). 24 Accurate unit-cell parameters were determined by a least-squares fit of 12 093 reflections of highest intensity, chosen from the whole experiment. The calculations were mainly performed within the WinGX program system. 25 The structures were solved with SIR92 26 and refined with the full-matrix least-squares procedure on F 2 using SHELXL97. 27 Scattering factors incorporated in SHELXL97 were used. The function ∑w(|F o | 2 − |F c | 2 ) 2 was minimized, with w −1 = [σ 2 (F o ) 2 + (0.0313P) 2 + 2.2712P], where P = [Max(F o 2 , 0) + 2F c 2 ]/3. All non-hydrogen atoms were refined anisotropically, hydrogen atoms from methyl groups were placed geometrically, in idealized positions, and refined as riding group with their U iso 's set at 1.2 (1.5 for methyl groups) times U eq of the appropriate carrier atom. Disorder was detected during structure refinement:
Conclusions
New silsesquioxyl ruthenium complexes (3 and 4) have been synthesized and their structures were confirmed by spectroscopic and X-ray methods. These complexes were proved to be intermediates in the silylative coupling of vinylsilsesquioxane with styrene. The reaction between vinylsilsesquioxanes and styrenes in the presence of ruthenium hydride complex 1 was confirmed to proceed via the insertion-elimination mechanism. Moreover, the obtained complex 3 exhibits higher catalytic activity than its parent hydride complex 1. | v3-fos-license |
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} | pes2o/s2orc | Zinc-modified Calcium Silicate Coatings Promote Osteogenic Differentiation through TGF-β/Smad Pathway and Osseointegration in Osteopenic Rabbits
Surface-modified metal implants incorporating different ions have been employed in the biomedical field as bioactive dental implants with good osseointegration properties. However, the molecular mechanism through which surface coatings exert the biological activity is not fully understood, and the effects have been difficult to achieve, especially in the osteopenic bone. In this study, We examined the effect of zinc-modified calcium silicate coatings with two different Zn contents to induce osteogenic differentiation of rat bone marrow-derived pericytes (BM-PCs) and osteogenetic efficiency in ovariectomised rabbits. Ti-6Al-4V with zinc-modified calcium silicate coatings not only enhanced proliferation but also promoted osteogenic differentiation and mineralized matrix deposition of rat BM-PCs as the zinc content and culture time increased in vitro. The associated molecular mechanisms were investigated by Q-PCR and Western blotting, revealing that TGF-β/Smad signaling pathway plays a direct and significant role in regulating BM-PCs osteoblastic differentiation on Zn-modified coatings. Furthermore, in vivo results that revealed Zn-modified calcium silicate coatings significantly promoted new bone formation around the implant surface in osteopenic rabbits as the Zn content and exposure time increased. Therefore, Zn-modified calcium silicate coatings can improve implant osseointegration in the condition of osteopenia, which may be beneficial for patients suffering from osteoporosis-related fractures.
Effects of Various Coatings on Cell Proliferation and Cytotoxicity. BM-PCs were seeded and grown
on Ti-6Al-4V (control), CaSiO 3 , and Zn-Ca 0.1 and Zn-Ca 0.3 coatings. After 1, 4, 7 and 14 days, cell proliferation was determined using the Cell Counting Kit-8 (CCK-8) assay (Fig. 1A). Cell proliferation increased with the culture time and Zn content, but no difference was observed on the initial day of culture. After 4, 7 and 14 days of culture, more BM-PCs were found on the Zn-Ca 0.3 coating than on the Zn-Ca 0.1 and CaSiO 3 coatings and the Ti-6Al-4V control. In addition, the cell cytotoxicity on the various coatings at 48 h was evaluated by a live/dead-staining assay. As shown in Fig. 1B, most BM-PCs were stained green in color and had almost no dead red-stained cells. This result was similar to that of the proliferation assay. Among all the samples, those with the Zn-Ca 0.3 coating had the largest number of living cells. Together, It has been well demonstrated that the release of suitable concentration of zinc could stimulate cell proliferation in vitro.
Effect of Zinc-modified Coatings on Osteogenic Marker Expression.
The expression of the specific osteogenic markers alkaline phosphatase (ALP), procollagen α1(I) (Col-I), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX-2) was examined using quantitative polymerase chain reaction (Q-PCR). A detailed presentation of the fold change in the expression of these markers is shown in Fig. 2. For each experiment, BM-PCs were cultured on Ti-6Al-4V with the growth medium as a negative control, which had no detectable levels of the osteogenesis-specific genes. In addition, osteogenic differentiation was induced on Ti-6Al-4V exposed to osteogenic medium, which served as a positive control and expressed osteogenesis-related genes. The expression of an early stage osteoblast differentiation marker, ALP, was upregulated in BM-PCs cultured on the Zn-Ca 0.3 coating compared to cells cultured on the other coatings and the control, with maximum upregulation at day 14 for all treatment groups. However, at day 21, the ALP levels were lower than the mRNA expression observed on other days, and no significant difference was found at day 1. Col-I is also an early marker of osteogenic differentiation. The Col-I mRNA expression level decreased from day 7 for all samples, but the levels were significantly higher in cells seeded on the Zn-Ca 0.3 coating than those on the Zn-Ca 0.1 coating, the CaSiO 3 coating and the control. The expression levels of the late osteogenic marker OCN increased in all groups with a time-dependent effect and significant upregulation for the Zn-Ca 0.3 coating compared with the Zn-Ca 0.1 coating, the CaSiO 3 coating and the control at all time points. A similar trend was observed for the RUNX-2 mRNA expression.
Zinc-modified Coatings Promote the Osteogenic Differentiation and Mineralization of BM-PCs. BM-PCs cultured on various coatings were subjected to an ELISA assay. The ALP activity levels had a similar profile for all samples, with increased activity during the differentiation of BM-PCs into osteoblasts at all time points (Fig. 3A). The expression of ALP was upregulated to its highest levels after 14 days and then downregulated after 21 days in all samples. However, the enzymatic activity of ALP was significantly increased in BM-PCs cultured on the Zn-Ca 0.3 coating compared to the other groups.
Col-I levels were determined at 1, 7, 14 and 21 days of culture (Fig. 3B). The Col-I level decreased with time for all groups from 7 to 21 days, and no significant difference was found between all groups on the first day of culture. However, higher levels of Col-I were observed in cells cultured on the Zn-Ca 0.3 coating than in the other coating groups and the control group, and the level increased with the Zn/Ca ratio.
OCN was also used to evaluate the effect on the osteogenic differentiation of BM-PCs on different coatings in vitro (Fig. 3C). In the BM-PCs grown on all coatings, the OCN level increased with culture time. Moreover, cells on the Zn-Ca 0.3 coating at 21 days had significantly greater OCN levels than the other coating groups and the control group, although there was no significant difference in OCN levels in all groups at day 1. All the results showed striking similarities with the Q-PCR analysis, which suggested that the Zn-Ca 0.3 coatings have higher potential to induce the differentiation of BM-PCs into osteoblasts than all other groups.
To evaluate the osteogenic mineralization of the BM-PCs on the various coatings, Alizarin red S staining was employed to assess calcium deposition using a semi-quantitative analysis of cells at day 21 (Fig. 3D). The amount of calcium deposition was greatly increased in the Zn-Ca 0.3 coating group compared to all other groups. In addition, the level of mineralized deposition increased as the Zn-Ca ratio increased.
Enhancement of the Osteogenic Differentiation of BM-PCs Cultured on Zn-modified Coatings through the TGF-β/Smad Signaling Pathway.
To understand the molecular mechanisms by which Zn-modified coatings promote osteogenic differentiation in BM-PCs, we first screened several signaling pathways associated with BM-PCs differentiation by Q-PCR. Several signaling pathways play significant roles in (1, 7, 14 and 21 days). Data were calculated relative to the expression of the housekeeping gene GAPDH, which was used as the internal control. The results are presented as the mean ± SD for triplicate measurements. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: GAPDH, glyceraldehyde phosphate dehydrogenase; ALP, alkaline phosphatase; Col-I, procollagen α1(I); OCN, osteocalcin; RUNX-2, runt-related transcription factor 2. Negative control: BM-PCs on Ti-6Al-4V with growth medium. Positive control: BM-PCs on Ti-6Al-4V with osteogenic medium. the regulation osteogenic differentiation on different surface coatings, including insulin-like factor-1 (IGF-1), mitogen-activated protein kinase (MAPK) and transforming growth factor-β1(TGF-β1) signaling [24][25][26][27][28][29][30][31][32][33][34] . In our study, the TGF-β/Smad signaling pathway played an essential role in the regulation of BM-PCs osteogenic differentiation. The Q-PCR results showed that the mRNA levels of TGF-β1, Smad2 and Smad3 all increased with incubation time for all coatings. Moreover, the associated genes were significantly increased in the Zn-Ca 0.3 coating compared with that of the other groups (Fig. 4A). However, no substantial enhancement with culture time was observed for the expression of genes in the classical MAPK signaling pathway, including insulin-like growth factor (IGF-I), extracellular signalregulated kinase1/2 (ERK 1/2) and protein kinase C-δ (PKC-δ), for any of the coatings ( Figure S1). Therefore, our data indicated that Zn-modified coatings appeared to specifically and significantly activate the TGF-β/Smad signaling pathway during the osteogenic differentiation of BM-PCs.
The effect of Zn-modified coatings on TGF-β/Smad signaling pathway activation was further verified by Western blotting assays (Fig. 4B). Typically, receptor-regulated Smad (R-Smad) is activated by phosphorylation upon TGF-β/Smad signaling. The expression levels of key proteins involved in the TGF-β/Smad signaling pathway, including Smad2/3 and p-Smad2/3, were increased in the Zn-Ca 0.3 coating group compared to the other groups. In addition, SB431542, a highly selective inhibitor of Smad2/Smad3, was used. As expected, treatment with SB431542 decreased the Smad2/3 and p-Smad2/3 protein levels in a manner consistent with the effects of SB431542. This result suggests that the TGF-β/Smad signaling pathway plays a direct and significant role in regulating BM-PCs osteogenic differentiation on the different coatings, while neither the IGF-1pathway nor the MAPK pathway appear to be involved.
Effects of Zinc-modified Coatings on Bone Regeneration In Vivo. Micro-CT Evaluation.
To investigate the effects of Zn-modified coatings on bone regeneration in osteopenic rabbits, bare (control) and hydroxyapatite (HA)-, CaSiO 3 -, Zn-Ca0.1-and Zn-Ca0.3-coated Ti-6Al-4V with a diameter of 2.0 mm and a length of 10 mm was implanted into the femur defects of osteopenic rabbits subjected to bilateral ovariectomy (OVX) in combination with methylprednisolone sodium succinate (MPS) ( Figure S2). 3-D micro-CT was used to evaluate the differences in the bone-implant interface and trabecular microstructure of peri-implant bone tissue between the control and treated groups (Fig. 5A). At 1 month post-implantation, almost no new bone formation was apparent in the control or the HA and CaSiO 3 coating groups, and a better performance of bone repair and integrity was found in the Zn-modified coating groups with increases in the Zn/Ca ratio. From 2 to 3 months post-implantation, all groups showed enhanced bone formation with implant time. 3D images indicated that the bone density on the surface of the Zn-Ca 0.3 coating was enhanced compared to that of the Ti-6Al-4V control and other coating groups. The osteogenic effect was in the order Zn-Ca 0.3 > Zn-Ca 0.1 > CaSiO 3 > HA > control. The comprehensive quantitative analysis of all the micro CT parameters is shown in Fig. 5B. At 1 month post-implantation, the Zn-modified coating resulted in slight changes in all micro-CT parameters. From 2 to 3 months post-implantation, the HA, CaSiO 3 , Zn-Ca 0.1 and Zn-Ca 0.3 coating groups displayed a significant increase in all structural bone parameters, including bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) and a lower trabecular separation (Tb.Sp) than the control. Moreover, the Zn-Ca 0.3 coating had the strongest effect on these parameters, and significant differences appeared within all five groups, which provided support for the results of the micro-CT images.
Histological Analysis. The micro-CT analysis of the segmental defects was further supplemented by histological analysis. The photomicrographs of longitudinal sections of the bone defect sites of all coatings show the details of the bone-to-implant interface and peri-implant bone tissue (Fig. 6). The Ti-6Al-4V-implanted group did not form new bones along the integrated surfaces at 1 and 2 months post-implantation but did have a small amount of new bone formation away from the implant interface. Moreover, almost no osteointegration was observed around the implants at 3 months post-implantation. For the HA coating, a few new bones started to form at 1 month, more regenerated bone formed at some sites of the coating surfaces at 2 months, and limited osteointegration was found at 3 months post-implantation. Although new bone started to be induced in the CaSiO 3 coating at 1 month, more new bones were regenerated along the integrated surfaces at 2 months, and a few showed osteointegration at 3 months. The Zn-Ca 0.1 and Zn-Ca 0.3 coating groups showed a prominent induction of new bone formation Quantitative results of implant osseointegration and peri-implant microstructural parameters, such as bone mineral density, bone volume/total volume of bone, trabecular number, trabecular thickness and trabecular separation, on the surface obtained by micro-CT assessment 1, 2, and 3 months after implant insertion. All data are represented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. at 1 month, and a large amount of the new bone surrounding the implant was characterized by prominent osteoid seams at the surface of newly formed mineralized bone at 2, 3 months. In particular, we found that most of the new bone integrated with the coating surfaces in the Zn-Ca 0.3 coating group at 2 months, and all but the most recently formed bones were tightly integrated with the implantation surfaces at 3 months. Collectively, our results suggested that the Zn-Ca 0.3 coating improved osseointegration with the formation of a direct bone interface in vivo.
Discussion
Calcium silicate (Ca-Si) ceramics have attracted great attention in bone tissue engineering because of their bioactivity and good osseointegration properties 35,36 . However, there are still some drawbacks, including a relatively high dissolution rate in biological environments that affects long-term clinical performance especially in osteoporotic patients 37,38 . Thus, various surface modification methods have been used to enhance the chemical stability and biological properties [39][40][41][42][43] . Some studies have suggested that incorporation of dopant Zn ions into bioactive coatings with appropriate concentrations positively affected the cellular responses 21,44 . Higher zinc content could result in reduction in cell proliferation 45,46 . In this study, we successfully prepared Zn-modified calcium silicate coatings with different Zn contents on Ti-6Al-4V by the plasma-spray method 47,48 . We found that Zn modified titanium surface improved the osteogenic activity as the Zn/Ca mol ratio increased in vitro and in vivo.
Much research in bone tissue engineering has been devoted to adult stem cells. BM-PCs were used in the present study due to their high osteogenic capacity and clinical significance 49 . Our results showed that Zn-modified coatings significantly enhance proliferation in BM-PCs and are non-cytotoxic compared with CaSiO 3 coatings and uncoated Ti-6Al-4V at all tested time points. Moreover, the cell proliferation rate correlated with the increase in the Zn/Ca mol ratio, consistent with our previous study and other studies reporting the stimulatory effect of zinc on osteoblastic cell behavior 41,47,50 . Therefore, the enhanced cell viability on the Zn-Ca 0.3 coating may be attributed to proper Zn release.
Previous research has demonstrated that osteoblast-specific factors might be important at several stages of bone regeneration 51,52 . In the present study, the osteogenic markers ALP, Col-I, OCN and RUNX-2 were chosen to examine osteogenic differentiation in vitro by Q-PCR. Compared to the Zn-Ca 0.1 coating, the CaSiO 3 coating and the control, the Zn-Ca 0.3 coating significantly upregulated osteogenesis-specific genes, such as ALP, Col-I, OCN and RUNX-2. ELISA analysis also indicated that the ALP activity and the Col-I and OCN secretion were stronger for BM-PCs on the Zn-Ca 0.3 coating than on the other coatings and the uncoated Ti-6Al-4V. Thus, notable differences in ALP, Col-I and OCN activity were related to the osteogenic effects of zinc. Similar results showing that various Zn concentrations have different effects on osteogenic differentiation were previously reported 53 . Furthermore, the effect of Zn-modified coatings on osteoblast differentiation was investigated by Alizarin red S staining. Bone nodules are osteoblastic phenotypic markers and represent the final stages of osteoblastic differentiation. Alizarin red S quantification revealed that the percentage of mineralized nodules increased with the Zn/Ca mol ratio and cells on the Zn-Ca 0.3 coating after 21 days had higher levels of calcium nodule formation than that in other groups. The results were in agreement with previous studies showing that Zn plays an important role in osteoblastic bone formation and mineralization and that Zn-containing implants could improve osteoblastic function through proper Zn-ion release 54,55 . However, little is known about the mechanism underlying the Zn-modified coating promotion of osteogenic differentiation and bone regeneration.
To further investigate the underlying molecular mechanisms, we searched for potential target genes associated with signaling pathways of BM-PCs osteogenic differentiation on the different coatings. Our results showed that Zn-modified coatings upregulated genes of the TGF-β/Smad signaling pathway, such as TGF-β1, Smad2 and Smad3. No change was observed with culture time for the expression of genes involved in the classical MAPK signaling pathway, including IGF-I, ERK 1/2 and PKC, in all the coatings, which suggested that Zn-modified coatings can activate the TGF-β signaling pathway and promote differentiation and mineralization in BM-PCs. The TGF-β pathway is important for BM-PCs differentiation into the osteogenic and chondrogenic lineages 56,57 . TGF-β affects bone formation by activating their receptors to induce the phosphorylation of a group of intracellular transcription factors known as Smads 58 . Smad2/3 serve as substrates for TGF-β receptors 59 . The activated Smad complex can then move into the nucleus to trigger the transcription of a set of target genes. In this study, the TGF-β inhibitor SB431542 was used to investigate the direct role of TGF-β signaling during the process of BM-PCs differentiation on the different coatings. Blocking TGF-β signaling resulted in reduced Smad2/3 and p-Smad2/3 protein levels, which indicated that the Smad2/3-mediated TGF-β signaling pathway is strongly involved in the Zn-modified coatings promoting differentiation and mineralization in BM-PCs (Fig. 7). In addition, Runt-related transcription factor 2 (RUNX-2) is the earliest osteogenic marker (Runx-2) that binds to the osteocalcin promoter 60-62 , interacts with Smads and subsequently induces the osteogenic marker genes ALP and Col-I at early stages and OCN at later stages of differentiation 63,64 . Our results further support the fact that TGF-β signaling is an important event in BM-PCs proliferation and differentiation in a Smad-3-dependent manner 65 .
For biomedical implants, improving bone regeneration on implant surfaces is an important issue for the long-term success of the implant in both healthy and pathological conditions. The mains studies on biomaterial implants were performed in healthy animals 4,5,47 . However, the effects of titanium implants in the presence of an osteoporotic state are rarely reported. The most significant problem in treating osteoporotic fractures is unstable fixation arising from implant loosening because of the poor bone stock with low implant pull-out strength 66,67 . The major contribution of this study is that shows that Zn-modified titanium improves implant osseointegration in ovariectomized rabbits as a model of osteoporosis. In in vivo study, the micro-CT results showed that Zn-modified coatings with higher Zn content can promote new bone formation to repair an osteoporosis metaphysis bone defect at early (1 month) and late time points (3 months). This function is correlated with the Zn content of the coatings and the implant time. Coatings containing higher amounts of Zn significantly increased BMD, BV/TV, Tb.N and Tb.Th and significantly decreased Tb.Sp compared to those in the other groups. Furthermore, the histomorphometric results also showed tighter contact with the surrounding host bone tissue with no obvious adverse effects and with accelerated new bone formation around the coating surface for the Zn-Ca 0.3 coating compared to the other coatings and pure titanium. These findings suggest that the Zn ions released from the Ca-Si-based coatings could influence the surrounding bone tissue, specifically by regulating the osteogenesis-related parameters of the environment for better integration of the titanium substrate with the host bone tissue, which was consistent with the previous reports that Zn has a positive effect on osteoblastic activity and bone formation 68,69 . In other words, the Zn-modified coatings exhibited better properties for implant osseointegration in osteopenic rabbits and could have promising clinical potential for orthopedic implants, especially in the condition of osteoporosis.
Conclusion
Zn-doped calcium silicate coatings with two different Zn contents were successfully prepared by plasma spraying. Zn-modified coatings significantly promoted the proliferation and osteogenic differentiation of BM-PCs in vitro, showing a remarkable increase in osteogenesis-specific gene markers, such as ALP, Col-I, OCN and RUNX-2, and higher levels of ALP activity, Col-I and OCN secretion and calcium deposition with obvious dose-and time-dependent tendencies. Moreover, we confirmed that Zn-modified coatings activated the TGF-β/Smad signaling pathway to regulate BM-PCs osteoblastic differentiation. Furthermore, the in vivo study demonstrated that Zn-modified coatings with a higher Zn content significantly enhanced bone contact and regeneration with host bone tissue in osteopenic rabbits. These findings suggested that Zn-modified coatings are good candidates with high potential for clinical applications for treating defective osteoporotic bone tissues in the future.
Experimental Section
Preparation and Characterization of Ca-Si-based Powders and Coatings. Zn-modified calcium silicate ceramic powders were synthesized using a previously described method 41,47 . Briefly, zinc nitrate hexahydrate (Zn(NO 3
Isolation, Culture, and Identification of BM-PCs. All experiments involving animals complied fully
with guidelines for animal care and use and the experimental protocol was ethically reviewed and approved by the Institutional Animal Care and Use Committee of The Second Military Medical University. The BM-PCs were isolated according to previously described methods 70 . Briefly, thigh bones were isolated from Sprague-Dawley rats (body weight, 100 ± 10 g), and bone marrow containing mononuclear cells was flushed out with Dulbecco's modified Eagle medium: Nutrient Mixture F-12 (DMEM/F-12, Gibco Life Technologies, Carlsbad, CA, USA) using a 1 ml syringe. The cell suspension was filtered through a 40-μm strainer, and the cells were centrifuged (1000 rpm for 5 min) and washed with phosphate-buffered saline (PBS). The cells were resuspended in DMEM/ F12 containing 10% fetal bovine serum (FBS; Gibco Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Life Technologies). The harvested cells were seeded at a density of 1 × 10 6 /ml in a 75-cm 2 tissue culture flask. The media were changed every 2-3 days. After 7 days of primary culture, the cells were passaged and expanded for future use. Cells at passages 3-5 were used in this study.
For BM-PCs characterization, cell surface markers were quantified by flow cytometry as previously described 71,72 . BM-PCs of passage 4 were harvested by treatment with 0.25% trypsin-EDTA (Gibco Life Technologies). The trypsinized cells (1 × 10 6 cells) were washed twice with PBS and stained with fluorescein isothiocyanate (FITC)-conjugated mouse antihuman CD29 and CD31, phycoerythrin (PE)-conjugated mouse antihuman CD90 and allophycocyanin (APC)-conjugated CD45 (BD Biosciences, San Jose, USA). After 30 min of incubation at room temperature, the cells were washed twice with PBS and analyzed using a BD FACS Aria II (BD Biosciences, San Jose, USA).
Cell Proliferation and Cytotoxicity. Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8; Beyotime, China). BM-PCs were cultured on the Ti-6Al-4V control and the CaSiO 3 , Zn-Ca 0.1, and Zn-Ca 0.3 coatings placed individually in a 24-well culture plate at a density of 1 × 10 4 cells/cm 2 in growth medium. After 1, 4, 7 and 14 days, the CCK-8 stock solution (10% of the total volume) was added to each well of the 24-well plates and incubated for 4 h at 37 °C and 5% CO 2 . Then, 100 μl of the solution was transferred to 96-well plates, and the absorbance was read at 450 nm by a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo Fisher Scientific, USA). Additionally, cell cytotoxicity was confirmed by a live/dead assay kit (Invitrogen, Carlsbad, CA, USA) after culturing for 48 h. The assay was performed according to the manufacturer's instructions. Afterward, the stained cells were visualized using confocal laser scanning microscopy (CLSM, LeicaTCSSP5, Germany).
ALP Assay and Col-I and OCN Secretion. The stable p-nitrophenol phosphate substrate was used to quantify ALP activity. BM-PCs were cultured on the Ti-6Al-4V control and the CaSiO 3 , Zn-Ca 0.1, and Zn-Ca0.3 coatings in osteogenic medium for 1, 7, 14, and 21 days. At each time point, the culture medium was removed, and the cells were washed with PBS and harvested in 1 ml of universal ALP buffer (100 mM citric acid, 100 mM KH 2 PO 4 , 100 mM sodium tetraborate.10H 2 O, 100 mM Tris, and 100 mM KCl; pH 11). The cells were centrifuged at 3000 rpm for 5 min at 4 °C. The ALP activity in the supernatants was determined following the addition of p-nitrophenyl phosphate substrate, and the reaction was stopped using 100 µl of 0.1 N NaOH. The absorbance was read with a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo, USA) at a wavelength of 405 nm.
Alizarin Red Staining (ARS).
Osteogenesis was confirmed by Alizarin red S staining. The cells were seeded on different coatings and cultured in osteogenic medium. At the indicated time point, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 0.1% Alizarin Red S (Beyotime, China) at room temperature for 30 min. Afterward, the cells were washed twice with PBS and air dried before the ARS staining was eluted with 5% perchloric acid (SCRC, Shanghai, China). The 100 μl solution from each well was then transferred to a 96-well plate, and the optical density (OD) was measured at 490 nm using a spectrophotometer (SPECTRA MAX PLUS 384 MK3, Thermo, USA). (Table S1). Q-PCR analysis was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Life Technologies, USA). The expression of all target genes was normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shown as the expression relative to that of the negative control group using the comparative CT method (2 −ΔΔCT ). Each experiment was repeated in triplicate for each individual sample.
Western Blotting Analysis.
A Western blot can be used to determine the specific protein expression in a given sample. BM-PCs in each group were lysed in RIPA buffer containing 1 mM phenylmethane sulfonyl-fluoride (Beyotime, China). The total protein concentration of the supernatant was measured using a bicinchoninic acid (BCA) assay kit (Beijing CoWin Biotech Co. Ltd., Beijing, China) in accordance with the manufacturer's protocol. 10 μg protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.22 μm polyvinylidene difluoridemembranes (Millipore, Bedford, Mass, USA). After the membrane was blocked, the blots were incubated with the appropriate primary antibodies: rabbit anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell Signaling Technology, Danvers, USA), rabbit anti-Smad2/3 (Cell Signaling Technology), and anti-GAPDH monoclonal antibody (Novus, CO, USA) overnight at 4 °C. All primary antibodies were applied at a 1:1000 dilution. The membranes were washed three times in phosphate-buffered saline +0.1% Tween 20 (PBST) buffer and then further incubated with anti mouse/rabbit HRP conjugated secondary antibodies at 1: 4000 dilutions for 1 h at room temperature and detected with the ECL Kit (Beijing CoWin Biotech Co. Ltd., Beijing, China). The protein bands were visualized using an Image Quant Las4000mini (GE Healthcare, UK). GAPDH served as an internal control.
Inhibition of TGF-β/Smad Signaling.
To assess the role of TGF-β/Smad signaling in the regulation of osteogenic differentiation of BM-PCs cultured on the Zn-modified coatings, on the second day of culture, BM-PCs on the different coatings were treated with or without 10 μM SB431542 (Sigma-Aldrich, St. Louis, USA). SB431542 is a selective inhibitor of activin receptor-like kinase ALK5 (TβRI), whereas Smad2 and Smad3 are substrates for ALK5. SB431542 has been demonstrated to be a specific inhibitor of the TGF-β/Smad pathway 73 . After 21 days, the BM-PCs were collected, and Smad2/3and phospho-Smad2/Smad3 proteins were detected by Western blotting.
Ovariectomized Rabbits. Forty-five mature female New Zealand white rabbits (90-100 days old, 2-2.5 kg) were used for this experiment. The use of animals and the surgical procedures were approved by the Institutional Animal Welfare Committee of The Second Military Medical University. To induce an osteopenic model, the rabbits were subjected to bilateral ovariectomy (OVX) through a ventral incision under general anesthesia with an intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight). The incision was closed layer by layer. After the surgery, the rabbits were injected intramuscularly with methylprednisolone sodium succinate (MPS) (Pfizer Manufacturing Belgium NV) dissolved in 0.9% benzyl alcohol at a dosage of 1.0 mg/kg/day for 4 consecutive months. Sham-operated rabbits were surgically operated on similarly to OVX rabbits except that the ovaries were not cauterized. At the end of the treatment, the osteopenic state in ovariectomized rabbits was confirmed by micro-computed tomography (micro-CT) imaging of the proximal tibia.
Surgical Procedure. After osteopenic animal models were created, each ovariectomized rabbit underwent general anaesthesia by the intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight). A linear skin incision that was approximately 2 cm long in the distal femoral epiphysis was made laterally, and the lateral femoral condyle was exposed by blunt dissection of the muscles. Then, circular holes (2-mm diameter, 10-mm depth) were created using a surgical electric drill at a slow speed. During drilling, physiological saline was supplied to remove bone shards and rinse the wounded area to stop the bleeding. Forty ovariectomized rabbits were randomly divided into five groups (8 rabbits per group), and the rabbits were implanted with (1) uncoated Ti-6Al-4V (control), (2) HA-coated Ti-6Al-4V, (3) CaSiO 3 -coated Ti-6Al-4V, (4) Ca 2 ZnSi 2 O 7 -coated Ti-6Al-4V (Zn-Ca 0.1) and (5) Ca 2 ZnSi 2 O 7 -coated Ti-6Al-4V (Zn-Ca 0.3). A total of four implants were implanted into the femur of each rabbit. Two implants were inserted into the femur of the left hind leg and another two into the femur of the right hind leg. When the implants were then gently placed to fill the grilled defects according to group allocation. Subsequently, the incision was closed with absorbable sutures (Marlin, Germany), and antibiotics and analgesics were injected intramuscularly. At each time point (1, 2 and 3 months after surgery), ovariectomized rabbits were euthanized and specimens were harvested for micro-computed tomography (micro-CT) analysis and histological examination.
Micro-CT Examination. The femoral heads were fixed with 4% paraformaldehyde for 24 h at 4 °C. A micro-CT imaging system (SkyScan1076, Brukermicro CT, USA) was used to evaluate the new bone formation within the defect region. The acquisition settings were 49 kV and 200 μA with a spatial resolution of 35 μm. A consistent volume of interest (VOI) with a diameter of 3 mm and a height of 4 mm was chosen to evaluate the level of bone regeneration and was reconstructed three-dimensionally using Micview software. The bone mineral density (BMD), bone volume/total volume of bone (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were automatically determined to evaluate the new bone formation within the defect region of the femoral heads. All the morphometric analysis was performed using SkyScan CTVOX 2.1.
Histopathological Examination. After micro-CT scanning, the defective femoral tissue was fixed in 4% paraformaldehyde for 3 days, dehydrated in a series of graded concentrations of ethanol from 70 to 100% for 1 day at each concentration, exposed to dimethyl benzene and then embedded in methyl methacrylate without decalcification., Each specimen was then cut perpendicular to the long axis of implants into 3 μm thick sections along the central axis of the implant using a cutting machine (EXAKT 300 CPB and System, Norderstedt, Germany). The sections from each group were stained with 1% toluidine blue and observed by light microscopy (BX51; Olympus, Japan) for histomorphometry.
Statistical Analysis. For statistical analysis, Levene's test was performed to determine the homogeneity of variance for all data, and then one-way analysis of variance followed by Tukey's or Tamhane's T2 posthoc test for multiple comparison was performed for the comparisons between different groups. All statistical analysis was performed using GraphPad Prism 5.0 (San Diego, CA, USA). The results were considered significant at *p < 0.05, very significant at **p < 0.01 and extremely significant at ***p < 0.001. All data were expressed as the mean ± standard deviation (SD). | v3-fos-license |
2018-08-23T13:03:33.586Z | 2018-08-23T00:00:00.000 | 52069932 | {
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} | pes2o/s2orc | Intravenously Injected Amyloid-β Peptide With Isomerized Asp7 and Phosphorylated Ser8 Residues Inhibits Cerebral β-Amyloidosis in AβPP/PS1 Transgenic Mice Model of Alzheimer’s Disease
Cerebral β-amyloidosis, an accumulation in the patient’s brain of aggregated amyloid-β (Aβ) peptides abnormally saturated by divalent biometal ions, is one of the hallmarks of Alzheimer’s disease (AD). Earlier, we found that exogenously administrated synthetic Aβ with isomerized Asp7 (isoD7-Aβ) induces Aβ fibrillar aggregation in the transgenic mice model of AD. IsoD7-Aβ molecules have been implied to act as seeds enforcing endogenous Aβ to undergo pathological aggregation through zinc-mediated interactions. On the basis of our findings on zinc-induced oligomerization of the metal-binding domain of various Aβ species, we hypothesize that upon phosphorylation of Ser8, isoD7-Aβ loses its ability to form zinc-bound oligomeric seeds. In this work, we found that (i) in vitro isoD7-Aβ with phosphorylated Ser8 (isoD7-pS8-Aβ) is less prone to spontaneous and zinc-induced aggregation in comparison with isoD7-Aβ and intact Aβ as shown by thioflavin T fluorimetry and dynamic light scattering data, and (ii) intravenous injections of isoD7-pS8-Aβ significantly slow down the progression of institutional β-amyloidosis in AβPP/PS1 transgenic mice as shown by the reduction of the congophilic amyloid plaques’ number in the hippocampus. The results support the role of the zinc-mediated oligomerization of Aβ species in the modulation of cerebral β-amyloidosis and demonstrate that isoD7-pS8-Aβ can serve as a potential molecular tool to block the aggregation of endogenous Aβ in AD.
INTRODUCTION
The deposition of amyloid-β peptides (Aβ) as extracellular polymeric aggregates (so-called amyloid plaques) abnormally enriched with Zn, Cu, and Fe ions in specific brain regions is one of the hallmarks of Alzheimer's disease (AD) (Cummings, 2004). The formation of brain amyloid plaques (cerebral β-amyloidosis) is Zn-dependent (Friedlich et al., 2004;Frederickson et al., 2005) and closely associated with neuronal loss and cognitive impairment (Musiek and Holtzman, 2015).
Endogenous Aβ is generated by the sequential cleavage of the amyloid precursor protein by βand γ-secretase, producing peptides with lengths that vary from 37 to 43 amino acids (Benilova et al., 2012). It is a normal component of biological fluids of humans and other mammals at picomolar concentration levels (Masters and Selkoe, 2012). The 42-mer variant (Aβ42) is one of the most aggregation-prone Aβ isoforms and is found as the main component of amyloid plaques (Finder and Glockshuber, 2007). In transgenic animal models of AD, pathological conversion of the intact Aβ from its monomeric state into fibrillar supramolecular aggregates can be induced by exogenous injections of chemically or structurally modified Aβ species present in the AD amyloid plaques (Meyer-Luehmann et al., 2006). The origins of such Aβ modifications are ostensibly linked to AD-associated stresses, e.g., aging, neurotrauma, and neuroinflammation (Barykin et al., 2017).
It was suggested that the prominent amyloidogenic features of isoD7-Aβ42 in comparison with the native Aβ42 (Kozin et al., 2013) are linked to the potential capacity of isoD7-Aβ42 to more readily generate zinc-induced oligomers relative to Aβ42 (Kulikova et al., 2015;Kozin et al., 2016;Kugaevskaya et al., 2018). This suggestion originated from the studies on interactions between zinc ions and the metal-binding domains of native Aβ and of the various naturally occurring Aβ species. Here, we focus on Aβ with the following post-translational modifications: isomerization of Asp 7 (isoD7-Aβ) and phosphorylation of Ser 8 (Tsvetkov et al., 2008;Kulikova et al., 2014;Istrate et al., 2016). It is worth noting that while the presence of isoD7-Aβ in amyloid plaques has been described in 1993 (Roher et al., 1993a), Aβ species with phosphorylated Ser8 (pS8-Aβ) have been found in AD patients relatively recently (Kumar et al., 2011;Rijal Upadhaya et al., 2014). The accumulation of isoD7-Aβ in amyloid plaques can be associated with the processes of spontaneous protein aging (Moro et al., 2018), while the formation of pS8-Aβ was attributed to the ecto-PKA activity (Kumar et al., 2011). Both isoforms of Aβ were suggested to play a role in AD pathogenesis Jamasbi et al., 2017;Kugaevskaya et al., 2018;Zatsepina et al., 2018).
Notably, Ser8 phosphorylation causes Aβ16 with such modification (pS8-Aβ16) to form zinc-bound homodimers but prevents it from further oligomerization in the presence of zinc ions (Kulikova et al., 2014). Moreover, insights into the molecular mechanism of zinc-mediated Aβ16 oligomerization indicate that pS8-Aβ16 can form zinc-driven heterodimers with Aβ species containing the primary zinc recognition site 11EVHH14, and such heterodimers might lack the ability to oligomerize (Istrate et al., 2016;Mezentsev et al., 2016;Polshakov et al., 2017). Based on the above, we have hypothesized that incorporating phosphorylated Ser8 into isoD7-Aβ42 resulting in the peptide with both isomerized Asp7 and phosphorylated Ser8 residues (isoD7-pS8-Aβ42) could safeguard such species from zinc-induced oligomerization in vitro and in vivo. To test this hypothesis, in the present study we have examined by thioflavin T (ThT) fluorometry and dynamic light scattering the ability of isoD7-pS8-Aβ42 to undergo spontaneous and zinc-induced aggregation in vitro, in comparison with synthetic peptides Aβ42 and isoD7-Aβ42 respectively. Subsequently, we have studied the effect of intravenous injections of isoD7-pS8-Aβ42 on the modulation of cerebral amyloidosis in the AβPP/PS1 transgenic mice model of AD.
Host Mice
Animals of mouse strain B6C3-Tg(APPswe,PSEN1dE9)85Dbo/j (stock number 004462), received from the Jackson Laboratory (JAX, ME, United States), were used in the study. Pedigree animal breeding and control genotyping procedures were conducted in compliance with the manufacturer's recommendations. Laboratory animals were produced and housed under specific pathogen-free conditions at the AAALAC-accredited Animal Breeding Facility, Branch of the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences (Pushchino, Moscow Region, Russia). In the course of experiments, the animals were kept under standard housing conditions in the barrier area according to the Institutional Animal Care and Use Program and the IACUC-approved Study Protocol. The experimental procedures were approved by the local Animal Care and Use Committee (Reg#126/15 from March 31, 2015).
Reagents
All chemicals and solvents used throughout this study were of HPLC-grade or better and were obtained from Sigma-Aldrich Biopeptide (San Diego, CA, United States). Amino acid sequences of the peptides were confirmed on an ultrahighresolution Fourier transform ion cyclotron resonance mass spectrometer 7T Apex Qe (Bruker Daltonics, Billerica, MA, United States) by using a de novo sequencing approach based on a collision-induced dissociation (CID) fragmentation technique as described in an earlier work .
Preparation of Aβ Peptide Samples for Aggregation Tests
To prepare monomeric solutions of Aβ42, isoD7-Aβ42, and isoD7-pS8-Aβ42, the peptides were treated with hexafluoroisopropanol, dried, and dissolved in 10 mM NaOH at a concentration of 0.5 mM. The solutions of Aβ isoforms in 10 mM NaOH were adjusted with 100 mM HEPES-buffer (pH 5.0) to pH 7.4 and subjected to centrifugation (10 min, 16,000 g, 4 • C) to remove insoluble peptide aggregates. Peptides in the supernatant were quantified spectrophotometrically based on the molar extinction coefficient 280 = 1490 M −1 cm −1 (Jan et al., 2010) and diluted with appropriate buffers to provide 30 µM Aβ solutions in buffer containing 10 mM HEPES (pH 7.4) and 150 mM NaCl (further referred to as buffer H). Aβ solutions were kept on ice before further use. To prepare zinc-induced Aβ aggregates, an aliquot of 30 µM Aβ solution was mixed with buffer H supplemented with 300 µM ZnCl 2 to provide the final Aβ concentration of 25 µM and zinc/Aβ molar ratio of two. The mixture was used for dynamic light scattering (DLS) measurements after 20 min incubation at room temperature.
Dynamic Light Scattering
DLS measurements were carried out on a Zetasizer Nano ZS system (Malvern Instruments Ltd., Malvern, United Kingdom) at 25 • C. The system is able to measure particle sizes ranging from 0.6 nm to 10 µm. Autocorrelation functions were collected at specified time intervals with acquisition times of 120 s per measurement and converted by the instrument software into particle size distributions, assuming the viscosity and the refractive index of the buffer to be equal to those of water (0.89 cP and 1.333, respectively). The instrument software provides the particle size distribution and the average particle diameter, approximating a heterogeneous population of zincinduced Aβ aggregates by a population of spherical particles with identical distributions of diffusion coefficients. Consequently, the characteristic size of Aβ aggregates is expressed in terms of the average "diameter." Number particle size distributions were used to calculate average diameters.
Fluorimetry
Fluorescence measurements were carried out on the Infinite M200 PRO microplate reader (TECAN, Switzerland) using Corning 96-well microplates. The excitation and emission wavelengths were set to 450 and 482 nm, respectively. To test the self-aggregation of Aβ peptide isoforms, 100 µl aliquots of Aβ solutions (peptide concentration: 30 µM) were mixed in wells with 20 µl of the ThT solution in buffer H (ThT concentration: 150 µM), followed by incubation at 37 • C with constant agitation.
The final Aβ and ThT concentrations were 25 µM. The fluorescence measurements were started immediately after the preparation of Aβ/ThT mixtures. All measurements were made in triplicates. The values of fluorescence in the wells containing buffer alone were subtracted from those in wells containing ThT. The relative changes of ThT fluorescence intensity were calculated as (F-F0)/F0, where F and F0 are the fluorescence intensities of ThT in the presence and absence of Aβ peptides, respectively.
Synthetic isoD7-pS8-Aβ42 Preparations for Injections
Two-thousand micrograms of isoD7-pS8-Aβ42 were dissolved in 2,000 µl of sterile physiological saline (PS), and the prepared solution was filtered through a 0.22 µm filter (Millex-GV, Millipore), aliquoted to 125 µl and frozen. For injection, one aliquot was thawed, and sterile PS was added to obtain 1,500 µl of solution with a peptide concentration of 0.08333 µg per µl ("administration solution"). Then, 150 µl of the "administration solution" were withdrawn and 125 µl of this solution were injected into one animal. Thus, with a single injection into the mouse blood, 10 µg of the peptide were injected. Each injection sample was prepared immediately before its introduction to the animals.
Intravenous Injections
Retro-orbital injections in female mice were performed according to Yardeni et al. (2011). Mice received one intravenous injection with 1-month intervals between injections. The compositions of injections for each group of mice are presented in Table 1. The mice were assigned to the various groups randomly.
Histology and Immunohistochemistry
The euthanasia procedure was applied to 8-month-old mice. Mouse euthanasia was carried out using CO 2 according to the IACUC-approved protocol with the use of automated CO 2 -box (Bioscape, Germany). Mice were transcardially perfused with 50 mL of PBS, followed by 50 mL of 4% paraformaldehyde (PFA). Mouse brains were fixed in 10% formalin. Processing for paraffin embedding was scheduled as follows: 75% ethanol overnight, 96% ethanol 5 min, 96% ethanol 10 min, 100% ethanol 10 min (two changes), ethanol-chloroform (1:1) 30 min, and chloroform overnight. Paraffin embedding was performed at 60 • C for 3 h (three changes). The embedding of tissues into paraffin blocks was performed using a Leica EG1160 device. Serial brain sections (8 µm) were cut using a Leica RM2265 microtome mounted onto slides. For deparaffinization, hydration, and staining of the sections, the following steps were performed. Slides were consistently placed in xylene with three changes (10 min each), 96% ethanol (5 min), 90% ethanol (2 min), 75% ethanol (2 min), H 2 O with three changes (5 min each), Congo red solution (5 min), potassium alkali solution, and water. The Immu-Mount medium (Thermo Scientific) was used for mounting.
Immunostaining was carried out as described elsewhere (Kozin et al., 2013). Briefly, sections were deparaffinized and
Quantitative Assessment of Cerebral β-Amyloidosis
The sections spanning the brain from 0.48 to 1.92 mm relative to the midline in lateral stereotaxic coordinates (Franklin and Paxinos, 2008) were used to quantify the congophilic amyloid plaques in the dentate gyrus and the CA1, CA2, and CA3 regions of the hippocampus. Every 15th section was analyzed, yielding 10 sections per animal. Amyloid plaques were identified by Congo red staining and manually counted as described previously (Kozin et
In vitro Spontaneous and Zinc-Induced Aggregation Behavior of isoD7-pS8-Aβ42
Significantly Differs From That of Aβ42 and isoD7-Aβ42 ThT is known to fluoresce upon binding to amyloid aggregates and the increase of ThT fluorescence is widely used as a qualitative measure of the aggregates' β-sheet content. As seen from Figure 1, the incubation of Aβ42 solutions at 37 • C with constant agitation leads to the increasing occurrence of β-sheetrich Aβ42 aggregates. The aggregation is completed in about 5 h of incubation, when the values of ThT fluorescence apparently approach a plateau (Figure 1). The isomerization of D7 residue notably alters the kinetics of aggregation. Though the extent of aggregation for both Aβ42 and isoD7-Aβ42 peptides appears to be similar, a rapid rise of ThT fluorescence was observed later in the course of aggregation for the isoD7-Aβ42 isoform (within the incubation time interval of approximately 200 to 290 min) in comparison with that for Aβ42 (approximately between 130 and 230 min of incubation, Figure 1). This observation suggests that the isomerization of D7 residue alters the aggregation kinetics for this Aβ isoform by slowing the formation of isoD7-Aβ42 aggregates characterized by the high β-sheet content. The occurrence of the second modificationthe phosphorylation of Ser8 residue -further reduces the ability of Aβ peptides to form β-sheet-rich aggregates, as exemplified by the absence of a substantial increase of ThT fluorescence observed for the isoD7-pS8-Aβ42 isoform within the 5 h incubation interval tested (Figure 1). However, the initial values of ThT fluorescence in the case of isoD7-pS8-Aβ42 were found to lie above those for Aβ42 and isoD7-Aβ42 peptides. This suggests either a higher initial degree of aggregation for the isoD7-pS8-Aβ42 isoform or a different ability to bind ThT molecules, compared to Aβ42 and isoD7-Aβ42 peptides.
Using DLS, we measured the characteristic diameter of zincinduced aggregates of Aβ42, isoD7-Aβ42, and isoD7-pS8-Aβ42 isoforms. The corresponding representative size distributions are presented in Supplementary Figure S1 and the mean values of the characteristic diameters in Figure 2. Prior to zinc addition, only aggregates 15-25 nm in size was detected. No statistically significant differences between the sizes of the preexisting aggregate were found for the Aβ isoforms under study (data not shown). After 20 min of incubation with Zn 2+ (the incubation time is due to the data presented in Supplementary Figure S2), the characteristic diameter of isoD7-pS8-Aβ42 aggregates was found to be remarkably different (45 ± 19 nm) from the diameters of Aβ42 and isoD7-Aβ42 aggregates that were statistically indistinguishable and equal to 1362 ± 180 nm and 1290 ± 128 nm, respectively (Figure 2). This observation suggests that the isoD7-pS8-Aβ42 isoform is substantially less susceptible to zinc-triggered aggregation, compared to Aβ42 and isoD7-Aβ42 isoforms. It should be noted that we observed no discernable changes in the characteristic size of Aβ oligomers for the peptide isoforms studied when incubating 25 µM Aβ solutions for 20 min under quiescent conditions at room temperature in the absence of zinc ions.
Intravenous Injections of isoD7-pS8-Aβ42 Decrease the Amyloid Burden in Transgenic Mice
We investigated the ability of synthetic isoD7-pS8-Aβ42 peptide to reduce cerebral β-amyloidosis in the APP/PS1 doubly transgenic mouse model of AD. These mice demonstrated cognitive features of AD-like pathology and possessed significant amounts of dense-core congophilic amyloid plaques starting from 4 to 6 months age regardless of sex (Borchelt et al., 1997;Garcia-Alloza et al., 2006). The experimental groups included female animals subjected to intravenous injections of isoD7-pS8-Aβ42 (10 µg in 125 µl of PS) starting from the age of 2 months. After serial inoculations (at 1-month intervals) with the peptide, the host mice were sacrificed at the age of 8 months. Female mice injected with PS (125 µl) were used as control.
The brains were extracted, and sagittal brain sections (8 µm thick) were analyzed histochemically using Congo red staining (Figure 3). The hippocampus was chosen as the target region for manual counting of stained congophilic amyloid plaques by using bright-field and polarized light microscopy in the sections representing the brain layer located from 0.48 to 1.92 mm relative to the midline in lateral stereotaxic coordinates. The congophilic plaques found in the brains of all experimental animals were similar in terms of their localization and size distribution in the brain parenchyma (Figure 3). Additionally, congophilic amyloid plaques were characterized by immunohistochemical analysis, which showed the presence of Aβ (Supplementary Figure S3). However, quantitative analysis revealed a significantly lower number of congophilic amyloid plaques per section in the isoD7-pS8-Aβ42-inoculated 8 month-old transgenic mice (p < 0.01) compared to control littermates (Table 1).
DISCUSSION
The presence of dense congophilic amyloid plaques in the patient's brain is a mandatory postmortem criterion for the final diagnosis of AD (Cummings, 2004). Studies in animal models of AD have shown that human Aβ and zinc ions are a prerequisite for the formation of amyloid plaques (Friedlich et al., 2004;Frederickson et al., 2005). However, neither intact Aβ itself nor its combination with zinc ions is amyloidogenic in vivo (Meyer-Luehmann et al., 2006). From transgenic mice studies, it has been suggested that structurally and/or chemically modified isoforms of Aβ can act as aggregation seeds, compelling endogenous molecules of Aβ to form soluble neurotoxic oligomers and insoluble extracellular aggregates (Meyer-Luehmann et al., 2006;Jucker and Walker, 2011). Indeed, isoD7-Aβ42 had been demonstrated to accelerate cerebral β-amyloidosis in transgenic mice upon serial intravenous injections (Kozin et al., 2013). Its amyloidogenicity was later attributed to the metal-binding domain isoD7-Aβ16 (Kulikova et al., 2016). The ability of peripherally derived Aβ species to directly contribute to AD pathogenesis has been recently confirmed in an independent study (Bu et al., 2017).
Based on the results of our previous studies (rev. in Kozin et al., 2016), we can hypothesize that the possible mechanism of isoD7-Aβ16 and isoD7-Aβ42 amyloidogenicity in vivo lie in their specific ability to form zinc-linked oligomers, which in the case of zinc-mediated interaction with endogenous Aβ molecules cause the latter to lose their native conformation. As a result, the modified Aβ molecules accumulate on the surface of zinc-dependent neurons in the form of amyloid plaques. The goal of this study was to test our hypothesis regarding the role of zinc-bound oligomers of isoD7-Aβ42 as aggregation seeds for endogenous Aβ in the transgenic model of AD. To achieve this, we have constructed an artificial peptide, isoD7-pS8-Aβ42, which in our opinion should block the formation of such zinc-induced oligomers, and as a consequence significantly slow down the formation of amyloid plaques in transgenic mice.
In contrast to Asp7 isomerization, Ser8 phosphorylation inhibits zinc-induced oligomerization of Aβ16 (Kulikova et al., 2014). In the present work, we have shown that the spontaneous aggregation (resulting in the formation of Aβ aggregates with high β-sheet content) of isoD7-pS8-Aβ42 is decreased in comparison with Aβ42 and isoD7-Aβ42 (Figure 1). This decrease in the propensity of isoD7-pS8-Aβ42 to undergo spontaneous aggregation is quite unusual in the light of previously obtained data showing that pS8-Aβ42 is much more prone to spontaneous aggregation than Aβ42 (Kumar et al., 2011). We have also found that zinc-induced oligomerization of isoD7-pS8-Aβ42 appears to be less prominent than that of the other peptides under study (Figure 2). This effect most likely appears due to the fact that isoD7-pS8-Aβ42 interacts with zinc ions similarly to pS8-Aβ16; i.e., after the formation of zinclinked dimer through sites 11-14 of the interacting molecules, further zinc-dependent oligomerization of isoD7-pS8-Aβ42 is stopped because of the absence of the second interface on the surface of such a dimer (Istrate et al., 2016). Taken together, observations in vitro demonstrated a significant effect of the phosphorylation of Ser8 on the aggregation properties of isoD7-Aβ42.
Experiments on transgenic mice revealed an approximately fourfold reduction in the number of congophilic amyloid plaques in animals injected with isoD7-pS8-Aβ42 in comparison with the control littermates (Figure 3 and Table 1). To the best of our knowledge, such an anti-amyloid effect has been shown for the first time for intravenously administered synthetic peptides. Earlier we showed that in the animal model of AD similar to the one used in this study, intravenous injections of isoD7-Aβ42 significantly accelerated the process of amyloidogenesis (Kozin et al., 2013). Overall, we have established that for isoD7-pS8-Aβ42, there is a direct correlation between its low zinc-mediated oligomerization in vitro and its ability to suppress cerebral β-amyloidosis in vivo. Thus, Ser8 phosphorylation substantially neutralizes the pathogenic features of isoD7-Aβ42. These data support the role of modified Aβ peptides as key factors regulating cerebral amyloidosis in AD. | v3-fos-license |
2016-11-04T21:38:15.820Z | 2011-11-10T00:00:00.000 | 15788928 | {
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} | pes2o/s2orc | Chemistry and Biology of Vision*
Visual perception in humans occurs through absorption of electromagnetic radiation from 400 to 780 nm by photoreceptors in the retina. A photon of visible light carries a sufficient amount of energy to cause, when absorbed, a cis,trans-geometric isomerization of the 11-cis-retinal chromophore, a vitamin A derivative bound to rhodopsin and cone opsins of retinal photoreceptors. The unique biochemistry of these complexes allows us to reliably and reproducibly collect continuous visual information about our environment. Moreover, other nonconventional retinal opsins such as the circadian rhythm regulator melanopsin also initiate light-activated signaling based on similar photochemistry.
Visual perception in humans occurs through absorption of electromagnetic radiation from 400 to 780 nm by photoreceptors in the retina. A photon of visible light carries a sufficient amount of energy to cause, when absorbed, a cis,trans-geometric isomerization of the 11-cis-retinal chromophore, a vitamin A derivative bound to rhodopsin and cone opsins of retinal photoreceptors. The unique biochemistry of these complexes allows us to reliably and reproducibly collect continuous visual information about our environment. Moreover, other nonconventional retinal opsins such as the circadian rhythm regulator melanopsin also initiate light-activated signaling based on similar photochemistry.
Our visual system operates over an extremely broad dynamic range, detecting variations in light intensity of over 8 orders of magnitude, from single photons to more than one-hundred million photons/s (1). This dynamic range is attributed to adaptation processes in rods and cones, with the remainder arising from pupil contractions, processes within inter-retinal neurons, and the production rate of visual chromophore. The rod cell saturates at several thousand photons/s, whereas cones continue to function at several millionfold higher light intensities (2). The central foundation of our vision is the photochemical isomerization of the vitamin A-derived visual chromophore (11-cis-retinal) from its cis-to trans-configuration. A single photon of light isomerizes a single 11-cis-retinal bound to rod or cone opsins. A photon carries ϳ2.5 eV energy (at 500 nm), but only a fraction (1.5 eV/opsin molecule) is utilized to elicit changes in retinal conformation and subsequently protein conformational changes, whereas the remaining energy is dissipated. The high excess of energy ensures that photoisomerization occurs with high fidelity (3). To renew a functional receptor after photoactivation, the chromophore must be regenerated metabolically through a series of enzymatic processes that include isomerization and oxidation of all-transretinyl ester to 11-cis-retinal. Enzymatic re-isomerization of alltrans-retinoid to 11-cis-retinoid requires only 3-4 kcal/mol energy (or 0.13-0.17 eV/molecule) (4).
The retina is a layered sensory organ containing all necessary functional and structural proteins to support human vision. How this remarkable tissue develops and operates over such an incredible dynamic range and how retinoids are recycled are some of the most stirring questions in biology. Blindness is one of the most feared and debilitating illnesses affecting humans, and without a detailed understanding of the basic events in vision, rational approaches to treating blinding diseases will not be possible. With the current methodology, it is now possible to identify all components of the retina and trace mutations to retinopathies.
Complete Mouse Transcriptome in Eye and Retina
The eye is a complex organ composed of specific tissues that carry out different functions to maintain continuous visual responsiveness. The main players are the cornea and lens in the front of the eye and the retina and retinal pigmented epithelium (RPE) 2 in the back. The primary light absorption events take place within the retina, a 0.24-mm thick tissue (mouse) composed of multiple cell layers (Fig. 1). Retinal development and maintenance, as well as light-sensitive visual functions, are highly regulated. Physical dissection of different ocular tissues followed by global analysis of their gene expression by massively parallel RNA sequencing (RNA-seq) allowed the assignment of a complete comprehensive transcriptome to the ocular tissue (5). The completeness of such analysis is an important prerequisite to understand the structure and physiology of the retina by identifying all players involved. Using RNA-seq of mature WT mouse ocular tissues (the retina and whole eye), we recently determined the complete composition of these transcriptomes (5). Retinal tissue yielded 13,406 unique transcripts, and as expected, many transcripts from WT retinal tissues had annotated functions that could be linked to specific metabolic processes or structural and regulatory functions (Fig. 2, upper) (5). In addition, analysis of WT whole eye tissues revealed a large number of genes with unknown functions that await further careful analysis (Fig. 2, lower). These studies complement and greatly expand earlier gene chip-based expression analyses in both accuracy and quantification (6,7). This new depth of knowledge of the retinal transcriptome will facilitate large-scale analyses of the functional consequences of manipulating photoreceptor gene expression (i.e. using gene transfer by retinal electroporation) (8). In addition to protein-coding mRNAs, a large number of microRNAs (miRNAs) and other noncoding RNAs are expressed in the eye (9,10). Together with many metabolites and dietary components, these RNAs regulate developmental and circadian control over the translation of proteins in each cell type. At least 78 miRNAs are preferentially expressed in the mouse retina from 689 identified miRNAs (miRBASE Sequence Database Release 13.0, March 10, 2009) (11), suggesting the importance of miRNAs in modulating gene expression profiles in retinal cells. Moreover, inactivation of Dicer (an essential RNase III endonuclease required for miRNA maturation) leads to progressive functional and structural degeneration of the mouse retina (12). Regulating miRNA levels could be an important approach to treat human retinal dis-eases, as demonstrated in a mouse model of light-induced retinal degeneration (13).
Photoreceptor Structure
Proteins involved in visual phototransduction are located predominantly in the photoreceptor outer segments (OS) of rods and cones. Photoreceptor OS of these highly differentiated neurons are actually specialized cilia (Fig. 1A). Structural studies of these cilia were first carried out with guinea pig and frog rod outer segments (ROS) (14,15). More recently, mouse tissue has been favored because of the ease of genetic manipulation (5,16).
The average mouse ROS length and diameter were estimated to be 23.8 Ϯ 1.0 m and 1.22-1.32 Ϯ 0.12 m, respectively (17). A mouse ROS contains ϳ800 membranous disks stacked on top of each other (Fig. 1A). These internal disk membranes increase the total membrane surface area by ϳ1500-fold compared with the plasma membrane surface alone (18), promoting a high density of the rod visual pigment rhodopsin. Cryo-electron tomography of vitrified mouse retina provided reliable threedimensional morphological information about this structure ( Fig. 1B) (19). Fig. 1C presents a diagram of this ROS structure with distances between different membrane components obtained from cryo-electron tomograms. Based on these and the abovementioned electron microscopy data, the ROS interior volume, including both the intradiskal and cytoplasmic space, is 32 ϫ 10 Ϫ12 ml, and the cytoplasm occupies 10 ϫ 10 Ϫ12 ml in the ROS (19). Thus, it is amazing that the cytoplasmic space used for phototransduction represents only ϳ30% of the space inside a ROS, underscoring the importance of internal membrane structures in phototransduction. This phototransduction cascade occurs as catalytic processes on the interface of disk membranes and the cytoplasm (interfacial catalysis). Cryo-FIGURE 1. Structures of rod and cone OS and ROS internal membranes. A, neuronal organization of a typical mammalian retina. A cross-sectional representation of rod and cone photoreceptors is presented, illustrating their connections to the RPE distally and to relaying cells (bipolar, horizontal, amacrine, and ganglion cells) proximally. The rod structure has a longer OS with membrane-enclosed disks tightly packed without connections to the plasma membrane. Cone disks are continuously connected with the plasma membrane. This figure was reprinted from Ref. 101 with permission. B, electron tomogram of vitrified ROS. The electron tomogram is represented in three orthogonal slices through the ROS volume. An x-y slice (right) and a y-z slice (left) display the high order and regular arrangement of stacked disks. Red represents the high concentrations of rhodopsin found in disk membranes; spacer structures (pillars) are colored green. Scale bar ϭ 200 nm. C, blueprint of ROS. A schematic of a plasma membrane and two disks with measured distances between membrane components is shown. Green cylinders represent monomeric rhodopsin, which forms a larger cluster in native ROS. B and C were reprinted from Ref. 19 with permission. electron tomograms also show spacers that keep the disks separate from one another and maintain appropriate distances between adjacent disks and the plasma membrane (19). Spacers consist of complexes of proteins with estimated molecular masses of ϳ500 kDa distributed at a mean density of ϳ500 molecules/m 2 throughout the disks (19). Intuitively, the presence of proteins responsible for maintaining this structure could be predicted because structural components are essential for maintaining the complex architecture of these fluid internal membranes. The intervening spacers are likely occupied solely or in part by glutamic acid-rich proteins and a membranebound retinal tetraspanin protein called peripherin/RDS (20).
Rhodopsin occupies ϳ50% of the membrane volume within the disks of ROS (21). This high density of photoreceptor opsin could be needed to increase the probability of photon absorption. In addition, it appears that rhodopsin could play a critical structural role in establishing ROS morphology, as opsin knock-out mice form only small ROS appendices early in life, before the cells degenerate (22). The size of the ROS is dictated by the expression level of rhodopsin (17), as heterozygous knock-out mice for the opsin gene possess ϳ50% smaller ROS (23), and overexpression of this protein leads to rod cell degeneration (24). Rhodopsin is not uniformly distributed throughout disks (25). For example, cryo-electron microscopy images of vitrified unstained native mouse ROS reveal high density regions on the disk surface. This difference in density could arise only from an uneven distribution of rhodopsin, which is the main protein in these disks, representing Ͼ90% of all disk proteins. Moreover, patches of disk membrane containing rows of rhodopsin dimers have been observed by atomic force microscopy, a finding supported by other biochemical methods summarized previously (3). Paracrystalline patches within carefully isolated fresh disks from photoreceptors of mouse retina, wherein the building blocks consist of rhodopsin dimers (26), imply functional significance in rhodopsin biosynthesis or function (27) and remain a topic of considerable interest (reviewed in Ref. 28). Interestingly, Corless et al. (29) found that crystalline structure is formed from visual pigments in cone cells when frog retinas are exposed to light. Because both the mouse rhodopsin level (ϳ520 pmol/eye) (16,17) and total cell number (6.4 ϫ 10 6 rods) (30) can be measured precisely, rhodopsin is calculated to have a concentration of 4.62 mM in disk membranes and 8.23 mM with respect to the ROS cytoplasm. The density of rhodopsin in the disk membrane is estimated to be 2.4 ϫ 10 4 molecules/m 2 on average or up to ϳ3.4 ϫ 10 4 molecules/m 2 in high density patches. Atomic force microscopy measurements yielded a density of 30,000 -55,000 rhodopsin molecules/m 2 and ϳ10 8 rhodopsin molecules/rod, partially organized in paracrystalline arrays (16,26). As the whole retinal transcriptome has now been analyzed and the majority of the ROS proteome has been identified by mass spectrometry, attention is now focused on the interactions of these proteins, their effects on function, and their regulation. More structural studies are required to answer these questions.
Structures of Phototransduction and Visual Cycle Components
Further molecular understanding of phototransduction inevitably focuses on the structures of phototransduction and retinoid cycle components and their complexes because the spatial organization of photoreceptor proteins underlies their functional ability to harvest light and generate a neuronal signal. Great progress has already been made by defining structures of a number of full-length proteins or fragments, either alone or in complex with effector proteins (see Ref. 31). A few interesting examples are listed below, but it is likely that more will be known in the near future about the structures of different components involved in this G protein-mediated process than about most other signal transduction systems in nature.
Structures of multiple forms of the G protein-coupled receptor (GPCR) rhodopsin (Fig. 3) (32-37), as well as various forms of G proteins (38 -40), the receptor-capping protein arrestin (41), or likely G protein partners involved in intracellular translocation between photoreceptor compartments (42), have been determined (43). Rhodopsin has been extensively studied as a prototypical GPCR (3), and insights derived from comprehensive biochemical and biophysical studies of rhodopsin and its cognate G protein, transducin, have significantly improved our understanding of GPCR signaling in general (44).
An enhanced insight into the dynamics of rhodopsin activation and interaction with ligand and G protein has been obtained more recently by NMR techniques that show confor- mational flexibility of this receptor and the G protein upon activation (45)(46)(47)(48). Several methods demonstrated that membrane proteins (49), including rhodopsin (50), contain integral ordered water molecules that play important roles in both structure and function. These water molecules could be key to the initial folding of these proteins as they insert into membranes, facilitating their assembly into functional entities, as well as playing roles in the activation process. Using radiolytic footprinting techniques, we found that water molecules are associated with highly conserved and functionally important residues (50). In all sub-3 Å resolution GPCR crystal structures determined to date, the observation of "conserved" waters in similar locations supports the notion that these waters are likely to be as important to receptor function as the conserved amino acid residues (32,37,51).
Key myristoylated Ca 2ϩ -binding proteins involved in phototransduction, namely guanylate cyclase-activating proteins (52), have been visualized at high resolution by NMR and crystallographic methods to reveal their internal architecture, but only in their Ca 2ϩ -bound forms (53)(54)(55). More advanced studies have been performed on another myristoylated photoreceptor protein called recoverin. In recoverin, Ca 2ϩ induces the N-terminal extrusion of a myristoyl group that interacts with a lipid membrane bilayer (56,57). This transition, termed a calcium-myristoyl switch, could allow a protein to translocate from the cytoplasm to membranes in a calcium-dependent manner (56). In contrast, GCAP1 has its myristoylated group bound within a cavity formed by the polypeptide chain, but this does not exclude the possibility that this acyl group is mobilized in complexes with targeted guanylate cyclases.
Other important structures of phototransduction proteins include rhodopsin kinase (GRK1) (58) and RGS-9 (regulator of G protein signaling 9), the latter alone or in complex with the activated ␣-subunit of the photoreceptor G protein transducin and/or an inhibitory subunit of phosphodiesterase 6 (59,60). These studies provide specific information about the termination of signal transduction on photoactivated rhodopsin and the activated G protein transducin.
In addition to high resolution crystal structures, complementary methods have proven to be informative about complex proteins that are not yet amenable to crystallographic approaches. Among these methods are cryo-electron microscopy and singleparticle analysis. For example, single-particle analysis and modeling provided the first views of phosphodiesterase organization (60,61) and of the complex of dimeric rhodopsin and heterotrimeric transducin (Fig. 3)(62). However, many additional proteins whose atomic level structural details are critical to understanding the regulation and precise mechanism of phototransduction continue to escape structural interrogation.
In addition to these functional receptors, enzymes, and structural proteins, the chemical transformation of retinoid metabolites, i.e. the retinoid cycle, is critical for proper visual function. The structure of retinoid isomerase RPE65, the key enzyme of this metabolic pathway, has been determined (63). This crystal structure reveals a seven-bladed -propeller motif with single-strand extensions on blades VI and VII and a two- Once rhodopsin in its dark 11-cis-retinal-bound state (a) is exposed to light, it immediately goes through a series of photointermediate states, including metarhodopsin I (Meta I; b), and eventually progressing to the Rho* (metarhodopsin II (Meta II)) activated state (c). All images shown were taken upon exposure with standard room lighting (10 and 40 s). Upon treatment with hydroxylamine, the chromophore is hydrolyzed, resulting in a largely colorless solution (d). C, model of the G protein rhodopsin complex based on singleparticle reconstruction of the negatively stained native entity. A model based on solved x-ray structures was built into constraints imposed by the map provided from single-particle analysis (62). This orientation of a G protein and its N and C termini is recapitulated only to the same degree by the  2 -adrenergic receptor-G s -nanobody structure (100). strand extension on blade III (Fig. 4). This crystal structure provided a basis for understanding RPE65 membrane binding and enzyme-catalyzed retinoid isomerization. The structure of an important 11-cis-retinal-binding protein called cellular retinaldehyde-binding protein has a defined hydrophobic core that is responsible for sequestering 11-cis-retinal (64). Additionally, the structure of the R234W mutant of cellular retinaldehydebinding protein, which is associated with Bothnia dystrophy and compromises visual pigment regeneration, identified the structural basis of that disease (4). Despite these advances, many questions remain with regard to the chemistry of the retinoid cycle.
Regenerating Spent Chromophore: Retinoid Cycle
For the retina to remain responsive to light and maintain vision, 11-cis-retinal, which is isomerized to all-trans-retinal, must be continuously and efficiently regenerated (65). The time constant for rhodopsin regeneration is ϳ400 s, and that for cone pigment regeneration is ϳ100 s (66). The pioneering work of Kühne and Wald (67)(68)(69) laid the foundation for our current understanding of the photochemistry of vision. This process takes place in two cellular systems, retinal photoreceptors and the adjacent RPE (Fig. 4). From a chemical perspective, enzymatic isomerization of the chromophore appears to be a formi-dable problem in regioselectivity. What regulates the specificity of the conversion of an all-trans-retinol to a specific 11-cisisomer, when this molecule has only one functional group (-OH) and several possibilities for single or multiple cisisomerizations? This reaction also must occur continuously in a membranous/aqueous environment at body temperature. Moreover, the chromophore has other chemical properties that must be cleverly utilized. First, it contains five or six conjugated double bonds that allow light absorption in the visible range of the spectrum when conjugated with protein via a Schiff base. Second, as predicted by Pauling (70), the repulsion between two methyl groups makes 11-cis-retinal an unstable isomer, which encourages its isomerization to all-trans-retinal. Third, retinol easily forms one of the most stable carbocations in biology (71), allowing reshuffling of double bonds. Fourth, the isomerization of retinol has a relatively low activation energy (72). Three chemical mechanisms for isomerization of conjugated doublebond polyisoprenoids in biological systems have been identified. (a) A transition state carbocation product is formed from retinyl esters by alkyl cleavage; this carbocation then adjusts to an 11-cis-retinyl-like conformation to fit the active site of the enzyme, and double bonds are re-established when water is added (reviewed in Ref. 73). (b) A specific double bond is satu- Absorption of a photon of light by rhodopsin causes photoisomerization of 11-cis-retinal to all-trans-retinal and productive signaling, eventually leading to release of all-trans-retinal from the chromophore-binding pocket of this opsin. All-trans-retinal is reduced to all-trans-retinol in a reaction catalyzed by NADPH-dependent all-trans-retinol dehydrogenases. Then, all-trans-retinol must diffuse into the adjacent RPE cell layer. This process is enabled by esterification of retinol with fatty acids in a reaction catalyzed by lecithin:retinol acyltransferase. In the RPE, these all-trans-retinyl esters tend to form intracellular structures called retinosomes. These esters serve as substrates for the RPE65 retinoid isomerase, which converts them to 11-cis-retinol (structure taken from Ref. 63), which is further oxidized back to 11-cis-retinal by retinol dehydrogenases. 11-cis-Retinal formed in the RPE diffuses back into the ROS because this reaction is virtually irreversible. This last step also completes the cycle by recombining 11-cis-retinal with opsin to form rhodopsin. The concept embodied in this figure was taken from Ref. 102. rated; the resulting transition state intermediate rotates, and the double bond is re-established by desaturation (74), as observed in tomato and Arabidopsis carotenoid isomerase, CRTISO (75). (c) An oxidative cleavage of carotenoids generates two retinal molecules in cis-and trans-forms, as in the case of NinaB (76). The structural explanation of these disparate dioxygenase and isomerase activities is critical to understanding the molecular mechanisms employed by this class of enzymes.
Remarkable progress has increased our knowledge of the retinoid cycle, expanding the work so brilliantly started over a century ago (Fig. 4). Several extensive reviews have provided a current update of this progress (4,65,66,73). Although the cycle's unique photochemistry maintains vision, a high flux of photons by light exposure can lead to elevated levels of toxic retinal metabolites that accumulate throughout life and induce photoreceptor degeneration (77). Blocking the accumulation and action of these toxic intermediates and preventing such photoreceptor degeneration can alleviate major human visual diseases such as Stargardt disease and age-related macular degeneration.
As mentioned before, the broad dynamic range of our vision also raises the intriguing question of how much chromophore is consumed during one's lifetime. This estimate requires several assumptions (see, for example, Ref. 78), but the high sensitivity of the visual system, the large Avogadro number, and the low molecular mass of the chromophore suggest that a realistic exposure to light would equate to consumption of ϳ1 mmol or only 284 mg of 11-cis-retinal during a life span! Although it is unclear why cell types other than photoreceptors are employed for chromophore regeneration per se, the adjacent RPE is vital for maintaining photoreceptor architecture and function. Thus, two cellular compartments are primarily associated with the retinoid cycle, the photoreceptor OS of rods and cones and the closely associated RPE (65). RPE cells are essential for chromophore regeneration in both rods and cones (79,80). In addition, cones appear to be supplemented with 11-cis-retinol by Müller cells (81,82).
The outflow of retinoids from photoreceptors to the RPE requires RPE-expressed lecithin:retinol acyltransferase, which esterifies retinol with fatty acid to form retinyl esters (Fig. 4) (83). Because retinyl esters have a propensity to self-aggregate and they form oil droplet-like structures (84) called retinosomes in the RPE (85,86), a flow of retinol out of rods and cones to the RPE would be expected based on thermodynamic considerations. The flow of 11-cis-retinal back from the RPE to rods and cones is governed by diffusion facilitated by an opsin "sink", i.e. the virtually irreversible reaction of opsins, especially rod opsin, with the chromophore that re-establishes the protonated Schiff base (21). The chromophore undergoes cyclic regeneration for each absorbed photon that causes isomerization of visual pigments, but occasionally retinoids condense with lipids or between themselves to form harmful byproducts of the retinoid cycle (87) that require photoreceptor cell regeneration.
Photoreceptor Renewal
Rods and cones are extensively exposed to light in the presence of high oxygen levels throughout the life of an animal. This environment would inevitably lead to rapid retinal degeneration if this damaging process was not countered by protective biochemical mechanisms and continuous renewal of these cells. Photoreceptor OS are particularly vulnerable to damage, as they contain highly reactive retinoids and high levels of unsaturated phospholipids such as esters of docosahexaenoic acid (88). However, as terminally differentiated post-mitotic cells, rods and cones do not divide. Thus, they have developed a unique mechanism of renewing photoreceptor OS content by shedding OS tips (Fig. 1A), which are then phagocytosed by the RPE. The apical processes of RPE cells encircle the distal 1/3-2/3 ends of photoreceptor OS (89). In the case of mammalian rods, ϳ10% of ROS disks are shed every day, and the same amounts of membrane and protein components are produced at the base of ROS (89). This process necessitates the synthesis of up to 10 7 new rhodopsins/ROS/day, or a half-million rhodopsins/cell/h. In addition, the membrane support must also be synthesized at a rate of ϳ77 cm 2 /day (18). This incredible load of GPCR and membrane synthesis strains the capacity of this system such that a minimal aberration could lead to disruption of photoreceptor OS disk renewal and related rod degeneration. When photoreceptor OS disk morphology and renewal are affected by mutations in the opsin genes, degeneration ensues, as is the case for the P23H mutation in the opsin gene (90) and over 100 other documented defects in production and transport of rhodopsin caused by rhodopsin gene mutations associated with retinitis pigmentosa (3).
Interestingly, photoreceptor OS disk recycling occurs in a circadian manner, with the peak of rod shedding in the morning and cone shedding after dark (91). The components involved in this recycling process are only partially known (Fig. 2). When ingested by the RPE, a photoreceptor OS is surrounded by the plasma membrane, producing a "phagosome." This structure undergoes a series of fusion events with endosomes and lysosomes, where several elements such as unsaturated lipids and retinoids are recycled back to photoreceptors and incorporated into new photoreceptor OS disks. Perhaps a number of genes with unknown function found in the total retina/RPE transcriptome will be shown to play roles in this process and its regulation (92).
Thus, photoreceptor cells absolutely require an extremely metabolically active RPE for their maintenance and survival. Genetic and age-related degenerative processes in RPE cells subsequently lead to degeneration of photoreceptors. For example, at the most metabolically active region of the retina around the fovea, each RPE cell must engulf 4 ϫ 10 8 rhodopsin molecules/day. It is likely that photoreceptors around the fovea place the greatest demand on the RPE, and as a consequence, this region is the first to degenerate during age-related macular degeneration, initially sparing the fovea.
Melanopsin: An Invertebrate-like Opsin in Retina
Patients with inherited retinal degeneration retain light-dependent sleep pattern regulation even when almost all of their photoreceptors have degenerated, but this is not the case when eyes are missing or in advanced stages of glaucoma when the optic nerve that connects the retina to the brain is severed (93). Two possible explanations of this phenomenon are that (i) only a small number of surviving photoreceptors are needed to regulate the sleep cycle, and (ii) the retina contains other types of light-sensitive cells. Using physiological and molecular tech-niques with the help of mouse genetics, it was unequivocally established that the retina contains a small subset of ganglion cells that are sensitive to light (94 -96). These ganglion cells (intrinsically photosensitive retinal ganglion cells) express a rhodopsin-like molecule, melanopsin, with characteristics of an invertebrate opsin. Intrinsically photosensitive retinal ganglion cells consist of distinct subpopulations that innervate the hypothalamus to control circadian photoentrainment, and the olivary pretectal nucleus and other brain targets involved, e.g. pupillary, produce other specific light-induced functions (97). Use of melanopsin, which has a stably associated chromophore, rather than a member of the opsin subfamily that recycles the chromophore enzymatically is likely dictated by the need to avoid the canonical retinoid cycle. Ganglion cells are located too far from the RPE to be readily supplied with new chromophore. Thus, a bistable pigment evolutionarily conserved from amphioxus (protochordate) would represent a useful solution.
We do not yet have structural information on melanopsin, but it is similar to other invertebrate rhodopsins. Significant insight into the function of invertebrate rhodopsin has been derived from crystallographic studies. The 2.5 Å resolution crystal structure of an invertebrate rhodopsin (squid Todarodes pacificus) displays a prototypical seven-helical bundle structure with the chromophore located about two-thirds away from the cytoplasmic surface (98). Notably, invertebrate phototransduction uses a G q -type G protein that is involved in regulating inositol 1,4,5-trisphosphate production. In contrast to bovine rhodopsin, however, helices V and VI extend into the cytoplasmic medium and comprise part of the G protein recognition surface. It has been suggested that invertebrate rhodopsin can oscillate between cis-and trans-retinal conformations upon photon absorption by one of these forms (99). In physiological native membranes, invertebrate rhodopsin is organized in hexagonally packed microvillar membranes of photoreceptors, and in crystals, it is tightly associated in a dimeric form (98).
Extraordinary progress made over the last 2 decades has allowed the development of multiple approaches targeted at understanding blinding diseases. This marriage of basic and translational investigation exemplifies the highest standard of current progress in biology. | v3-fos-license |
2019-05-26T13:18:26.110Z | 2019-04-26T00:00:00.000 | 164259271 | {
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} | pes2o/s2orc | SELECTION OF FLAVORING AGENTS AND PRIMARY PACKAGING FOR THE COMBINED ORAL
Мета. На основі теоретичних та експериментальних досліджень обрати коригенти смаку і первинне пакування для комбінованого орального розчину під умовною назвою «Магліцимет» на основі солей магнію аспарагінату, магнію глутамінату, гліцину і метилкобаламіну. Методи. У дослідженнях використовували органолептичні, фізико-хімічні, фармако-технологічні методи згідно вимог Державної Фармакопеї України та Європейської Фармакопеї. Результати. Для надання препарату приємних органолептичних характеристик нами були досліджені серії отриманого розчину з різними коригентами та їх концентраціями. При виборі підсолоджувача вивчали натрію цикламат у кількості 0.05–0.15 %; натрію сахаринат у кількості 0.05–0.15 %; натрію сахаринат і сорбітол в таких співвідношеннях: 0.1 і 5 %, 0.1 і 8.5 %, 0.15 і 10 %; сахарозу у кількості 20–30 %. Дослідження проводили на основі методик А.І. Тенцової та І.А. Єгорова в три окремих етапи. На першому етапі отримали 12 серій розчину з різними підсолоджувачами, з яких до другого етапу пройшли чотири. На третьому етапі на підставі оцінок групи добровольців обирали найкращий. Ним виявився натрію сахаринат у кількості 0.1 %. Для вибору ароматизатора досліджували такі: «вишня», «малина», «персик» в кількості від 0.1 до 1.0 %. В результаті досліджень було визначено, що найкращими смаковими якостями володів зразок з ароматизатором «вишня» у кількості 0.6 %. При визначенні первинного пакування досліджували дозволені в Україні флакони по 100 мл з помаранчевого скла і поліетилентерефталату (двох типів). Проводили контроль основних показників якості досліджуваних зразків після 3, 6, 9, 12, 18 місяців зберігання у флаконах. Встановлено, що всі види пакування не викликали змін основних показників якості розчину, що виходять за межі проекту методів контролю якості. Це дозволяє рекомендувати всі види досліджуваних флаконів. Висновки. На підставі теоретичних та експериментальних досліджень для комбінованого орального розчину під умовною назвою «Магліцимет» обрані підсолоджувач натрію сахаринат у кількості 0.1 % і ароматизатор «вишня» у кількості 0.6 %. В якості первинного пакування рекомендуються всі види досліджених флаконів: з помаранчевого скла і поліетілентерефталату (двох типів) Ключові слова: оральний розчин, солі магнію, корекція смаку, первинне пакування
Introduction
Each medicine contains not only active pharmaceutical ingredients (API), but also auxiliary substances (AS). Many factors can affect the stability of a medicine, including pH, temperature, solvent, light, oxygen, carbon dioxide. The purpose of AS utilization is determined by their physico-chemical properties and is associated with complex interactions within the composition of the dosage form, the influence of the external environment on it, the characteristics of the production process. The exact choice is established in the context of each particular medicine, taking into account all the above mentioned factors.
One of the most important characteristics of a medicine is its taste and smell, especially for syrups and oral solutions. Various unpleasant taste of the active ingredients in the composition of medicines -bitter, salty, sour, sweet, alkaline, metal -significantly reduce the compliance of patients to treatment. Therefore, an important technological aspect in the development of medicines in these dosage forms is giving them pleasant organoleptic characteristics with the help of special auxiliary substances -corrective agents of taste and smell.
Sweeteners and flavoring agents are used as corrective agents. At the stage of medicine development, along with the correction of taste and smell, it is necessary to consider the stability of the dosage form as a whole, i.e. compatibility of all active and auxiliary substances. Corrective agents should have a pleasant taste, smell and color; mix well with other substances of the composition, do not reduce medicine activity and stability, be indifferent or beneficial to the body, stable in a certain pH range, resistant to light, oxidation and reduction.
We have developed a combined oral solution based on magnesium aspartate and magnesium glutamate. The amino acid glycine and vitamin B 12 methylcobalamin were also added. Magnesium is a macroelement and plays a fundamental role in many cellular functions, including energy production, neurochemical transmission, skeletal and cardiac muscle excitability, normal intracellular calcium, sodium and potassium levels, insulin secretion, bone formation, synthesis of carbohydrates, proteins, lipids, nucleic acids and others. Magnesium deficiency is one of the factors for the development of many diseases [1,2]. The tendency to an increase of an element insufficiency throughout the world is a topical issue of world society [3,4].
The choice of the dosage form -oral solutionpossess several advantages: high bioavailability of API, ease of usage compared to tablets and injection forms, the ability to mask the unpleasant smell and taste of ingredients, which is a benefit in pediatric and geriatric practice. Moreover, such liquid dosage forms are convenient for manufacturing.
Formulation of the problem in a general way, the relevance of the theme and its connection with important scientific and practical issues
In the process of medicines development an integrated approach to the choice of corrective agents is necessary: on the one hand, they should provide their function in the form of imparting pleasant organoleptic characteristics to the medicine, and on the other hand, they should be harmless and compatible with other components. Taste masking is based on suppressing the undesirable taste of individual components by introducing substances with stronger taste impulses.
The APIs of the developed medicine are magnesium salts with aspartic and glutamic acids, glycine. They possess specific bitter taste. Therefore, the selection of corrective agents -sweeteners and flavoring agentswas an important stage of technological research.
Another important aspect for the oral solution development was the selection of suitable primary packaging. For this purpose, we should take into account the physicochemical properties of the combination of active and auxiliary substances in the selected dosage form. Primary packaging should ensure the stability of the medicine during storage, be sealed, convenient during transportation and utilization.
Analysis of recent studies and publications in which a solution of the problem are described and to which the author refers
A group of scientists from Duke University in North Carolina investigated the taste sensations caused by the application of various medicines on the surface of the patient's tongue. Among over 60 tested medicines were drugs against AIDS, hypertension and depression treatment. Scientists identified those groups of medicines that need emergency measures to improve their taste [5]. The article by Susan S. Schiffman summarizes various data on the frequency of chemosensory disorders that occur after the use of different medicines. Such phenomena as ageusia (complete loss of taste), dysosmia (distortion of smell) and many others may occur after taking medicines with unpleasant organoleptic characteristics [6]. In the investigation of oral gels with an unpleasant aftertaste (gel of glycine based on a derivative of cellulose) and a pronounced unpleasant odor and bitter taste (gel based on ibuprofen derivative of acrylic acid) using the method of A.I. Tentsova next corrective agents were selected: in the first case -mannitol and vanillin, in the second -aspartame and a combination of flavoring agents [7]. Literature data suggest that the introduction of corrective agents is a necessary technological aspect and is solved in each specific case.
Several authors provide an overview of the various primary packaging for liquid dosage forms, and indicate factors affecting their choice and stability [8,9]. There are also studies of primary packaging for specific oral medications. For example, for ammonium chloride and diphenhydramine chloride syrup, the suitability of polyethylene, polypropylene and glass vials was studied based on quality indicators after accelerated aging [10]; for an oral solution, based on arginine asparaginate, studies on stability in orange glass, glass and polyethylene vials were provided [11].
The field of research considering the general problem, which is described in the article
An important task in the process of pharmaceutical development is the correction of taste of each specific medicine, since it has its own specific composition of active pharmaceutical ingredients, auxiliary substances and the dosage form. The choice of corrective agents in the case of oral solutions requires not only correcting its organoleptic characteristics, but also the simultaneous study of the effect of corrective agents on the biological activity of the composition and the stability of the dosage form during storage.
The choice of primary packaging must be justified by data on the study of the medicine stability during the shelf life. Each product contains a specific composition of the main active and auxiliary substances, which requires the individual selection of packaging in each case.
Formulation of goals (tasks) of article
To choose corrective agents and primary packaging for the combined oral solution named «Maglycimet», which consist of magnesium aspartate, magnesium glutamate, glycine and methylcobalamin, based on theoretical and experimental investigations.
Presentation of the main research material (methods and objects) with the justification of the results
The objects of research were the obtained series of the combined oral solution named «Maglycimet». The medicine composition includes the following APIs: magnesium aspartate, magnesium glutamate, glycine, methylcobalamin. All substances met the requirements of the State Pharmacopoeia of Ukraine (SPHU) [12] and the European Pharmacopoeia (EP) [13]. Methylcobalamin met the requirements of the Japanese Pharmacopoeia [14]. As auxiliary substances antioxidants, sweeteners, and preservatives that are generally accepted in the technology of oral solutions preparing were used. Their quality corresponded to the requirements of the EP [13]. Food flavoring agents «cherry», «raspberry», «peach» corresponded to TU U 15.8-23788752-001-2001. Solventpurified water -met the requirements of SPHU [15]. The preparation of the experimental series of the «Maglycimet» oral solution was carried out in the research laboratory of parenteral and oral liquid medicines of the National University of Pharmacy.
Organoleptic, physicochemical, and pharmacotechnological methods according to the requirements of SPHU [12] and EP [13] were used during the research. The pH of the medium was measured by a potentiometric meth-od according to SPhU, 2.2.20. [12]. Quantitative determination of magnesium was performed by the complexometric method, SPHU, 2.5.11. [12], glycine and methylcobalamin -by HPLC, SPHU, 2.2.29. [12]. All the laboratory and analytical equipment passed metrological certification.
Corrective agents belong to substances that are added to medicines in order to give them pleasant taste and smell. Such organoleptic properties of medicines are acceptable, which flavoring effect appears quickly and to the fullest extent, do not have aftertaste and unpleasant sensations.
One of the most commonly used in the pharmaceutical industry groups of corrective agents is sweeteners. Sweeteners are substances that make medicines taste sweet. They are classified according to their origin (natural or synthetic), caloric level (high-caloric, low-caloric, practically non-caloric), degree of sweetness (high or low sweetness factor K s ), chemical composition, etc. [16][17][18][19]. The most common are traditional sweeteners, such as sucrose, glucose, fructose, sorbitol, and more intensesodium cyclamate, saccharin sodium, etc. The table shows the sweeteners, based on their sweetness ratios (Table 1). Sorbitol is currently the most common sweetener in pharmaceutical practice. But it possesses low potential of sweetness, and that is why sorbitol is added to dosage forms in large quantities, which causes certain difficulties in industrial production.
We have developed samples of oral solution with magnesium aspartate and magnesium glutamate, glycine, methylcobalamin and antioxidant sodium metabisulphite to conduct research on the choice of sweetener. Sweeteners were introduced into the samples in various concentrations. The assessment of taste was made by the method of A. I. Tentsova (determination of numerical indexes). The method is characterized by the basic taste of the substance. The degree of basic taste is determined in points from 0 to 5. Also the method for taste assessing using alphabetic and numeric indices (method of I.A. Egorov) was used. In this acse the most important qualitative signs of the medicine are evaluated by alphabetic and numeric indices constituting the «taste panel», which later helps to record the general formula of medicine taste. The sense of taste is conventionally denoted by letters: S stands for sweet, B -bitter, S -salty, SRsour; and digital indices: 1 -not sweet, not bitter, not salty, not sour; 2 -slightly sweet, slightly bitter, slightly salty, slightly sour; 3 -sweet, bitter, salty, sour; 4 -very sweet, very bitter, very salty, very sour [20,21].
In order to choose the sweetener were carried out next investigations in several separate stages. The best composition was proved according to the given methods. Table 2.
Based on the data presented in Table 2, the composition of the oral solution with different sweeteners was similar in taste characteristics. However, in terms of numerical value, composition No.3 was preferred. Saccharin sodium in the amount of 0.1 % was chosen as a sweetener. It belongs to the group of intense synthetic sweeteners and is 300 times sweeter than sucrose. Its concentration in the composition of oral dosage forms varies from 0.04 to 0.25 %. The main advantage of saccharin sodium utilization is the possibility of using the developed medicine for patients with diabetes.
Flavoring agents were also investigated to improve the taste of the oral solution dosage form based on amino acid salts of magnesium.
As flavoring agents «cherry», «raspberry» and «peach» was chosen for research. According to the previous studies the composition of the oral solution with saccharin sodium (0.1 %) as sweetener was used as a sample to determine the most suitable flavoring agent and its quantity. We investigated the above mentioned substances in the range of concentration from 0.1 % to 1.0 %.
The food flavoring agent was selected using an organoleptic taste assessment from the point of view of objective sensations according to this system: 5 -very pleasant, 4 -pleasant, 3 -not bad, 2 -bad, 1 -very bad. As a result of research, based on the findings of a group of 20 volunteers, the sample of the oral solution with the addition of «cherry» flavoring with concentration 0.6 % had the best taste. Table 3 shows the composition of the oral solution, taking into account the selected corrective agents. The safety of the developed medicine in the course of storage, transportation and use by the patient is to a great extent ensured by the correctly chosen primary packaging. The choice of primary packaging must be confirmed in the process of scientific research for each medicine individually, since none of the types of materials is universal and indifferent to all substances and sol-vents. The main requirements for the design of primary packaging are the following: the ability to protect medicine from adverse environmental factors, protect it from mechanical effects, ensure tightness and stability, and protect against microbial contamination. Packaging materials and closures must be non-fragile, chemically inert, compatible with the components of the preparation.
We have investigated the samples of primary packaging and closures that are available on the pharmaceutical market of Ukraine, registered in the Ministry of Health of Ukraine and are approved for use, namely: orange glass bottles of type FV 100-20-OS according to TU U 26.1-00480810-004: 2011, sealed with screw-on plastic lids of type 1.1-20 (from a mixture of high and low pressure polyethylene in proportions that prevent cracking at low temperatures and preserve rigidity and elasticity at high temperatures); polyethylene terephthalate (PET) bottles: type FP-100 according to TU U 25.2-34014330-001: 2008, complete with lids with first opening control (made of high-pressure polyethylene) and type FVP-100 according to TU U 26.1-19046619-007: 2007, complete with a lid, providing tightness and control of the first opening.
The suitability of the primary packaging was checked after 3, 6, 9, 12, 18 months of storage.
Control was carried out according to the main indicators of quality according project of methods of quality control (MQC). The main results are presented in Table 4. It was established that the solution in all typed of bottles did not change the critical quality indicators and the analysis data corresponded to the limits of the MQC project.
7. Conclusions 1. The research for choosing corrective agents of taste and smell and primary packaging for the combined oral solution named «Maglycimet» was provided.
2. Corrective substances were selected as a result of investigation. Studying of various sweeteners and their quantitative combinations led to the selection of best option: saccharin sodium with concentration 0.1 %.
Among the investigated flavoring agents «cherry» agent with concentration 0.6 % was selected.
3. Studies of oral solution in three types of primary packaging (bottles of orange glass and polyethylene terephthalate (two types)) showed the suitability of all the proposed options. This fact was confirmed by the results of determining the basic quality indicators after 3, 6, 9, 12 and 18 months of storage.
4. The research revealed the perspective of regulatory and technical documentation developing and, subsequently, of introducing the combined oral solution named «Maglycimet» into industrial production. | v3-fos-license |
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} | pes2o/s2orc | Modification of in situ Biofilm Formation on Titanium by a Hydroxyapatite Nanoparticle-Based Solution
Oral biofilms play an essential role on peri-implant disease development. Synthetic hydroxyapatite nanoparticles (nHAP) are a bioinspired material that has structural and functional similarities to dental enamel apatite and may provide preventive properties against biofilm formation. This study aimed to investigate the effects of an experimental nHAP solution on biofilm formation on polished and non-polished titanium under oral conditions. Five volunteers carried maxillary splints with non-polished and polished titanium and followed a 48 h rinsing protocol with the proposed nHAP solution, and with chlorhexidine 0.2% (CHX) and water, as controls. Samples were analyzed by fluorescence microscopy (FM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). FM showed a significant reduction of biofilms on polished samples treated with nHAP (p = 0.0485) compared with water, without differences between nHAP and CHX (p > 0.9999). Analyzing biofilm viability, polished samples rinsed with nHAP showed significantly fewer dead bacteria than CHX (p = 0.0079), but there was no significant difference in viability between polished samples rinsed with water and nHAP (p = 0.9268). A significantly higher biofilm coverage was observed on the non-polished surfaces compared to the polished surfaces when nHAP was applied (p = 0.0317). This difference between polished and non-polished surfaces was not significant when water (p = 0.1587) or CHX (p = 0.3413) rinsing were applied. SEM and TEM analysis supported the FM findings, that polished samples rinsed with nHAP presented fewer biofilm coverage compared to samples rinsed with water. In conclusion, the nHAP solution reduced the biofilm formation on polished Ti surfaces without altering bacterial viability, providing a novel approach for the management of biofilm formation on biomaterials.
INTRODUCTION
Dental implants are one of the greatest advancements in dentistry. They are a well-established and predictable method for partial and total oral rehabilitation. Titanium (Ti) is the most common material used for dental and medical implants due to its biocompatibility and mechanical properties (Muddugangadhar et al., 2015). However, there are still multiple factors that can affect the clinical success of implants, including the risk of bacterial colonization around the titanium device (Veerachamy et al., 2014). Biofilm formation usually occurs after Ti implants exposure to the oral cavity and may lead to persistent infection, playing an essential role for peri-implant disease development (Fürst et al., 2007;Veerachamy et al., 2014).
Periimplantitis is an inflammatory disease that affects the soft and hard tissue around an implant. It starts with pellicle formation followed by bacteria adhesion to the titanium surface, leading to biofilm formation. Once a mature multi-layered biofilm is formed, the bacteria are extremely resistant to conventional antimicrobial therapies and immune system lines of defense and may lead to an inflammatory response. The inflammatory process starts on the soft tissue surrounding the dental implant (peri-implant mucositis) and can evolve causing progressive loss of supporting bone, leading to implant failure (Smeets et al., 2014;Schwarz et al., 2018).
Until the present date, chlorhexidine (CHX) is still the first-choice adjunct solution for prevention of dental biofilm formation. It is widely used as a broad-spectrum antiseptic, being the gold standard in dentistry (James et al., 2017). However, despite its antimicrobial effect, CHX is not recommended for long-term use, due to various adverse effects such as teeth staining, oral mucosal erosion, and transient taste disturbance (James et al., 2017). Therefore, to achieve reasonable biofilm control and less adverse effects as possible, the search for new biomimetic materials is of utmost importance.
Research concerning the application of hydroxyapatite nanoparticles (nHAP) in dentistry has increased within the past few years. Hydroxyapatite is a calcium phosphate ceramic and the main mineral component of the tooth. Synthetic nHAP has structural and functional similarities to dental enamel apatite. It can mimic the enamel crystallites, which are the smallest building units of dental enamel, constituting the enamel prisms (Sakae et al., 2011;Kensche et al., 2017). As a bioinspired material, hydroxyapatite is non-toxic and non-immunogenic when applied in adequate doses (Epple, 2018). Kensche et al. (2017) observed that a mouthwash containing hydroxyapatite particles could reduce the number of adherent bacteria on enamel specimens, having comparable effects to chlorhexidine. Recently, Nobre et al. (2020) observed that hydroxyapatite nanoparticles could adhere not only to enamel but also to dental material surfaces, such as titanium, under oral conditions. Thus, nHAP may have preventive properties against biofilm formation also on titanium.
In this study, an in situ experimental model has been applied due to its suitability to reproduce the intraoral conditions and to understand the influence of nHAP on oral biofilm formation (Hannig et al., 2007;Hannig and Hannig, 2009).
The objective of this in situ study was to investigate the effects of a biomimetic pure nanohydroxyapatite solution on biofilm formation on titanium. The hypothesis was that this bioinspired nHAP solution would reduce biofilm formation on Ti surfaces.
Subjects
This in situ experiment evaluated the biofilm formation on titanium in five healthy volunteers aged between 28 and 35 years, who are members of the laboratory staff of the Clinic of Operative Dentistry, Periodontology and Preventive Dentistry, Saarland University. The subjects had to fulfill the following inclusion criteria: good oral health with no signs of gingivitis, caries or unphysiologically salivary flow rate; no systemic diseases; no use of antibiotics or any kind of periodontal treatment within the past 6 months; non-smoker; not pregnant or breastfeeding and absence of orthodontic appliances, confirmed after an intraoral examination and a questionnaire.
The study protocol was approved by the Medical Ethics Committee of the Medical Association of Saarland, Germany (no 283/2009-2016. Informed written consent concerning the participation in the study was obtained from all subjects.
Titanium Samples
Titanium discs (5 mm diameter; 1 mm height) with microstructured surfaces and treated with the sandblasted and acidetched (SLA) technique (Ra = 2 µm, grade 2), were obtained from Dentsply Implant Systems (Dentsply Sirona, Bensheim, Germany). Half of the samples remained unpolished, while the other half was polished by wet grinding with abrasive paper (800 to 4000 grit). To remove the resulting smear layer and for disinfection purpose, Ti discs were immersed in isopropanol (70%) for 10 min, followed by ultrasonic bath in distilled water.
Tested Solution
Hydroxyapatite nanoparticles (Kalident powder 100 nm) were supplied by Kalichem Srl, Italy. The containing nHAP test solution was prepared mixing 0.5 g powder in 10 ml bidistilled water. Chlorhexidine mouthwash [0.2% (w/v) chlorhexidine digluconate in 7% (v/v) ethanol (Saarland University Hospital Pharmacy, Homburg, Germany] and distilled water rinse (10 ml) served as positive and negative controls, respectively. The subjects used different rinsing solutions in different weeks to avoid interferences between test and control solutions, preventing a possible cross-over effect. The first solution used by each volunteer was the water control. One week later, the nHAP test solution was introduced. The volunteers performed a final rinse with the CHX control solution after an additional 2 weeks clearance period.
Oral Exposure
Titanium samples were mounted in customized maxillary splints to evaluate in situ biofilm formation (Figure 1). They were prepared from 1.5 mm thick methacrylate foils, extending from premolars to the second molar. Perforations in the buccal aspects of the splints were prepared to fix the polyvinyl siloxane impression material, in which the Ti discs were placed. Initially, four samples (2 polished and 2 non-polished) were mounted in each upper quadrant, totalizing eight samples per volunteer for each rinsing solution (Figures 1, 2).
Before intraoral exposure of the splints, the volunteers brushed their teeth without toothpaste and rinsed with tap water only to avoid possible interferences from the compounds of the toothpaste. Each volunteer performed the rinse with 10 ml of the nHAP-based solution during 30 s four times during a period of 48 h. The mouthwashes were performed 3 min, 12, 24, and 36 h after the insertion of the splint. During this experiment, the participants did not use toothpaste or any other kind of mouthwash for oral hygiene purpose. Volunteers also took off their splints during meals, brushed their teeth without toothpaste after eating to avoid any interference from the toothpaste compounds, and placed the splints again after 10 min. Splints were stored in a plastic box at 100% humidity and room temperature.
After 48 h, Ti discs were removed and immediately rinsed with distilled running water to remove non-adsorbed salivary components. Then, two samples (one each side) were prepared for fluorescence microscopy (FM) and another two (one each side) for scanning electron microscopy (SEM). The same protocol was used to prepare two additional polished samples (one each side) for transmission electron microscopy (TEM) (Figure 2).
BacLight Viability Assay
The BacLight viability assay differentiates living from dead bacteria based on two nucleic acid stains: SYTO 9 and propidium iodide. While the first one stains green all bacteria (with intact or damaged membranes), the last one stains red cells with compromised membranes. When mixed, propidium iodide reduces SYTO 9 fluorescence, enabling a viability evaluation between live and dead bacteria (Stiefel et al., 2015).
The BacLight viability assay was carried out at room temperature in a 6-well-plate. One micro-liter SYTO 9 and 1 µl PI were mixed in 1 ml saline solution (0.9% NaCl). This staining solution was vortexed before use. Ti samples were covered with 10 µl staining solution and left 10 min in a dark chamber. Subsequently, Ti discs were washed in saline solution two times, fixed to a glass slide and mounted in BacLight oil.
Fluorescence Microscopy
The detection of bacteria and their viability was conducted with FM at 1000-fold magnification (Axioskop II, ZEISS MicroImaging GmbH, Göttingen, Germany), using AxioVision 4.8 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) for image processing. Nine random representative pictures per sample were taken. Bacteria coverage was evaluated with Sefexa Image Segmentation Tool. For live and dead cell correlation, a scoring system was used.
Scoring System
The viability correlation between live and dead cells observed in the biofilm on the titanium discs samples were assessed and scored by two calibrated examiners (90% agreement rate) and it was based on the following scoring system table ( Table 1) described by Rupf et al. (2012).
Scanning Electron Microscopy
Titanium samples were also prepared for an SEM analysis to investigate biofilm coverage and to detect adherent nHAP particles. After 48 h oral exposure, Ti discs were washed with sterile water. After washing, samples were fixated with 1 ml 2% Glutaraldehyde in 0,1 M cacodylate buffer during 1 h at 4 • C. Next, samples were washed five times, 10 min each, with 1 ml of cacodylate buffer. A series of ethanol dehydration followed this procedure. Samples were immersed in various ethanol solutions accordingly: ethanol 50% (2 × 10 min), ethanol 70% (1 × 5 min), ethanol 80% (1 × 5 min), ethanol 90% (1 × 5 min) and ethanol 100% (2 × 10 min). Finally, the samples were dried in 1,1,1,3,3,3hexamethyldisilazane (HMDS, Acros Organics, Geel, Belgium). HMDS was vaporized at room temperature in a clean bench. Finally, Ti discs were left to airdry overnight at room temperature TABLE 1 | Scoring system for the assessment of biofilm viability detected with the BacLight viability assay.
1
Mainly red fluorescence; ratio between red and green fluorescence 90:10 and higher.
2
More red fluorescence; ratio between red and green fluorescence 75:25 and higher.
4
More green fluorescence; ratio between red and green fluorescence 25:75 and lower.
5
Mainly green fluorescence; ratio between red and green fluorescence 10:90 and lower.
in the air chamber. Samples were attached to aluminum stubs, sputtered and coated with carbon. SEM and EDX evaluations were made in an XL30 ESEM FEG (FEI, Eindhoven, Netherlands) at 5 and 10 kV, consecutively, at up to 20,000-fold magnification.
Transmission Electron Microscopy
To better understand the test solution effects on biofilm formation, it was also decided to investigate the ultrastructural characteristics of the obtained biofilm by TEM. Immediately after volunteers took off their splints, Ti discs were washed with sterile water to remove not adhered bacteria. Then, the specimens were placed in 1,5 ml tubes with 1 ml 1% Glutaraldehyde fixing solution at 4 • C during 1 h. After primary fixation, samples were washed with cacodylate buffer 0.1 M 4 times, 10 min each, and stored at 4 • C in cacodylate buffer. Ti discs were subjected to a secondary fixation in osmium tetroxide during 1 h in a dark chamber at room temperature, followed by five times 10 min wash in distilled water and immersion in 30% ethanol overnight. Following the TEM preparation procedures, dehydration was performed at room temperature. Samples passed through series of 50% (2 × 10 min), 70% (2 × 20 min), 90% (2 × 30 min), and 100% (2 × 30 min) ethanol. Finally, Ti discs were further immersed in 100% acetone two times, 30 min each, and stored overnight in an acetone/Araldite (Electron Microscopy Sciences, United States) mixture plus 3% accelerator (mixture A) at room temperature. On the following day, mixture A was poured out, and a second mixture, mixture B was prepared (Araldite mixture with 2% accelerator). Samples were left again overnight in mixture B at room temperature in the air chamber. Next, a new mixture B was used to fill half of the embedding forms. Notes with identification number were placed at the bottom side. Ti discs were placed, and the embedding forms was filled until the top again with mixture B. Then, samples were incubated for polymerization for 48 h at 65 • C. After polymerization, Ti was removed by treatment with hydrofluoric acid (5%) during 48 h, and the specimens were re-embedded in Araldite.
Finally, samples were cut in ultra-thin sections in an ultramicrotome with a diamond knife (Leica EM UC7, Germany) and mounted on Pioloform-coated copper grids and contrasted with aqueous solutions of uranyl acetate and lead citrate at room temperature. After an intensive wash with distilled water, biofilm inner and out layers could be then analyzed with a TEM Tecnai 12 BioTwin (FEI, Eindhoven, Netherlands) under a magnification up to 100.000-fold.
Statistics
The mean values were analyzed using GraphPad Prism 6. Mann-Whitney test was performed to evaluate the differences between polished and non-polished titanium samples for each solution used, and Kruskal-Wallis test with Dunn's correction for multiple comparisons test to access the differences between all polished samples in all solutions. Statistical significance was considered for p < 0.05.
Fluorescence Microscopy BacLight Assay
Biofilms were formed within 48 h in all samples, regardless of the solution used. However, there were variations concerning the quantity and viability of biofilms that covered at the Ti surfaces and the bacterial viability.
Biofilm Coverage
As shown in Figures 3, 4, samples rinsed with water presented a thick biofilm layer, covering the majority of the Ti surfaces after 48 h. There was no significant difference between polished and non-polished surfaces when rinsed with water (p = 0.1587) or with CHX (p = 0.3413). However, the difference was significant between polished and non-polished samples rinsed with nHAP (p = 0.0317). Furthermore, another predictable result was the significantly lower biofilm coverage after treatment with CHX 0.2% when compared with the water rinse (p = 0.0215) on polished samples. Similar results were achieved comparing nHAP test solution and water (p = 0.0485) but with no significant difference between nHAP and CHX (p > 0.9999).
Biofilm Viability
Scoring was performed to analyze the biofilm viability ( Figure 5 and Table 1). There was no significant difference between polished and non-polished samples rinsed with water or nHAP concerning the bacteria viability, presenting a majority of live bacteria. However, it could be observed significantly more live bacteria on non-polished samples rinsed CHX than on polished samples rinsed with the same solution (p = 0.0476). Regarding the polished samples, significantly more live bacteria could be seen after water rinsing, when compared with CHX (p = 0.0037). However, no significant difference between polished samples rinsed with water and with nHAP could be detected (p = 0.9268). Samples rinsed with CHX showed a significant reduction in the number of vital bacteria compared with nHAP rinsed samples (p = 0.0079).
Scanning Electron Microscopic Analysis
Scanning electron microscopy analyses were performed on two types of surfaces: polished and non-polished titanium (Figure 6). The 48-hour biofilm presented on titanium surfaces samples from healthy volunteers were associated predominantly with coccoid or rod-shaped bacteria. These bacteria were distributed randomly on the titanium surfaces as individual bacteria or colonies (Figure 7). These observations were independent of the surface type.
Independent of the surface topography, Ti samples from the water control had thicker biofilm covering compared to nHAP and CHX samples (Figure 8). Polished samples treated with hydroxyapatite or chlorhexidine solutions presented areas with thin biofilm layers and areas without biofilm (Figure 8). Therefore, SEM analysis corroborated the FM results, indicating that the nHAP rinsing solution reduced the amount of mature biofilm formed on polished Ti surfaces. FIGURE 4 | Biofilm coverage of the polished samples (P): chlorhexidine 0.2% (CHX) and hydroxyapatite nanoparticles-based solution (nHAP) reduced the bacterial adherence on Ti discs surfaces compared to water. There was no significant difference between the gold standard CHX and the nHAP test solution. Comparing polished and non-polished samples (NP), the only significant difference was between P and NP samples when nHAP solution was used.
FIGURE 5 | Biofilm viability: chlorhexidine (CHX) significantly reduced the number of live bacteria compared to the negative control on polished (P, p = 0.0037) and on non-polished (NP, p = 0.0079) samples. Non-significant reduction of viability between hydroxyapatite nanoparticles-based solution (nHAP) and water samples was present on P (p = 0.9268) and NP (p = 0.1667) titanium discs. Furthermore, Figure 9 shows that hydroxyapatite nanoparticles and aggregates could be detected randomly on the pellicle surface after 48 h of intraoral exposure when rinsing with the nHAP test solution. Energy-dispersive X-ray spectroscopy (EDX) analysis endorsed the SEM results, confirming the presence of hydroxyapatite through elements quantification.
Transmission Electron Microscopic Analysis
Transmission electron microscopic micrographs at 30.000-fold also show a higher number of bacteria in samples rinsed with water (Figure 10). On Figures 10B, 11, some small black spots scattered randomly on the samples rinsed with hydroxyapatite nanoparticles-based solution were detected.
DISCUSSION
This study evaluated the differences between biofilm formation under the influence of water, chlorhexidine, and an experimental hydroxyapatite nanoparticles-based solution as oral rinsing adjunct treatment on polished and non-polished titanium surfaces. Applications of the 5% watery nHAP solution could reduce the biofilm coverage of the polished titanium surfaces. In contrast to rinsing with CHX, live bacteria were present on nHAP rinsed samples, pointing to a rather biofilm modifying than an antibacterial effect.
An in situ experimental biofilm model was applied because of its capacity to reproduce the intraoral in vivo biofilm formation, which comprehend a dynamic and multifactorial process. According to Hannig and Hannig (2009) the biofilm formation process occurs differently under in vitro and in vivo conditions. In vitro and in situ models are selected according to the research question. For the topic investigated here, an in situ model was more suitable, since the active rinsing process cannot be performed in vitro. Furthermore, the antimicrobial defense mechanisms of saliva in an in situ model correspond to the real situation. Another advantage of the in situ approach is that it is possible to analyze vital biofilms with fluorescence microscopy (Hannig et al., 2007). Therefore, the in situ model seems to be suitable to understand the intraoral biofilm formation process properly. Intraoral removable splints were applied to proceed with the in situ investigation. This methodology has been used in many previous studies with excellent results (Hannig et al., 2007;Hertel et al., 2016Hertel et al., , 2017Kensche et al., 2017;Nobre et al., 2020). FIGURE 6 | SEM figures at 5,000-fold magnification from original titanium specimens not exposed to the oral cavity polished by wet grinding with abrasive paper from 800 to 4000 grit (A) and without polishing (B). Additionally, the acrylic appliance is a convenient method for subjects, since it is easily removable during mealtimes or for oral hygiene purposes, not affecting the biofilm formation on titanium samples (Hannig, 1999). To avoid the influence of oral hygiene and diet on biofilm formation, the participants were instructed to remove the splints from the oral cavity and store them in a humid atmosphere. This avoided drying of the biofilms and minimized the influence of this interruption of the experiment. The rinsing solutions were applied in the order of their assumed effect on the biofilm: first water, then nHAP and finally chlorhexidine. In contrast to chlorhexidine, no comparable substantivity has been described for nHAP so far.
According to the SEM results, no differences in biofilm density were visible when comparing polished and non-polished samples after water rinsing. Some subjects had a slightly lower biofilm amount on polished samples, but the Ti discs presented a complex and multilayer biofilm for both types of surfaces. These small variations in biofilm formation may be due to individual factors, such as salivary flow rate, saliva composition, or dietary habits (Marsh, 2012). The biofilm could develop to advanced stages, because there were no external factors like antibacterial agents or mechanical cleaning to disturb it (Kreth et al., 2009).
Fluorescence microscopy and SEM results (Figures 3, 8) revealed a significantly denser and multilayered biofilm on the non-polished rough samples rinsed nHAP solutions when compared to the polished samples. Other in vivo studies have already reported this relationship between surface roughness and biofilm formation (Quirynen et al., 1990;Bollen et al., 1996;Rimondini et al., 1997;Al-Ahmad et al., 2010;Burgers et al., 2010). The increase of titanium surface roughness is directly related not only to a higher rate of biofilm formation but also to a better osseointegration (Teughels and Van Assche, 2006;do Nascimento et al., 2008). Increased roughness of titanium surfaces provides better growth of fibroblasts on the Ti substrate, establishing better osseointegration with substantial epithelial soft tissue seal around the implant Quirynen et al., 1996). However, this irregular topography facilitates bacterial adhesion and colonization (Dhir, 2013). To solve this problem, previous studies suggested a surface roughness threshold Ra value of 0.2 µm: when Ra > 0.2 µm, the biofilm formation is facilitated, whereas an Ra < 0.2 µm does not promote the biofilm formation but still supply an irregular surface proper to fibroblast fixation (Buser et al., 1991;Bollen et al., 1996;Quirynen et al., 1996). The Ra value of the nonpolished titanium samples used in this study was 2 µm according to the manufacturers' information. This is the typical roughness of the endosseous parts of many dental implants. It may explain the higher biofilm coverage of the non-polished titanium samples mainly due to their roughness, offering an attractive microstructured surface to bacteria.
Concerning the microbial morphology, similar morphological patterns were found on polished and non-polished titanium samples in all volunteers. Coccoid shaped bacteria were present in a higher proportion, but rods could also be seen. This result agrees with the literature since gram-positive cocci and rods are the early colonizers on titanium surfaces (Steinberg et al., 1995). As observed in previous studies, there was no difference between rods and cocci proportions on both rough and smooth titanium surfaces, but a difference in thickness and biofilm density was visible (Foster and Kolenbrander, 2004;Al-Ahmad et al., 2010).
Interesting results are detected in the present study when the polished samples were analyzed. As expected, positive control samples rinsed with chlorhexidine presented a thin biofilm layer and areas without microorganism, endorsing the well-consolidated antibacterial properties of the gold standard chlorhexidine (James et al., 2017). Similar biofilm distribution was also visible in samples rinsed with the watery hydroxyapatite solution. This result may be due to a significant biofilmformation reducing effect already shown by Kensche et al. (2017) on enamel surfaces. SEM and TEM results also confirm that hydroxyapatite nanoparticles are randomly distributed over the titanium surface, which was also demonstrated by Kensche et al. (2017). Hydroxyapatite crystallites measuring 90 to 500 nm could be identified even 12 h after the last rinse. Previous studies had also observed the hydroxyapatite particle accumulation, but on enamel surfaces and not for such a long time after the last rinsing (Hannig et al., 2013a;Kensche et al., 2017). In a recently published study using the same nHAP-based mouthrinse, it was observed that HAP nanoparticles could adhere to titanium surfaces, forming a heterogeneous layer within 2 h of intraoral exposure. In the present study the heterogeneous pattern continued to exist when the samples were removed 48 h after the beginning of the experiment (Nobre et al., 2020).
Fluorescence microscopic results of the present study demonstrated that application of CHX as a mouthwash revealed the most effective bactericidal effects, significantly reducing the vital biofilm bacteria on titanium surfaces compared to water and hydroxyapatite solutions, which is in accordance to previously published data (Hannig et al., 2013a,b;Kensche et al., 2017). This finding is related to the various chlorhexidine properties, such as broad-spectrum bacteriostatic and bactericidal effects, and substantivity, which makes it the gold standard solution on reducing bacterial vitality and biofilm formation (Hannig et al., 2013b;James et al., 2017).
Concerning biofilm coverage, nHAP was as effective as the CHX mouthrinse in decreasing biofilm formation, with no significant difference between both groups (p > 0.9999), demonstrating that oral rinsing with a watery solution of hydroxyapatite nanoparticles significantly reduces the number of bacteria which adhered on polished titanium surfaces.
On the other hand, fluorescence microscopic images also revealed a significantly higher number of dead bacteria after treatment with CHX compared with nHAP rinsed samples (p = 0.0079). Similar results were already shown by Kensche et al. (2017) on enamel surfaces. The presence of live bacteria observed in the FM investigation suggests that nHAP has a rather modifying and reducing than an antibacterial effect on biofilm formation. However, when considering the present results, it is important to mention that the effect of reduced biofilm formation by the nHAP was observed on polished titanium surfaces, but not on non-polished surfaces. The less pronounced effects on rough surfaces point to a limited clinical application of nHAP in biofilm management on complex implant prosthodontics suprastructures.
According to the scarce literature in this matter, the biofilm reducing effect of hydroxyapatite is related to its particle sizes (Sakae et al., 2011;Kensche et al., 2017). This size effect facilitates the direct interaction with the bacteria, meaning that nano and sub-micron hydroxyapatite particles can interact with adhesins on the bacterial membrane, reducing the bacterial adherence (Venegas et al., 2006;Kensche et al., 2017). Kensche et al. (2017) also suggested another mechanism of action for the anti-biofilmformation properties of hydroxyapatite particles. Hydroxyapatite particles accumulated on the pellicle-covered titanium surface could hamper the bacterial attachment to pellicle receptors blocking the interaction with cell wall adhesins from bacteria. This effect would decelerate bacterial adhesion, reducing the biofilm formation, as shown in this study (Kensche et al., 2017). In addition, it was recently observed that HAP nanoparticles can interact with the pellicle formed on titanium surface through bridge-like structures (Nobre et al., 2020). Thus, both mechanisms of nano/microparticle accumulation and receptor sites inhibition could be the reason for the higher number of dead bacteria observed by FM after nHAP solution rinsing when compared with the control water rinse.
Despite being the gold standard adjunct solution for biofilm control, long-term use of CHX is not indicated due to its well-known side effects such as teeth staining, oral mucosal erosion, and transient taste disturbance (James et al., 2017). On the other hand, after rinsing with the nHAP solution, volunteers reported an acceptable taste and no side effects during the study. Furthermore, hydroxyapatite nanoparticles mimic the dental enamel structure, and for being a biocompatible solution, side effects are unlikely to occur (Epple, 2018). Additionally, results suggested that nHAP had biofilm-formation modulating but not antimicrobial effects, therefore, the tested solution is less likely to impact on the oral cavity homeostasis. Finally, literature states that hydroxyapatite particles are dissolved in gastric fluid in case of ingestion, not being harmful to the human body (Kensche et al., 2017).
The small number of subjects involved in the present study was a limitation of this investigation. The complexity of the in situ methodology and the electron microscopic techniques used for biofilm analyses were the reasons to select a small number of volunteers, such as in previous studies with similar methods (Al-Ahmad et al., 2009;Jung et al., 2010;Kensche et al., 2017).
Another interesting question is the influence of nHAP on the microbial diversity of oral biofilms. Studies on this issue would also require a higher number of volunteers. Depending on the methodology, culture or sequencing techniques, sufficient biofilm mass would have to be generated. This should also be a topic of a follow up study.
The results of this investigation indicated that the experimental 5% solution of pure hydroxyapatite nanoparticles reduced in situ oral biofilm formation on titanium surfaces, representing a novel bioinspired approach for biofilm management without altering bacterial viability. Additionally, independent from the rinsing solution used, a thicker and multilayer biofilm coverage was present on the non-polished samples. Thus, titanium surface morphology reveals a strong impact on bacterial colonization.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Medical Association of Saarland, Germany. The participants provided their written informed consent to participate in this study. | v3-fos-license |
2019-08-31T14:20:00.379Z | 2019-08-31T00:00:00.000 | 201675910 | {
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} | pes2o/s2orc | Viral-mediated gene delivery of TMBIM6 protects the neonatal brain via disruption of NPR-CYP complex coupled with upregulation of Nrf-2 post-HI
Background Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress play a major role in the pathogenesis of neonatal hypoxic-ischemic (HI) injury. ER stress results in the accumulation of unfolded proteins that trigger the NADPH-P450 reductase (NPR) and the microsomal monooxygenase system which is composed of cytochrome P450 members (CYP) generating reactive oxygen species (ROS) as well as the release of inflammatory cytokines. We explored the role of Bax Inhibitor-1 (BI-1) protein, encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, in protection from ER stress after HI brain injury. BI-1 may attenuate ER stress-induced ROS production and release of inflammatory mediators via (1) disruption of the NPR-CYP complex and (2) upregulation of Nrf-2, a redox-sensitive transcription factor, thus promoting an increase in anti-oxidant enzymes to inhibit ROS production. The main objective of our study is to evaluate BI-1’s inhibitory effects on ROS production and inflammation by overexpressing BI-1 in 10-day-old rat pups. Methods Ten-day-old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5 h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48 h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). BI-1 and Nrf-2 silencing were achieved by icv injection at 48 h pre-HI using siRNA to elucidate the potential mechanism. Percent infarcted area, immunofluorescent staining, DHE staining, western blot, and long-term neurobehavior assessments were performed. Results Overexpression of BI-1 significantly reduced the percent infarcted area and improved long-term neurobehavioral outcomes. BI-1’s mediated protection was observed to be via inhibition of P4502E1, a major contributor to ROS generation and upregulation of pNrf-2 and HO-1, which correlated with a decrease in ROS and inflammatory markers. This effect was reversed when BI-1 or Nrf-2 were inhibited. Conclusions Overexpression of BI-1 increased the production of antioxidant enzymes and attenuated inflammation by destabilizing the complex responsible for ROS production. BI-1’s multimodal role in inhibiting P4502E1, together with upregulating Nrf-2, makes it a promising therapeutic target. Electronic supplementary material The online version of this article (10.1186/s12974-019-1559-4) contains supplementary material, which is available to authorized users.
Background
Hypoxia-ischemia (HI) in the perinatal period is associated with long-term disabilities affecting 1-4 infants per 1000 births [2,13,40]. The most common cause of HI is intrauterine asphyxia, which may be brought on by placental artery clotting, abruption, or inflammatory processes [7]. In the event of prolonged abruption and extended period of HI, the neonate develops hypoxic-ischemic encephalopathy (HIE), causing irreversible brain injury [7].
A main contributing injury mechanism post-HI is the disruption of correct protein folding that subsequently triggers reactive oxygen species (ROS) accumulation, microglia activation, and inflammation [8,14,44]. Previous reports have shown inflammatory cytokine release to be significantly elevated in the full-term infant post-HIE, which is linked with further exacerbating the damage and results in poor neurodevelopmental outcome [7]. Therefore, inhibition of inflammation is an attractive target for new therapeutic strategies.
Correct folding of transmembrane proteins takes place in the endoplasmic reticulum (ER) which is composed of an elaborate system of chaperones and enzymes [37]. However, under stressful conditions, such as after HI injury, the number of unfolded proteins exceeds the capacity of the chaperones leading to the accumulation of unfolded proteins and ER stress [33]. The ER responds to this by activating the unfolded protein response (UPR), which triggers sensor proteins to recognize and ameliorate ER stress [33,47,50]. However, persisting ER stress leads to the over-activation of the UPR, a highly redox-dependent pathway that causes accumulation of ROS [15]. ROS are a natural byproduct of signaling pathways; however, during stress, ROS production is increased significantly, which causes oxidative stress and damage to the cell [15].
A major source of ROS production at the ER is from the microsomal monooxygenase (MMO) system which is composed of cytochrome P450 (CYP), NADPH-P450 reductase (NPR), and phospholipids [12,15,32]. Specifically, cytochrome P4502E1, a member of CYP, is associated with the production of large amounts of ROS, due to the leakage of electron transfer between P4502E1 and NPR, thus indicating an important role of these cytochromes during ER stress [15,32].
Bax Inhibitor-1 (BI-1) is an evolutionary protein, encoded by the TMBIM6 gene, which mainly resides on the ER membrane. BI-1 is a member of the TMBIM family, associated with cytoprotection [45,49], and has been suggested to regulate ER stress-induced ROS production and subsequent inflammatory cytokine production via two essential mechanisms [19]. First, BI-1 can directly inhibit ROS by disrupting the NPR-CYP complex, a major generator of ROS. BI-1 alters the electron flow from P4502E1 to NPR, thus destabilizing this complex and attenuating ROS accumulation [12,15,17]. The P4502E1 member was found to be significantly reduced and its upregulation attenuated after ER stress in BI-1 overexpressing cells [15]. Second, BI-1 can directly increase the production of anti-oxidant transcription factors such as nuclear factor erythroid 2-related factor 2 (Nrf-2) [17,19]. Nrf-2 stimulates the production of antioxidant enzymes, heme oxygenase-1 (HO-1), which in turn blocks ROS, thereby attenuating inflammation and promoting cell survival. Cells that overexpressed BI-1 increased Nrf-2 and HO-1 expression while its protective effects were abrogated after inhibition of HO-1 [17].
The specific objective of this study was to establish that overexpression of BI-1 protein, with adenoviral-TMBIM6 vector, can attenuate the morphological and neurological consequences postneonatal HI via disruption of the NPR-CYP complex coupled with upregulation of Nrf-2 and HO-1, thus attenuating oxidative stress. HO-1 is a rate-limiting enzyme shown to be able to attenuate ROS accumulation. Increased levels of HO-1 may limit oxidative dysregulation which causes misfolding of ER proteins thereby decreasing the UPR. Our main novelty lies in the ability to upregulate BI-1 protein using an adenoviral vector carrying the TMBIM6 gene to induce overexpression of BI-1 in an in vivo HIE model and the mechanisms involved.
Given the lack of effective treatment options for neonatal HI injury, we hope to establish a novel role for BI-1 protein and ER stress in the pathophysiology of neonatal HI injury and help leverage this new understanding to design interventions that affect the outcome of neonatal HI patients. This work is essential and may help to change the clinical management for HI patients and provide a foundation for future research in other related diseases with similar pathologies.
In vivo experiments
All protocols were approved by the Institutional Animal Care and Use Committee of Loma Linda University and with NIH guide for the Care and Use of Laboratory Animals. The animals were cared for and all studies conducted in accordance with the US Public Health Service's Policy on Humane Care and Use of Laboratory Animals. Sprague-Dawley rat mothers, with litters of 10~12 pups, were purchased from Harlan Labs (Livermore, CA). All experiments adhere to the ARRIVE guidelines for reporting animal studies. A total of 166 unsexed rat pups weighing 15-20 g were used and kept in a temperature-controlled environment with regular light/ dark cycle until they were ready for surgery at 10-day-old post-birth (P10). All rats were randomly assigned to the experimental groups which are shown in Additional file 1: Experimental Design.
Hypoxic-ischemic rat model
Hypoxic-ischemic (HI) injury was induced as previously described following the well-established Rice Vannucci model [41,42]. Briefly, P10 unsexed rats were anesthetized with 3% isoflurane gas in the air and maintained at 2.5% isoflurane during surgery. Once the rat was fully anesthetized and unresponsive, the rat's neck was prepared and draped using standard sterile techniques. A small midline neck incision on the anterior neck was made with a No. 11 blade surgical knife (approximately 3-5 mm in length). The right carotid artery was dissected, isolated from surrounding structures, and double ligated with 5-O surgical suture and cut between the ligations. The animal's skin was sutured back to close the incision. Throughout the surgical and postoperative period, the temperature was controlled with heating blankets and incubators. Rats were then allowed to recover for 1 h on a heated blanket and then placed in a 2 L Erlenmeyer airtight flask, which was partially submerged in a 37°C water bath to maintain a constant thermal environment. Rat pups were exposed to a gas mixture of 8% oxygen and 92% nitrogen for 90 min. Thereafter, the animals were returned to their mothers and monitored daily.
Time course evaluation of proteins
Time course expression of endogenous BI-1, P4502E1, NPR, pNrf-2, and HO-1 levels were measured at 6 h, 12 h, 24 h, and 72 h post-HI by western blot. Rats were randomly divided into groups, and the ipsilateral brain hemispheres were collected for western blotting. Sham animals underwent surgery; however, the artery was only isolated without being ligated or cut, and pups were euthanized at 72 h post-HI.
Viral administration
Human adenoviral-TMBIM6 vector (Ad-TMBIM6) (Vector Biolabs) was injected intracerebroventricularly (icv) at 2 μL containing 1.6 × 10 11 PFU/mL or 1.7 × 10 11 PFU/mL per injection [3] at 48 h pre-HI. For ICV administrations, rat pups were anesthetized with isoflurane and their heads placed in a stereotaxic head frame. Scalps were incised, and a burr hole (1 mm) drilled at 1.5 mm rostral and 1.5 mm lateral/right of bregma. A Hamilton syringe was inserted to a depth of 1.7 mm below the dura and a microinfusion pump infused 2 μL (0.1 μL/min) of the virus. The needle was then removed over 10 min after completion of the infusion, and the burr hole plugged with bone wax. As a control for the viral vector, rats were infused with Ad-GFP with similar concentrations. The control group is referred to as vehicle.
RNAi administration
Rats were anesthetized and placed on a stereotactic frame. Two microliters of BI-1 siRNA (300 pmol/μL, Sigma-Aldrich) or Nrf-2 siRNA or scramble siRNA (300 pmol/μL, Santa Cruz) were administered icv using a Hamilton syringe (10 μL, Hamilton Co) into the right lateral ventricle (1.5 mm posterior, 1.5 mm lateral to bregma and 1.7 mm down from the surface of the brain) at 48 h pre-HI at a rate of 0.3 μL/min [34]. The needle was left in place for 10 min after administration was completed and was then withdrawn slowly over 5 min to prevent backflow. The hole was sealed with bone wax, and the skin was sutured.
Infarct area measurements TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was performed at 72 h post-HI to determine the percentage of the infarcted area as previously described [25]. Animals were anesthetized, the brains were removed and sectioned into 2-mm slices. A total of five to six slices were cut per brain and were then immersed in 2% TTC solution until the brains turned pink-red (~5 min) at room temperature [20,53]. Slices were then washed in PBS and fixed overnight in 10% formaldehyde solution and imaged. Image J software was used to calculate the percentage of the infarcted area.
The area of each slice was calculated using the formula: ((area of contralateral hemisphere − area of the non-infarcted ipsilateral hemisphere)/2 × (area of contralateral hemisphere))× 100 [5,48]. The average of all five slices was taken as a representative of the percentage of infarcted areas for each animal. All experiments were performed in an unbiased blinded fashion.
Western blotting of brain tissue samples
Western blotting was performed at 6 h, 12 h, 24 h, and 72 h post-HI as previously described [10,21,34,46]. Rats were anesthetized and transcardially perfused with 100 ml ice-cold PBS (pH 7.4). The brain was isolated and divided into contralateral and ipsilateral hemispheres, then immediately snap-frozen in liquid nitrogen, and stored in − 80°C for further use. Western blot samples were prepared by homogenizing the ipsilateral hemispheres in RIPA lysis buffer (Santa Cruz Biotechnology) for 15 min followed by centrifugation, 14,000×g at 4°C for 30 min. The supernatant was collected, and protein concentration was measured using a detergent compatibility assay (catalog number: 5000112, DC™ Protein Assay Kit II, Bio-Rad, USA).
Immunofluorescence staining Rats were anesthetized at 72 h post-HI and transcardially perfused with PBS and 10% formalin. Brain sections were then post fixated with formalin overnight and dehydrated in 30% sucrose solution for 3-5 days after which brains were frozen in OCT. Immunofluorescence staining was performed as previously described [34]. The tissue mounted on the slides was washed with 0.1 M PBS three times for 5 min and then incubated in 0.3% Triton X-100 in 0.1 M PBS for 30 min at room temperature. The tissue was then washed once more by 0.1 M PBS for 5 min, three times, and primary antibodies were applied overnight at 4°C: anti-BI-1 antibody (1:100, Abcam), anti-P4502E1 antibody (1:100, Abcam), anti-NPR (1:100, Abcam), anti-Nrf-2 (1: 100, Abcam), anti-HA tag (1:50, Abcam), anti-Iba-1 (1: 100, Abcam), anti-Il-1β (1:100, Abcam), and anti-MPO (1: 100, Abcam). After washing with PBS, sections were incubated with appropriate secondary antibodies: anti-rabbit IgG-TR, anti-mouse IgG-FITC, anti-goat IgG-FITC, and anti-rabbit IgG-FITC (1:200) for 2 h at RT. Finally, slides were mounted using Vectashield Antifade with DAPI (catalog number: H-1200, Vector Laboratories Inc., USA) and visualized under a fluorescent microscope (Leica DMi8, Leica Microsystems), and Magna Fire SP system (Olympus) was used to analyze microphotographs. Quantification of Il-1β and MPO positive cells was manually counted in the peri-ischemic regions. A total of six sections/brain were averaged and expressed as the ratio of positive cells to total cells (percentage).
DHE staining
To evaluate ROS production, we stained brain slices with dihydroethidium (DHE) stain [31]. Slides were prepared as described above.
A stock solution of DHE was prepared by diluting a total of 25 g of DHE (Invitrogen) solid in 1 mL methanol. The working reagent was prepared by taking 25.3 μL of stock solution and diluting it in 1 mL methanol. After washing slides in PBS 1 × 10 min, a working reagent of DHE was applied to each brain section and slides were let stand in a dark room for 30 min. Slides were then washed in PBS 3 × 10 min, dried off, and then mounted using DAPI and coverslip. A Leica DMi8 fluorescent microscope was used to visualize the slides.
Behavioral analysis
Neurobehavioral tests were performed at 4 weeks using rotarod and water maze tests.
Rotarod The rotarod assesses motor impairment. Animals were placed on a spinning bar to test their motor function. Rats underwent stationary, constant (5 rpm), and 5 rpm with 2 rpm acceleration tests. The latency to fall was recorded for each test [4,11].
Water maze Morris water maze test assesses learning, memory, and visual functions [4,11]. The activity of animals' swim paths was recorded and measured for quantification of distance, latency, and swimming speed for 5 days using a Video Tracking System SMART-2000 (San Diego Instruments Inc., CA).
In vitro experiments
Rat microglial immortalized cell line, HAPI (Millipore Sigma) [54], was used at passage six through nine. Cells were cultured in FK12 media enriched with 15% horse serum, 2.5% FBS and 5 mL of penicillin was added. Cells were then placed in an incubator at 37°C receiving 5% CO 2 and 95% oxygen and allowed to grow till they reached 70% confluency with replacing media every 3 days. Cells were passaged before use for experiments. For Western blot, cells were plated at a density of 200,000 cells/well in a six-well plate.
Primary microglial cells were prepared from newborn rats at postnatal day 1 according to protocol and a previous study [22,52]. Briefly, brain cortices were collected and kept in HBSS solution. Once dissected, tissue was placed in trypsin-EDTA for 15 min to digest it. F12 media with 15% FBS was used to neutralize the trypsin and stop the enzymatic reaction. The cell suspension was then strained through a 200-μm filter and plated on poly-ornithine-coated flasks and placed in an incubator at 37°C and 5% CO 2 and 95% oxygen. Cells were allowed to grow and attach with replacing media every 3 days. Primary microglial cells were harvested via mechanical agitation for 2 h at 10 days post initial plating. The isolated cells were then plated at the desired density in six-well plates coated with poly-ornithine for western blotting and immunofluorescence staining. For western blot, cells were plated at a density of 200,000 cells/well in a six-well plate. For immunofluorescence, cells were plated at a density of 2.5 × 10 4 cells/18-mm coverlid.
OGD Cells were grown in full growth media which was replaced with glucose-deprived media prior to being placed in a chamber. Cells were placed in a hypoxic chamber and flushed with 1% oxygen for 3 h. Exposure of cells to 3 h of OGD was chosen from our preliminary results (data not shown). Media was removed, and complete growth media was re-introduced to the cells. Cells were left in an incubator to recover for 18 h after which were prepared for cell death assay and western blotting [1,36].
Cell death assay
To determine the percentage of viable cells, we used trypan exclusion as previously described [36]. Briefly, cells were scraped from the plates, centrifuged for 5 min, and then re-suspended in 10 mL complete growth media. Equal volumes of cell suspension were added to trypan blue; cells were then vortexed and let stand for 3 min. Ten microliters from the mixture was placed on cell counter slides, and slides were read using an automated cell counter using an average of six counts [35].
Western blotting for cells
Cells were collected 18 h after OGD and stored for western blotting as previously described [6,36]. Briefly, cells were scraped, centrifuged, and then re-suspended in 1 mL PBS, after which they were centrifuged one more time at 14,000 rpm for 5 min. The remaining supernatant was removed after centrifugation, leaving only the pellet. RIPA lysis buffer was added with protease inhibitor cocktail and pipetted thoroughly until the pellet was fully dissolved. This suspension was then left on ice for 20 min following which it was centrifuged for 30 min. The supernatant was harvested while the pellet was discarded. Protein concentration was measured using Bradford assay, and SD-PAGE electrophoresis was performed as previously described [6]. Primary antibodies, BI-1 antibody (1:200, Abcam), anti-P4502E1 antibody (1:1000, Abcam), anti-Nrf-2 antibody (1:1000, Abcam), anti-TNFα antibody (1:1000, Abcam), anti-IL-6 antibody (1: 500, Abcam), anti-Il-1β antibody (1:500, Abcam), and actin (1:4000, Santa Cruz) were applied to the membrane and left overnight.
Calculating MOI
The Multiplicity of Infection (MOI) was calculated to determine the total amount of infectious particles needed to infect one cell. MOI was calculated as follows: no. cells × desired MOI = total PFU (or plaque-forming units) needed; (total PFU needed)/(PFU/mL) = total milliliters of virus needed to reach your desired dose. A 100 MOI of Ad-TMBIM6 virus was chosen as the optimal amount needed to infect cells and provide protection from our preliminary results (data not shown).
siRNA transfection
Cells were allowed to differentiate and reach 80% confluency in poly-D-lysine-coated six-well plates prior to transfection. Both BI-1 and Nrf-2 siRNAs were prepared according to the manufacturer's protocol (Sigma-Aldrich); a stock solution of 10 μM siRNA was prepared. From the siRNA stock, 4 μL was taken and mixed with 125 μL Opti-MEM. In a separate tube, 7 μL lipofectamine 3000 was mixed with 125 μL Opti-MEM. The solution from both tubes was combined and mixed well and left to sit at room temperature for 15-45 min. Growth media was removed from cells and replaced with 250 μL/well siRNA transfection solution as prepared above. An additional 1 mL Opti-MEM was added to each well and cells were then placed in an incubator for 5-7 h, following which media was removed and replaced with normal growth media.
Statistical analysis
Statistical analysis was performed using one-way analysis of variances (ANOVA), followed by Tukey's test using Graph Pad Prism (Graph Pad Software, San Diego). All values were presented as mean ± standard deviation (SD), with a p value less than 5% considered significant. Sample sizes were determined by the power of 0.8, α = 0.05, and a 20% standard deviation from the preliminary results. The estimated sample size was 6-8/group for in vivo studies and 4/group for cell culture studies. All rats were randomly assigned to each group, and all the investigators were blinded. Please refer to Additional files 4, 5, 6, 7, 8, 9, and 10 for detailed statistical analysis.
Time course expression of endogenous proteins post-HI
Endogenous expression of BI-1, NPR, P4502E1, pNrf-2, and HO-1 were measured at 6 h, 12 h, 24 h, and 72 h in the ipsilateral cerebral hemispheres post-HI. Results showed that BI-1 levels significantly increased at 24 h post-HI and returned to sham levels at 72 h post-HI (p < 0.05, Fig. 1a, b). There were no significant changes for NPR expression levels (Fig. 1b). P4502E1 significantly increased in a time-dependent manner, peaking at 72 h post-HI (p < 0.05, Fig. 1c). pNrf-2 showed a tendency to decrease in a time-dependent manner from 6 h to 72 h post-HI, and HO-1 levels significantly decreased from 6 h to 72 h, reaching significantly lower expression levels at 72 h post-HI compared to sham (p < 0.05, Fig. 1d, e). Please refer to Additional file 4 for detailed statistical analysis.
Low dose Ad-TMBIM6 vector reduced infarct area at 72 h post-HI Two doses of the viral vector (1.6 × 10 11 PFU/mL or 1.7 × 1011 PFU/mL) were injected intracerebroventricularly (icv) at 48 h pre-HI, and infarct percentage was measured at 72 h post-HI. TTC staining results indicated that the low dose (1.6 × 10 11 PFU/mL) viral vector was more effective as it significantly reduced the percent infarcted area when compared to either vehicle or the high dose vector (1.7 × 1011 PFU/mL, p < 0.05, Fig. 2a).
Quantification data of western blot bands showed that there was a significantly higher amount of Ad-TMBIM6 present at 72 h post-HI versus the sham or vehicle groups. To further confirm these findings, immunofluorescent staining results indicated high expression of Ad-TMBIM6 in the brain when stained with anti-HA tagged antibody and showed to co-localize with neurons, microglia, and astrocytes (p < 0.05, Fig. 2b). There was no staining seen for Ad-TMBIM6 in sham or vehicle (data not shown).
Please refer to Additional file 5 for detailed statistical analysis.
Ad-TMBIM6 improved long-term neurological outcomes at 4 weeks post-HI Neurological function was assessed by rotarod and water maze at 4 weeks post-HI. In both behavioral tests, the vehicle group performed significantly worse compared to sham animals. In the rotarod test, the Ad-TMBIM6treated group showed to significantly improve sensorimotor coordination as displayed by the longer distance traveled, on the rotating rod (p < 0.05, Fig. 2c), versus vehicle. In the water maze test, compared to the sham group, the vehicle group demonstrated substantial memory impairment and learning abilities in terms of the time it took to reach the platform (p < 0.05, Fig. 2d). However, Ad-TMBIM6 significantly improved both memory and learning function compared to vehicle (p < 0.05, Fig. 2d). Please refer to Additional file 5 for detailed statistical analysis.
Silencing BI-1 or Nrf-2 reversed Ad-TMBIM6's protective effects at 72 h post-HI To evaluate the effects of BI-1 or Nrf-2 siRNA on the percent infarcted area, animals were sacrificed at 72 h post-HI and brain samples harvested for TTC staining. Results showed that Ad-TMBIM6 + BI-1siRNA significantly reversed BI-1's protective effects as did the Ad-TMBIM6 + Nrf-2siRNA group. This is seen from the significant increase in percent infarcted area when compared to the Ad-TMBIM6 only group (p < 0.05, Fig. 3a, b). The control group, Ad-TMBIM6 + scramble siRNA, did not exacerbate the damage when compared to Ad-TMBIM6-treated group. Furthermore, BI-1 and Nrf-2 siRNA only groups (without treatment) showed similar percent infarcted areas as compared to HI + vehicle or HI + scramble siRNA groups. Please refer to Additional file 6 for detailed statistical analysis.
Overexpression of BI-1 disrupted the NPR-CYP complex at 72 h post-HI
Quantification data showed that BI-1 expression levels were significantly increased in the Ad-TMBIM6-treated and in the Ad-TMBIM6 + scramble siRNA groups, while BI-1 siRNA reversed those effects (p < 0.05, Fig. 4a, b). In addition, BI-1 expression levels were low in all three groups (without Ad-TMBIM6) BI-1 siRNA, Nrf-2 siRNA, and scramble. Ad-TMBIM6 significantly reduced the expression of P4502E1 (p < 0.05, Fig. 4c), while silencing BI-1 reversed those effects, as seen from the elevated expression levels of P4502E1 (p < 0.05, Fig. 4c). Please refer to Additional file 7 for detailed statistical analysis.
To observe changes in BI-1 and P4502E1 expression levels, as well as co-localization with microglia, after either Long-term neurobehavioral tests: total distance in rotarod test (c) and time to platform in water maze test (d) at 4 weeks post-HI (data expressed as mean ± SD; an asterisk indicates p < 0.05 vs sham; number sign p < 0.05 vs vehicle; @ p < 0.05 vs Ad-TMBIM6 (1.6 × 10 11 PFU); n = 6-9. One-way ANOVA followed by Tukey's multiple-comparison/or Holm-Sidak post hoc analysis) Ad-TMBIM6 or after silencing of BI-1 and Nrf-2, immunofluorescence staining was performed. Data showed increased expression of BI-1 and co-localization on microglia in Ad-TMBIM6 treatment group and scramble control group compared to sham or vehicle while BI-1 siRNA reversed those effects (Fig. 4d). On the contrary, P4502E1 expression levels were elevated in the vehicle, BI-1 siRNA, and Nrf-2 siRNA groups but not in sham or Ad-TMBIM6treated groups (Fig. 4e). There was also col-localization of P4502E1 on microglia.
BI-1 upregulated pNrf-2 expression and induced antioxidant enzyme production at 72 h post-HI
After treatment with Ad-TMBIM6, the expression levels of pNrf-2 were significantly increased compared to the vehicle. Either silencing BI-1 or Nrf-2 with siRNAs reversed those effects (p < 0.05, Fig. 5a, c). An increase in Nrf-2 levels was followed by an increase in the antioxidant enzyme, HO-1, in the Ad-TMBIM6 group when compared to vehicle (p < 0.05, Fig. 5a, d). Furthermore, inhibition of BI-1 or Nrf-2 significantly decreased HO-1 levels and its ability to block ROS. Please refer to Additional file 7 for detailed statistical analysis.
Double immunofluorescence staining was performed to detect co-localization of NPR or Nrf-2 with microglia (Iba-1) in the presence of Ad-TMBIM6, BI-1 siRNA, or Nrf-2 siRNA. Fluorescent staining showed that although NPR co-localized with microglia, its expression levels were unchanged among groups (Fig. 5e). On the contrary, Ad-TMBIM6 increased Nrf-2 expression and colocalization with microglia compared to sham or vehicle, while Nrf-2 siRNA abolished that effect (Fig. 5f).
BI-1 overexpression attenuated ROS production and inflammation at 72 h post-HI
To evaluate whether overexpression of BI-1 with Ad-TMBIM6 could inhibit ROS production and subsequently attenuate inflammation, western blot was done to quantify ROMO1 (a modulator that induces production of ROS), IL-6, TNFα, and IL-1β expression levels. Data showed that the Ad-TMBIM6 treatment group significantly reduced ROMO1 levels (p < 0.05, Fig. 6a, b) and attenuated pro-inflammatory markers, IL-6, TNFα, and IL-1β (p < 0.05, Fig. 6c-e). In addition, the administration of BI-1 siRNA and Nrf-2 siRNA reversed Ad-TMBIM6's protective effects as seen from the significantly higher upregulation of pro-inflammatory mediators and ROS. Please refer to Additional file 8 for detailed statistical analysis.
Since HI results in the induction of inflammation, we stained for IL-1β (a pro-apoptotic marker) with microglia. The counting of positively stained cells for IL-1β with microglia showed a higher percentage of IL-1β on microglia in the vehicle, BI-1 siRNA, and Nrf-2 siRNA groups compared to sham, Ad-TMBIM6, or scramble group (p < 0.05, Fig. 6f; scale bar 50 μm). Please refer to Additional file 9 for detailed statistical analysis.
To evaluate ROS production, we stained brain slices with dihydroethidium (DHE) dye. DHE is possibly the most specific and least problematic dye for detection of superoxide radicals. Cells with high ROS accumulation will have a red fluorescent light. Our data revealed a higher number of red positively stained cells in vehicle and siRNA groups versus sham, Ad-TMBIM6, and control group in the cortex region (Fig. 7a).
Furthermore, we also stained for ROS around the ventricles and observed trends similar to the cortex region (Fig. 7b). In addition, to determine whether ROS accumulation is correlated with increased inflammation, we stained for myeloperoxidase (MPO; a marker that shows the inflammatory activity) and detected significantly higher expression of MPO positively stained cells in vehicle and siRNA groups compared to sham or Ad-TMBIM6-treated group (p < 0.05, Fig. 7c), thus showing both a correlation between an increase in ROS production with an increase in inflammation, as well as BI-1's ability to inhibit ROS production and the increase in inflammation independently. Please refer to Additional file 9 for detailed statistical analysis. Fig. 4 The effects of silencing BI-1 on the NPR-CYP complex at 72 h post-HI. Representative bands of western blot data (a). Quantification of western blot bands for BI-1 (b) and P4502E1 (c) (data expressed as mean ± SD; an asterisk indicates p < 0.05 vs sham; number sign p< 0.05 vs vehicle; @ p< 0.05 vs Ad-TMBIM6 or scramble; n = 6/group using one-way ANOVA followed by Tukey's multiple-comparison post hoc analysis). Immunofluorescence staining of Iba-1 with BI-1 or P4502E1 (d, e). Green was for microglial staining, red was for BI-1 or P4502E1 staining, and blue was for DAPI. Merge (yellow) showed the co-localization of BI-1 or P4502E1 on microglia (scale bar = 50 μm; n = 3/group)
To investigate BI-1's effects on NPR-CYP antiinflammatory pathway in an in vitro OGD model
As BI-1 has been previously reported to disrupt the NPR-CYP complex as well as activate Nrf-2 resulting in inhibition of ROS production and subsequent attenuation of inflammation, we employed an in vitro OGD model using a microglial cell line and primary microglia cell culture (Additional files 2 and 3) to test the specificity of BI-1 related attenuation of ROS-induced inflammation in microglia. To test BI-1's pathway, we administered a BI-1 siRNA to knockdown BI-1 expression levels and Nrf-2 siRNA to silence Nrf-2 expression. Data showed that the administration of Ad-TMBIM6 vector significantly upregulated BI-1 levels in cells, both in treated and scramble control group, while BI-1 siRNA group reversed those effects; Nrf-2 siRNA did not affect BI-1's levels (p < 0.05, Fig. 8a, b). Intervening at either BI-1 level or Nrf-2 level with siRNAs significantly reversed BI-1's protective effects as seen from the increased expression levels of inflammatory markers, IL-6, TNFα, and IL-1β, when compared to Ad-TMBIM6-treated group, sham, or scramble groups (p < 0.05, Fig. 8c-e). Our data demonstrated HO-1 (c), and pNrf-2 (d). Immunofluorescence staining of Iba-1 with NPR or Nrf-2 (e, f). Green was for microglial staining, red was for NPR or Nrf-2 staining, and blue was for DAPI. Merge showed the co-localization of NPR or Nrf-2 on microglia (scale bar = 50 μm; n = 3/group). Data expressed as mean ± SD; an asterisk indicates p < 0.05 vs sham; number sign p < 0.05 vs vehicle; @ p < 0.05 vs Ad-TMBIM6 or scramble; n = 6/group using one-way ANOVA followed by Tukey's multiple-comparison post hoc analysis that Ad-TMBIM6 inhibited P4502E1, the primary source of ROS production, while simultaneously upregulating pNrf-2 levels (p < 0.05, Fig. 8f, g). Silencing BI-1 or Nrf-2 reversed those effects. Percent cell viability data showed that Ad-TMBIM6 was able to significantly improve the percent of viable cells while the knockdown of BI-1 and Nrf-2 reversed those effects (p < 0.05, Fig. 8h). Please refer to Additional file 10 for detailed statistical analysis.
Discussion
The endoplasmic reticulum (ER) is a major organelle that has an essential role in multiple cellular processes, such as the control of correct protein folding and function [47,50]. Hypoxia-ischemia, oxidative stress, calcium disturbances, and inhibition of protein glycosylation may contribute to a disruption in ER homeostasis and lead to stress [50]. Cells respond to ER stress by activating several pathways, including promoting the ability of proteins to refold correctly, inhibiting protein translation, increasing protein degradation, stimulating the transcription of genes, and enabling self-repair mechanisms [50]. All these processes are referred to as the unfolded protein response (UPR) which under prolonged activation result in cell death [47].
Bax Inhibitor-1 (BI-1) is a conserved evolutionary protein that resides on the intracellular membrane of the ER [12]. BI-1 was first named as Bax Inhibitor due to its ability to suppress cell death in yeast [49]. More recently, it is also referred to as TMBIM6 as it is part of the transmembrane Bax Inhibitor-1 containing motif 6 family [19]. Overexpression of BI-1 has been demonstrated to play a protective role in ER stress-induced cell death, and to a lesser extent in other ER related stresses, such as oxidative stress and inflammation. In the present study, we first measured endogenous expression levels of BI-1 and its downstream proteins. Our results showed that endogenous expression of P4502E1 (CYP2E1), a member of the MMO system and a major inducer of ROS generation, increased in a time-dependent manner after HI and remained elevated through 72 h post HI. This is expected as after HI injury, there is a significant increase in ROS production that is regulated mainly through P4502E1 (Fig. 1a, d). Hence, levels of P4502E1 Fig. 6 Overexpression of BI-1 attenuated ROS production and inflammation at 72 h post-HI. Representative picture of western blot bands (a). Quantification analysis of band intensities for ROMO1 (b), IL-6 (c), TNFα (d), and IL-1β (e). Immunofluorescent staining and quantification of the number of positively stained cells of Iba-1 with the inflammatory marker, IL-1β, in the peri-infarcted region (f). Green was for microglial staining and red was for IL-1β. Merge showed the co-localization of IL-1β on microglia. Scale bar 50 μm; n = 3/group. Data expressed as mean ± SD; an asterisk indicates p < 0.05 vs sham; number sign p < 0.05 vs vehicle; @ p < 0.05 vs Ad-TMBIM6 or scramble; n = 6/group using one-way ANOVA followed by Tukey's multiple-comparison post hoc analysis increase during times of injury. On the contrary, BI-1's role is to reduce ROS by disrupting P4502E1. In our results, we demonstrated an initial increase in BI-1 expression; however, at 72 h post HI, BI-1 expression levels significantly declined (Fig. 1a, b). This initial increase in BI-1 can be explained as a protective response to the injury. BI-1 levels increase with the attempt to reverse the damage caused by HI injury. However, the endogenous levels are not enough to reverse the damage and BI-1 declines significantly by 72 h as seen from our data. Therefore, the main goal in our study was to significantly increase BI-1 levels in the brain, using exogenous means, to be able to reverse damage caused after HI injury.
To demonstrate BI-1's protective effects after hypoxic-ischemic (HI) injury in the neonatal rat, we overexpressed BI-1 using Adenoviral-TMBIM6 (Ad-TMBIM6) vector. We tested two doses and showed that low dose Ad-TMBIM6 vector significantly reduced the percent infarcted area (Fig. 2a) and improved long-term neurobehavioral outcomes ( Fig. 2c-d). In addition, we quantified the amount of viral vector present in the brain using western blot and showed robust expression of Ad-TMBIM6 at 72 h post-HI (Fig. 2b).
ER stress is associated with the production of reactive oxygen species (ROS) through oxidative protein folding by the MMO system, composed of NADPH-P450 reductase (NPR) and cytochrome P450 (CYP) members such as P4502E1 [27,28,32]. It has been shown that BI-1 overexpressing cells can regulate UPR induction and inhibit ROS accumulation under ER stress [15], thus making BI-1 a crucial regulator of ROS inhibition. Cells respond to ROS by activating genes to encode antioxidative stress enzymes. A key transcription factor activated is nuclear factor erythroid 2-related factor 2 (Nrf-2), which regulates the production of several cytoprotective enzymes, such as heme oxygenase-1 (HO-1), a potent inhibitor of ROS [15,17]. Studies showed that in BI-1 overexpressing cells, inhibition of HO-1 attenuated BI-1-mediated protection against ER stress [17]. However, the role of HO-1 in BI-1's protective mechanism is mostly unknown and debatable.
Some studies have shown that HO-1 was unaffected in BI-1 deficient mice embryonic fibroblast cells [24]. In contrast, BI-1 overexpressed cells showed significant inhibition of P4502E1 expression [15] and suppression of ROS production in human embryonic kidney cells [16]. The P4502E1 member of the CYP ER heme proteins is a major contributor to ROS production. It acts by metabolizing and activating substrates into more toxic products, thus not only increasing ROS production but also stimulating inflammatory cascades and ultimately worsening HI pathology. Previous studies have shown P4502E1 to be upregulated during ER stress [15][16][17][18], thus playing an essential role in HI pathology.
In addition, other studies showed that P4502E1-induced oxidative stress played a role in the translocation of Nrf-2 to the nucleus followed by the upregulation of HO-1. Moreover, increased HO-1 levels may be dependent on P4502E1 [9], perhaps attempting to counteract P4502E1 effects and act as a survival signal. The same study showed that the inhibition of P4502E1 significantly reduced ROS generation in an acute kidney injury model [43]. The relationship between P4502E1 and HO-1 needs further research in order to fully understand the specifics covering the apparent connection. Since P4502E1 is a major regulator of ROS production and that Nrf-2 has a role in the response against P4502E1, inhibition of P4502E1 coupled with activation of Nrf-2 is a promising therapeutic target.
Although we are now beginning to understand the importance of BI-1 in the cell and in human physiology, its function and signaling mechanisms remain unknown. Given the importance of P4502E1 and Nrf-2, finding a key molecule to regulate both is of great interest. Here we identified BI-1 as one such potential molecule which may protect the cell against ER stress-induced ROS production as well as the subsequent increase in inflammatory response via two possible mechanisms. First, it may interact with NPR, thus destabilizing the NPR-CYP complex, which directly inhibits the formation of ROS by reducing the activity of P4502E1 [15]. BI-1 may interact through its C-terminus with NPR and to a lesser extent with P4502E1 [15]. This interaction induces destabilization of the NPR-CYP complex, thus blocking electron transfer and ROS production [32]. A recent study showed that BI-1 overexpressing cells caused P4502E1 degradation, leading to ER stress suppression, and subsequent reduction in ROS [18]. We observed similar findings from our western blot results; overexpression of BI-1 in the neonatal rat significantly upregulated BI-1 levels in the brain, while simultaneously reducing P4502E1 expression levels (Fig. 4a-c). Silencing BI-1 reversed those effects while silencing Nrf-2 did not significantly change P4502E1 levels, which indicates Nrf-2 to be downstream of P4502E1. This data was further supported by our IHC staining that demonstrated across groups that as BI-1 expression increased, P4502E1 decreased; they have converse expression patterns throughout (Fig. 4d, e). Our western blot data also demonstrated a decrease in BI-1 expression levels after silencing Nrf-2 with siRNA ( Fig. 4a). This may be explained due to BI-1's multimodal properties and hence targeting multiple signaling pathways. In our study, we focused on BI-1 and its role on the CYP-NPR complex and Nrf-2 signaling. Specifically, we showed that BI-1 may upregulate Nrf-2, an anti-oxidative molecule, thus attenuating ROS production and subsequent inflammation. However, BI-1 has other roles such as being able to reduce Bax via direct interaction with Bcl-2 [45,49]. This interaction with Bcl-2 showed to increase its levels while decreasing the pro-apoptotic protein, Bax [45,49]. Like BI-1, Nrf-2 levels may be affected by different signaling pathways as well. In this case, as previous studies have shown an interaction between BI-1 and Bcl-2, studies have also shown a direct connection between Nrf-2 and Bcl-2 [23,29,30,39,51]. Specifically, Nrf-2 has been shown to bind to Bcl-2 ARE and regulate the expression and induction of the Bcl-2 gene [30]. Since both Nrf-2 and BI-1 have a direct interaction with Bcl-2, this may explain as to why the decrease in Bcl-2 levels, due to knockdown of either Nrf-2 or BI-1, will indirectly affect each other's expression levels. In our study, we knocked down Nrf-2, using siRNA, and saw a decrease in BI-1 expression. This result may be explained, as mentioned above, by the fact that knocking down Nrf-2 affects Bcl-2 levels (Bcl-2 decreases) and since BI-1 and Bcl-2 have a direct interaction as well, it will also affect BI-1 expression levels acting like a negative feedback loop, which resembles the decrease we observed in our experiment (Fig. 4).
Second, BI-1 may upregulate pNrf-2 which in turn triggers HO-1 thus inhibiting ROS production and attenuating inflammation. Lee et al. showed that overexpression of BI-1 in cells increased Nrf-2 transcription factor, which then translocated to the nucleus where it stimulated the production of anti-oxidant enzymes, HO-1. HO-1 is known to block ROS production and accumulation thereby promoting cell survival [17]. Similar to Lee et al., we found that BI-1 overexpression, after neonatal HI, significantly upregulated pNrf-2 ( Fig. 5c) and HO-1 (Fig. 5d) while silencing of either BI-1 or Nrf-2 with siRNAs reversed those effects. In addition, our IHC staining showed changes in Nrf-2 expression patterns among groups that correlated with our western blot findings (Fig. 5f). Furthermore, inhibition of P4502E1 with activation of Nrf-2 and HO-1 was linked with a reduction in ROS accumulation and the subsequent release of pro-inflammatory mediators. Here, we used a ROS marker, Reactive Oxygen Species Modulator 1 (ROMO1), which is a protein-coding gene responsible for ROS generation. There was a significant reduction in ROMO1 expression levels (Fig. 6a, b) as well as in IL-6, TNF-α, and IL-β (Fig. 6c, d). Staining for IL-1β on microglia demonstrated a significantly higher expression and co-localization in the vehicle, BI-1 siRNA, and Nrf-2 siRNA groups compared to sham or Ad-TMBIM6-treated group (Fig. 6f). These results were confirmed to be via BI-1-induced inhibition of P4502E1 or activation of Nrf-2 as either inhibition of BI-1 or Nrf-2 reversed those effects. To further examine BI-1's inhibitory role on ROS accumulation, we used a dihydroethidium (DHE) dye to detect ROS production [31]. The dye binds with the superoxide anions, thus illuminating a red fluorescent image which is an indication of ROS presence. Our data indicated a higher amount of ROS accumulation in vehicle and siRNA groups versus sham or Ad-TMBIM6-treated groups, in both cortex region and around ventricles, while overexpression of BI-1 reversed those effects (Fig. 7a, b). To detect whether a reduction in ROS was associated with a reduction in inflammation, we performed immunofluorescence staining for myeloperoxidase (MPO). A similar pattern was observed in the MPO staining where Ad-TMBIM6 significantly reduced MPO positively stained cells compared to vehicle or siRNA groups (Fig. 7c), thus indicating a correlation between a reduction in ROS and the subsequent attenuation of inflammatory processes.
Conclusions
In summary, the overexpression of BI-1 attenuated inflammation and demonstrated to promote cell survival by increasing the production of antioxidant enzymes as well as destabilizing the complex responsible for ROS production. This was observed as either silencing BI-1 or Nrf-2 reversed BI-1's protective effects by altering P4502E1, Nrf-2, and HO-1 expressions. This is indicative of BI-1's ability to interact with the NPR-CYP complex and induce dissociation, thus disrupting the electron flow, as a physical association between NPR and CYP is required for the production of ROS. Furthermore, BI-1 was shown to upregulate pNrf-2 and HO-1 directly. These two mechanisms play a major role in BI-1's anti-inflammatory effects. In addition, our in vivo findings were translated to an in vitro OGD model to validate this pathway in microglial cells (Fig. 8, Additional files 2 and 3).
This study expands the current evidence on BI-1 as a protective protein and advances present in vitro studies to in vivo stroke model. A major challenge with any mechanism design lies in the existence of several parallel pathways that may act at the same time. Thus, focusing on only one may not be ideal for preserving cell survival. BI-1's multimodal properties suggest that it can target a wide array of pathophysiological consequences after HI, thus making it an ideal therapeutic candidate. | v3-fos-license |
2018-12-12T05:17:30.437Z | 2015-08-10T00:00:00.000 | 55620328 | {
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} | pes2o/s2orc | Compression deformation behaviors of sheet metals at various clearances and side forces
Modeling sheet metal forming operations requires understanding of plastic behaviors of sheet metals along non-proportional strain paths. The plastic behavior under reversed uniaxial loading is of particular interest because of its simplicity of interpretation and its application to material elements drawn over a die radius and underwent repeated bending. However, the attainable strain is limited by failures, such as buckling and inplane deformation, dependent on clearances and side forces. In this study, a finite element (FE) model was established for the compression process of sheet specimens, to probe the deformation behavior. The results show that: With the decrease of the clearance from a very large value to a very small value, four defects modes, including plastic t-buckling, microbending, w-buckling, and in-plane compression deformation will occur. With the increase of the side force from a very small value to a very large value, plastic t-buckling, w-buckling, uniform deformation, and in-plane compression will occur. The difference in deformation behaviors under these two parameters indicates that the successful compression process without failures for sheet specimens only can be carried out under a reasonable side force.
Introduction
For simulating metal forming processes, many new constitutive relations have been developed to describe materials behavior along non-proportional strain paths [1][2][3][4][5][6][7][8].In order to fit these new constitutive models, experimental methods are required to test materials in an accurate, reliable and reproducible manner.The uniaxial tension-compression testing is of particular interest because of its simplicity of interpretation and uniformity of deformation over the entire sampled volume.However, the attainable strain in compression testing is severely limited by failures, such as buckling and in-plane deformation.Thus, various side support devices were proposed to suppress the buckling [9][10][11].In these side support devices, the symmetric wedge device is the one that can completely eliminate the unsupported area [12].However, the gap is fixed and the side force is uncontrollable in this symmetric a Corresponding author: [email protected] is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.wedge design.Therefore it is necessary to research the deformation behaviors of sheet metals under compression at various clearances and side forces for successful large-strain compression without failures.
MATEC Web of Conferences
In this study, a finite element (FE) model was established for the compression process of sheet specimens, to probe the deformation behavior.The influences of different initial clearances and side forces to sheet metal deformation were studied.Finally reasonable conditions are obtained to ensure the uniform deformation in sheet metal compression testing.
Material and methods
Dual Phase (DP 600) steel sheet metal was used in this study, which has a thickness of 1.0 mm.Uniaxial tensile tests were conducted on normal specimens without fins using an MTS Sintech 20 g universal mechanical testing machine and a 25.4 mm MTS strain gauge.The elastic modulus of DP 600 is 182 GPa, and its plastic behavior obeying the Hollomon hardening law = 1085 0.1813 was determined from the uniaxial tensile tests by averaging from data in Fig. 1.
Since the implicit method is faster to obtain a steady-state and reliable solution than the explicit method is, the ABAQUS/standard software tool was used for finite element analyses for the compression test of the specimen with two pairs of asymmetry fins (Fig. 2) to probe the deformation behavior.Considering there are three directional deformations along the axial, width and thickness direction, three-dimensional (3D) analyses were performed.A 14 mm length region at each end of the specimen is clamped by a pair of clamps, and other regions of specimen are wrapped by four side supports (Fig. 2).The specimen, and four supports were modeled as separate parts.The specimen was defined as a 3D deformable solid body.The supports and clamps were defined as discrete rigid bodies.Each rigid body was assigned a reference point to represent its rigid motion in all degrees of freedom.The contact between the specimen and the supports was modeled with the CONTACT PAIR option, and the Coulomb friction formulation and penalty contact method were used.The supports were meshed by 3D bilinear rigid quadrilateral elements with four nodes.3D linear reduction integration continuum elements with eight nodes were used to discretize the specimen, and an enhanced hourglass control was employed to ensure computational precision.Initial geometric imperfections were imported to probe the buckling behaviors and other defects of sheet metals under compression.In the analyses, doubleprecision computations were carried out to improve simulation accuracy.To minimize the number of degrees of freedom and computation time, variable mesh densities were used for the specimen in this study.Using the meshing technology, the mesh size of 0.2 mm by 0.2 mm within the gauge region was used.Since this mesh density is close to that of Boger et al.'s study [13] and is much less than that of Cao et al.'s analysis (Cao et al. 2009, which is 0.75 mm by 0.75 mm), the mesh technology used in this study can be used to capture the buckling, stress and strain distribution for the compression test of DP600 sheets.Two parameters influencing the compression process are the initial clearance (C i ) between the specimen and the supports, and the normal side force (F n ) loading on the specimen.Deformation behaviors under various initial clearances (C i = 0-0.5 mm) and normal side forces (F n = 0.0-40 kN) during 4 mm compression displacement (Dis) are analyzed.
Deformation behaviors under various clearances
The simulations under various initial clearances C i show that the initial clearance has a significant influence on the deformation behaviors of the sheet specimen during compression.Under various initial clearances, four deformation modes, plastic t-buckling, micro-bending, w-buckling, and in-plane compression deformation (or barreling) will occur, as shown in Fig. 3.Under a given C i value, the occurrence of deformation mode and its transfer to others has been investigated as the compression displacement increasing.
When given an over-large clearance (such as C i = 0.3-0.5 mm), the compression in the beginning is similar to a free compression process.Therefore, with the increase of compression displacement, the axial compressive stress and strain increase.When the stress and strain reach their critical values, some mode plastic t-buckling (Fig. 3(a)) occurs with a very small increase in displacement according to compression bar buckling theory of Euler.Thus a decrease in the crosshead force and reaction forces on supports occurs (Fig. 4(a) and (b)).After the plastic t-buckling occurring, the unstable state and the decrease in forces will continue for some time till the specimen contacts with the supports, then the forces will increase resulting from supports until the next mode t-buckling occurs due to that the constraint from the normal direction is not sufficient to support this mode plastic stable deformation out of plane from normal direction (Fig. 4(a) and (b)).
As the clearance decreases a little from the occurrence condition of plastic t-buckling but still a large value (such as C i = 0.1 mm), the decrease in clearance will suppress the obvious plastic t-buckling and thus the loading force can be transfer smoothly.However, local micro-bending occurs in the early and midst period of compression (Fig. 3(b)).This local micro-bending is similar to the plastic t-buckling with very low wave.As the compression going on, w-buckling occurs (Fig. 3(c)) according to compression bar buckling theory of Euler.During the whole process, there is almost no abnormal variation in the crosshead force (Fig. 4(a)) but the abnormal variation in the reaction force (Fig. 4(b)) of the supports is still obvious.
As the clearance decreases continually to a small value (such as Ci = 0.035 mm), local microbending still occurs in the early and midst period due to that the small clearance in this period, and in-plane compression occurs in the last period due to the increase in thickness in this period make the real clearance becomes less and less, as shown in Fig. 3(d).This leads to negligible abnormal variation in the loading force (Fig. 4(a)) but still noticeable variation in the reaction force on the supports in the early and midst period, and a dramatic increase in these forces in the last period (Fig. 4(b)).
As the clearance continually decreases to an over-small value (such as C = 0 mm), in-plane compression deformation occurs in the beginning of compression and becomes more and more obvious until barreling occurs (Fig. 3(d)) due to that the normal constraint from supports will constrain excessively the thickness deformation.In the case, there is no abnormal variation in the crosshead force and the reaction force on the supports in the whole period, as shown in Fig. 4(a) and (b).However, the values of these forces are larger a lot than those under other clearances (Fig. 4(a) and (b)).
Deformation behaviors under various side forces
The simulations under various side forces show that the side force also has a significant influence on the deformation behaviors of the sheet specimen during compression.With the increase of the side force, four deformation modes, including plastic t-buckling, w-buckling, uniform deformation, and in-plane compression deformation, will occur successively, as shown in Fig. 5.
The plastic t-buckling occurs when the normal side force F n is such a small value (for example, F n = 0.03 kN), as shown in Fig. 5(a).Under this deformation behavior, the constraint from the normal direction is too small to support the plastic stable deformation out of plane.As the side force increases on the occurrence condition of plastic t-buckling (such as F n = 1.25-3.75kN), w-buckling will occur (Fig. 5(b)) due to that the increase in side force is enough to suppress the plastic t-buckling and thus the loading force can be transfer smoothly.With the increase in the side force, the thickness strain reduces and width strain increases, thus the ratio of width strain to thickness strain increases, as shown in Fig. 6. Figure 6 shows that when F n =5-10 kN, most of ratios are in the range of 0.95-1.1,when ICNFT 2015 (a) Plastic t-buckling under over-small side force (F n =0.03kN).
(b) w-buckling under small side force (F n =1.25kN) (c) In-plane deformation under over-large side force (F n =40.0kN).
(d) Uniform deformation behavior under reasonable side force (Fn =5kN).F n = 20 kN, most of ratios are in the range of 1.1-1.3,and when F n = 40 kN, most of ratios are in the range of 1.4-1.7.This means that as the side force increases excessively (such as F n = 20-40 kN), in-plane compression deformation even barreling will occur due to excessive constraint in the thickness deformation, as shown in Fig. 5(c).Uniform deformation occurs when a reasonable side force (such as F n = 5-20 kN) is applied, as shown in Fig. 5(d).In this case, the unstable deformation behaviors can be suppressed and the deformation along the thickness direction can't be constrained excessively, thus in-plane compression deformation also can be suppressed.Figure 7 shows that the side force when in-plane compression deformation occurring is larger a lot than other side forces.When the plastic t-buckling occurs under a small side force there is only a decrease in the axial loading force.This is different from that when the plastic t-buckling occurs under an over-large clearance, where there is usually an increase followed by the decrease in the force (Fig. 4(a)).This is due to that once the plastic t-buckling occurs under a small side force, the unstable state will continue since the side force is insufficient to suppress the unstable state.
Relationship of deformation behaviors with initial clearance and side force
Based on the above results, the relationship of deformation behaviors under various clearances and side forces can be schematized as Fig. 8.Under an over-large clearance, plastic t-buckling from low mode to high mode will occur as compression going on.Under a large clearance, micro-bending occurs in the early and midst period and w-buckling occur in the last period of compression.Under a small clearance, in-pane deformation occurs in the last period of compression.Under an over-small clearance, in-plane deformation will occur in the beginning and become more and more obvious with the process going on. Figure 8(b) shows plastic t-buckling, w-buckling, uniform deformation, and in-plane compression deformation that occur successively as the side force increasing.
Comparison in Fig. 8(a) and (b) shows that the difference of the two parameters is that uniform deformation behavior desired will only occur under a reasonable side force.This is due to that a reasonable side force can give the specimen a proper support to suppress the unstable deformation and make the supports move normally to let the specimen thicken "freely" which can avoid in-plane compression deformation.However, a given initial clearance is difficult to attain both of goals.
Conclusion
With the decrease of the clearance from a very large value to a very small value, four defects modes, including plastic t-buckling, micro-bending, w-buckling, and in-plane compression deformation will occur.While with the increase of the side force, plastic t-buckling, w-buckling, uniform deformation, and in-plane compression will occur.And uniform deformation occurs when there is no initial clearance
(a) Specimen with two pairs of fins (b) Assembly of specimen and side force
Figure 4 .
Figure 4. Crosshead forces and reaction forces on supports verse displacement at various clearances. | v3-fos-license |
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} | pes2o/s2orc | Location and characterisation of pollution sites by principal component analysis of trace contaminants in a slightly polluted seasonal river : a case study of the arenales River ( Salta , argentina )
Principal component analysis (PCA) was used to deduce the common origin of trace contaminants in a slightly contaminated, strongly seasonal river of low-average discharge, aiming to ascertain the type of the pollution. Splitting of data into categories according to specific conductance was essential to reach conclusions. Dry-season data allowed the pinpointing of polluting sites by means of the biplots resulting from the representation of the scores on the components. Concentrations corresponding to the wet seasons yielded no useful results probably due to the high percentage of data below detection limits for 2 of the 6 variables. The Arenales River in North-West Argentina was monitored by means of 19 sampling campaigns between 2003 and 2005 comprising two hydrological cycles, at seven locations along a 25 km section of the river course across the city of Salta. Pollution of the river was not severe, overall mean values in μg/l being: As 1.2; B 490; Cu 4; Fe 92; Pb 13; Zn 83. Simple correlation analysis revealed no significant correlation between these elements. The high positive loadings of variables B and As concentrations on the first principal component and the biplots indicate that their main common point sources are boron mineral deposits still existing in the urban area. Interpretation of the biplots shows that Cu, Fe and Zn contamination also originated at point sources, the contribution of the sewage treatment plant being negligible.
Introduction
River water quality monitoring is mandatory in present-day society, especially for rivers affected by urban effluents.The registration of their physico-chemical characteristics and of the concentration of their main components as well of any trace elements present is recommended to establish the level of contamination, the efficiency of the wastewater treatment facility when it exists, and the degree of recovery of the river water quality.
Even if the river water is not used as a source of drinking water, pollution with microbes and organic and inorganic substances can pose a health hazard to water biota and to humans as well.When the river water is used for irrigation, even low concentrations of certain elements like B, Cu, Fe and Zn can produce drastic effects on the yields, because, though needed for the normal development of plants, at the same time they can be poisonous when present at certain concentration levels.Abundant literature can be found on the determination of the concentration levels of numerous inorganic substances in river water, from the Mississippi (Meybeck et al., 1989) through the Danube (Marjanovic et al., 1985) and to the Huanghe (Zhang et al., 1993), since the first half of the 20 th century.In some instances the mineral contents reflect the main composition of the river basin (De Villiers, 2005), but when abnormally high concentrations are found, the origin of the pollution is identified so as to be in a position to take adequate protective action for the environment (Schmitz et al., 1994).
Data produced by the monitoring of river water contamination with heavy metals and other elements in trace level concentration are generally used to assess water quality according to concentration limits established by environmental protection authorities.But the sources of the different elements are not always clear, the hydrological system constituting a complicated background.Statistical analysis is essential for proper interpretation of these time-and location-dependent data, to establish correlations not only among the data, but between these and the geological and climatic parameters as well, so as to characterise the water system.When concentrations of a relatively high number of elements are recorded, factorial analysis is used to reduce the number of variables and to highlight any relationships among them.
Principal component analysis (PCA) is proving to be a valuable tool to establish the hydrochemistry of rivers, as shown in extensive studies carried out by Simeonov et al. (2003) in Greece; to typify pollution sources of surface waters (Wunderlin et al., 2001); or coastal seawaters (Morales et al., 1999); to study the temporal variation of groundwater (Helena et al., 2000); and for establishing water quality (Praus, 2005).
Trace element concentrations in a highly seasonal river at 7 sampling sites during 2 hydrological cycles were analysed to assess the nature and the origin of the pollution.However, the high background noise provided by the low concentrations involved (less than 1 mg/ℓ, except for B), the sporadic nature of the pollution at most of the sites, and the ample variations of the river flow hindered the simple statistical analysis by correlation coefficients.Even PCA did not yield a clear picture until the data were separated into two groups, not according to rainfall records, but to specific conductivity.Data corresponding to the dry seasons defined in this way proved to be useful to establish correlations among the polluting elements, and to point out the probable origin of the contamination along the river course.'QUIMIO', easy-to-use software created for statistical calculations in chemical analyses (Cela, 1994) was used to process the data.
The monitoring area
The city of Salta, with 500 000 inhabitants, lies at the northern extreme of the Lerma Valley, limited by the Andean cordillera in the West and the Mojotoro hills in the East, in north-west Argentina (Fig. 1).
The Valley is characterised by its proximity to the Tropic of Capricorn and by its elevation of about 1 100 m a.s.l.The climate is, in consequence, of a semi-desert nature, with a short rainy season in summer.The water flow of the Arenales River, which varies quite significantly naturally through a hydrological cycle (8.8 to 284 m 3 /s), has been so drastically altered upstream by its intensive use for irrigation that by the end of the dry season there is practically no superficial flow when it enters the western outskirts of the city.The flow gradually increases when the river course changes due south, by inflows from surfacing aquifers at the eastern border of the Valley, and by the effluents of the city's wastewater treatment plant.Consequently the water quality declines significantly between April and November, B being the main contaminant.This anthropogenic trace component has been introduced to the Lerma Valley from the Andean plateau as raw material used by boron mineral-processing plants, and a decade ago constituted a very serious contamination of the water system (Lomniczi et al., 1997 and1999), including the reservoir General Belgrano of 3 130 Hm 3 , fed mainly by the Arenales River.
Following the introduction of strict environmental protection laws the concentration of B has declined significantly (from a median of up to 3.90 mg/ℓ between 1991 and 1994 to 0.38 mg/ℓ in 2005), but it still frequently presents high values (over the 0.5 mg/ℓ maximum limit for irrigation water), the origin of which must be ascertained.Fish malformation was reported by news media in the river and the reservoir, so heavy metal concentration also had to be monitored.According to the only previous study of the river, sampled on 3 occasions at 7 sites of a 75 km section (including 2 sampling sites 50 km upstream from Salta city) in 1998 to 1999, Cu, Pb and Zn concentrations increased in the urban environment to values considered toxic to water biota (Musso, 2003).The two most probable sites of contamination seemed to be the industrial park and the inflow from the sewage-processing plant.More frequent monitoring of the concentrations of these, as well as of other elements, at an increased number of sites in the urban environment was needed to discern the origin of the pollution.
Sampling sites (Fig. 1) were selected according to suspected inflow of different kinds of effluents.Site No 1 lies on the Arias River, the only tributary of the Arenales River in the area under study, without suspected raw sewage discharges.This site could represent uncontaminated conditions, as according to former studies the natural content of the monitored trace elements is very similar to that of the nearest unpolluted site of the Arenales River, 50 km upstream of the city of Salta (Musso, 2003).Site No 7 lies 12 km south of the city, so that the quality of the river water here would provide a measure of the degree of its selfrecovery from the urban pollution with trace elements.
Experimental
Water samples were collected between September 2003 and March 2005 on 19 occasions, in 5 ℓ high-density polyethylene containers, previously washed with AR quality HNO 3 and rinsed afterwards with water distilled over glass, and delivered to the laboratory on the day they were collected.They were filtered through glass-fibre filter paper (Whatman 934-AH or similar) and preserved with HNO 3 , according to Standard Methods (1992).Total concentrations of Cu, Fe, Pb and Zn, in samples belonging to the 2003 sampling campaigns, were determined by Flame Atomic Absorption Spectrometry (FAAS) after a ten-fold preconcentration by extraction with methyl-isobutylketone and ammonium pyrrolidine-dithiocarbamate at pH 3.5, (regulated with sodium citrate/citric acid buffer solution).For the 2004 and 2005 sampling campaigns expanded instrumental response by FAAS to Cu, Fe, and Zn was applied to the original samples, once this method was proved to be able to yield repeatability (%RSD) of the same order by performing 12 instead of 5 absorbance readings.At the same time Pb determination was shifted to electrothermal graphite furnace AAS (ETAAS).FAAS preceded by continuous-flow hydride generation with sodium borohydride was used as analytical technique for the determination of As, while B concentration was determined by molecular absorption spectroscopy with azomethine-H, the detection limit being 0.02 mg B/ℓ, as established in previous studies (Lomniczi et al., 1995).Table 1 shows typical numerical values of DL expressed in µ/ℓ, calculated as 3 times the standard deviation of the concentration of reactive blanks.Repeatability of the analyses was assessed as percentage relative standard deviation (%RSD) of duplicates of samples systematically prepared with each batch of determination.Typical values of %RSD are listed in Table 1.Analyses of standard solutions as well as of blanks taken to the sampling sites in collector vessels of the type used for sampling assured that contamination during transport, as well as loss by adsorption on the sampling vessels, were negligible for all trace elements considered.Data were handled according to analytical quality assurance procedures (Bartram et al., 1996).Accuracy was controlled by spiking several samples of every batch under analysis, mean recovery for spiked samples being 83 to 117%.Validation requirements of trace-metal analyses are satisfied by external evaluation through national inter-laboratory comparative exercises practiced every 2 years since 1996.
Results and discussion
General mean concentrations of Cu, Pb and Zn in the Arenales River are high compared to maximum levels tolerated by National Environmental Law 24.051 for water biota protection, while that of B is very near the 500 µg/ℓ maximum recommended limit for irrigation water, confirming the persistence of the pollution previously detected (Musso, 2003).(Table 2) Variation of the concentrations along the river course through the city should point out the sites of clandestine sporadic inflow of urban waste, the efficiency of the local wastewater treatment system in eliminating them from the sewage, and the capacity for self-recovery of the river regarding these contaminants.
All data distributions were tested for normality, concentrations below detection limits considered as zero.The cumulative frequency distributions were compared with the Gaussian cumulative distributions corresponding to the average and standard deviation values of the data.Correlation coefficients between these were significant in every instance, the t value calculated according to Fisher (Sánchez de Peña, 1997) several times higher than the tabulated value of 2, so that r 2 values could be considered statistically significant at 0.95 confidence level.Consequently frequency distributions of all trace elements can be considered normal.
Preliminary examination of the data by means of correlation plots could not detect any correlation between the trace elements and climatologic or hydrologic data, nor between the elements, not even in the case of the As -B couple.As generally accompanies B as a trace component in the Andean minerals so that a definite correlation could be expected to exist between the concentrations of these two elements.In spite of this, the Pearson correlation coefficient is very low (r 2 = 0.31).A similar behaviour of trace element concentrations has been observed by Helena et al. (2000).But PCA of their time-and site-dependent concentrations should provide information regarding the type and location of the still existing pollution sources, while Cu, Fe, Pb and Zn could be introduced by unauthorised sewage dumping into draining canals as well as by leaching of contaminated soil by rain, or by air borne particles.Nevertheless, PCA applied to 720 data revealed no clear associations between the contaminants nor did it show any grouping of the sampling sites.
While the area is affected by a sharp separation between dry and rainy seasons, which evidently has a strong influence on the concentrations, the splitting of data into two groups proved to be difficult.The best parameter for establishing limits between data corresponding to different seasons should be the river flow, but data were not available for all sampling points.Surface velocity values could not be correlated with the existing flow data, so it was felt that they did not represent the flow fluctuations.When concentration data of each trace element were distributed into two groups according to rainfall data for the sampling day, no statistically meaningful correlation could be obtained.This can be due to the fact that precipitation data are registered at a location 10 km to the West from the monitored river section, so that it frequently does not coincide with the actual situation in the city.Accumulated rainfall data for the 5 d (or 10 d) preceding sampling proved to be only slightly better, Spearman correlation coefficients for Cu and B with rainfall being the only ones to give higher t values than the tabulated one for a 0.995 confidence level.Plots of mean specific conductivity at the 6 sampling sites on the Arenales River vs. sampling date were finally taken into account to define time limits between seasons (Fig. 2).
Specific conductivity data distributed into two categories according to this criterion show the notoriously different behaviour of the water system according to the two seasons (Fig. 3), which could explain the impossibility to find any correlations when applying PCA to the complete set of data.In the dry seasons conductivity suddenly increases at Site 3, an indication of the existence of illegal sewage discharge.In the wet seasons, on the other hand, conductivity remains low, the sudden rise corresponding to sampling Site 7, 12 km downstream from the urban environment, a sign of inorganic, diffuse type of pollution.Concentration data distributed following this parameter produced Spearman´s correlation coefficients with t values higher than the tabulated one for 0.995 confidence level for all elements except Pb.Concentration ranges for dry and wet seasons defined according to this criterion are presented in Tables 3 and 4.
Sampling campaign No
B is the only element with mean concentration roughly following mean specific conductivity when represented vs. sampling campaign as well as vs. sampling sites, presenting maxi- 360 data for each season were normalised and then subjected to PCA.The covariance matrix for the dry seasons shows a correlation between B and As concentrations (with a coefficient of 0.51).According to the matrix for the wet seasons, correlation was only observed between Cu and Pb concentrations.For each season, the first 3 eigenvalues explained more than 70% of the system´s variance, being very similar in magnitude.
The first 3 PCs for both seasons combine all 6 variables, so none of them can be discarded.To achieve a sharper separation of the variables, as well as a better clustering of the objects, the first 3 PCs were subjected to rotation.The software 'Quimio' offers the Oblimin method for rotation, a method with the possibility of orthogonal or oblique rotation, according to the value selected for the γ parameter.With γ = 0 oblique rotations are performed by iterative calculations (constituting the Quartimin method) without the requirement of non-correlation of the rotated components.
Composition of the rotated PCs can be observed in Fig. 5. Separation of the variables is quite clear in the dry seasons, coupling As with B in the first PC and separating these two from the heavier elements.The 60 objects (sets of the concentrations of the 6 elements corresponding to a given sampling site and date), when pondered by the inverse of the square root of their communality, form definite clusters on the biplots (Fig. 6 -next page).
In dry weather, when there is no possibility of leaching or draining from diffuse contamination sources, the clustering of the sampling dates according to sampling points, with high scores on a rotated principal component, can be considered as a sign of the existence of point sources of contamination espe-cially rich in the trace elements with high loadings on the component.The origin of these contamination sources could be illegal sewage discharges as well as the existence of contaminant-containing sediments at the bottom of the river.
On the Mahalanobis biplots (Fig. 6 a and c) all the objects of Site 1 are clustered at the quarter opposite the vectors of all trace-element concentrations so that this site can be considered uncontaminated by the 6 elements.
Most of the objects corresponding to Site 2 are found along R 1 in Fig 6 (a) and (b), an indication that the site is contaminated mainly with B and As, that is to say, with boron minerals.The only possible source of contamination here is Canal A located upstream, into which a boron-processing plant used to flush its effluents before it was closed down.
Site 3 presents most of its objects arranged at negative values of R 1, so that there is no B and/or As contamination at this site.As these elements are highly soluble, and Site 2 was found to be polluted with them, a drainage entrance between the 2 sites must exist which produces their dilution.The rise of the specific conductivity at Site 3 (Fig. 3 a)) points to sewage of domiciliary origin.
Sites 4 and 5 have their objects aligned along positive values of R1 in Fig. 6 (a) and (b), and at high values of R2 in Fig. 6 (c).This means a new inflow of B and As, along with Zn, Cu and Fe.Probable sources of this contamination are Canals B and C. As the objects of Site 5 do not score higher on the rotated axes than those of Site 4, there is no evidence of raw effluents of the Industrial Park entering the river.
Objects belonging to Site 6 are scattered at high as well as at low values of R2, but they score low on R1: occasionally Cu and Zn concentrations are high at this site, but B and As are not important here as contaminants.This means that the effluents of the city sewage treatment plant most of the time dilute the polluting elements existing at Site 5, and do not contain B (or As), as can be expected from predominantly household effluents.
Most of the objects of Site 7 score low on R1, and on R2 as well, but not always on R3.This means that the river seems to have recovered from the B and As contamination, but occasionally still scores high in the quarter containing Cu and Zn.The rise of specific conductivity at this site, during both seasons (Fig. 3), indicates the concurrence of pollution of the point as well as of the diffuse type.
The loadings of the concentrations of the trace elements on the first three rotated components found for the wet seasons are different from those for the dry seasons (Fig. 5) but no clear conclusions could be derived from the biplots, the objects showing no clustering.This can be ascribed to the unfavourable relation between concentration variation and noise, due to the remarkable dilution produced by the heavy rains, and also to the correlations existing between the original components which persist on the rotated ones.Lower DLs for the elements are needed to derive conclusions from the data system.
Conclusions
PCA of trace element concentrations was used to provide answers not only to the location of pollution sources in the Arenales River, but also to their type.Accurate splitting of data into sets of wet and dry seasons, before applying this statistical method, was essential to their interpretation because of the characteristics of the river: low-level contamination, highly seasonal flow and several suspected intermittent pollution sources.While the entire set of data neither led to separation of the variables nor to any noticeable grouping of the objects, once split into two groups according to the cyclic variation of specific conductance, conclusions could be drawn from those corresponding to the dry seasons.Data belonging to the wet seasons behaved in the same way as the entire data population, confirming that the low concentration of the pollutants compared to the DLs of the analytical techniques employed caused the failure of PCA to yield meaningful results.Data of the dry seasons, on the contrary, were found to be useful to characterise the pollution of the river with trace elements.Two of the variables, B and As concentrations, were coupled on the first rotated component.This association allowed to ascertain their common origin as boron The biplots also confirmed the previously unsuspected existence of two other point-type sources of contamination contributing mainly Cu, Fe and Zn to the river water.The distribution of the objects on the biplots also led to the conclusions that the municipal sewage treatment plant effluent does not affect the quality of the river water.No conclusive evidence as to the recovery of the river from urban pollution at the last sampling site could be derived from the biplots.
aknowledgements
Funding for this research was provided by the Consejo de Investigación de la Universidad Nacional de Salta (Argentina).The authors want to thank Dr José Ávila Blas for controlling the statistical treatment of data.
Figure 1
Figure 1Map of the sampling sites on the Arenales River and the city of Salta.Numbers increase according to the river flow direction
Figure 3
Figure 3 Variation of specific conductivity along the downstream sampling line with data separated into two seasons: a) dry seasons b) wet seasons.Box sizes give the average values of two consecutive seasons of the same type and whiskers represent the general average with the average standard deviation of the data in the two seasons.
Available on website http://www.wrc.org.zaISSN 0378-4738 = Water SA Vol.33 No. 4 July 2007 ISSN 1816-7950 = Water SA (on-line) 483 mum values at sites 2, 4 and 5. Mean concentration of As follows the behaviour of B concentration, but it shows a sudden rise at the end of the 2003 dry season which spoils their simple lineal correlation (Fig. 4).
Figure 4 Figure 5
Figure 4Mean B and As concentrations (in mg/ℓ and µg/ℓ respectively), and mean specific conductivity (in µS/cm) vs. sampling campaign (a) and sampling site (b) Figure 6Mahalanobis biplot representation of the scores of the objects corresponding to the dry seasons on the rotated principal components.Loadings of trace element concentrations on the components are represented by vectors.Groups of sampling sites are highlighted in black frames.
TaBLE 2 Overall mean concentration of trace elements in arenales River and maximum acceptable values recommended by argentine national environmental law Nº 24 051
Available on website http://www.wrc.org.zaISSN 0378-4738 = Water SA Vol.33 No. 4 July 2007 ISSN 1816-7950 = Water SA (on-line) | v3-fos-license |
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} | pes2o/s2orc | Substrate Specificity of δ Ribozyme Cleavage*
The specificity of δ ribozyme cleavage was investigated using a trans-acting antigenomicδ ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min−1, an apparent K m of 17.9 nm and an apparent second-order rate constant of 1.89 × 107 min−1 m −1. The substrate specificity of theδ ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of δ ribozyme cleavage.
M ؊1 . The substrate specificity of the ␦ ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of ␦ ribozyme cleavage.
␦ ribozymes derived from the genome of hepatitis ␦ virus (HDV) 1 are metalloenzymes. Like other catalytically active ribozymes, namely hammerhead and hairpin ribozymes, the ␦ ribozymes cleave a phosphodiester bond of their RNA substrates and give rise to reaction products containing a 5Јhydroxyl and a 2Ј,3Ј-cyclic phosphate termini. Two forms of ␦ ribozymes, namely genomic and antigenomic, were derived and referred to by the polarity of the HDV genome from which the ribozyme was generated. Both ␦ ribozyme forms exhibit selfcleavage activity, and it has been suggested that they are involved in the process of viral replication (1). This type of activity has been described as cis-acting ␦ ribozymes (2).
Like other ribozymes, ␦ ribozymes have a potential application in gene therapy in which an engineered ribozyme is directed to inhibit gene expression by targeting a specific mRNA molecule. It has been demonstrated that a very low concentration (Ͻ0.1 mM) of Ca 2ϩ and Mg 2ϩ is required for ␦ ribozyme cleavage (3). ␦ ribozymes have a unique characteristic in their substrate binding, namely that only the 3Ј-portion of the substrate is required for binding to the ribozyme. A short stretch of nucleotides (7 nt) located on the substrate is required for cleavage. Although one might suspect the specificity of ␦ ribozyme cleavages due to their short recognition site, we view this characteristic of the ␦ ribozyme as an advantage for the future development of a therapeutic means of controlling, for example, a viral infection.
Since little is known about the kinetic properties of ␦ ribozymes, study of the trans-acting system will enable us to answer some basic questions on both the structure required and the kinetic properties, including the substrate specificity, of ␦ ribozymes. Depending on the predicted secondary structures used, various trans-acting ␦ ribozyme systems were generated by separating the RNA molecule into ribozyme and substrate molecules at various positions (4 -6). Here, we generate a trans-acting ␦ ribozyme, based on the pseudo knot-like structure proposed by Perrotta and Been (2), by separating the single-stranded region located at the junction between the P1 and P2 stems (Fig. 1). Although, several investigations have been performed to address the questions related to the substrate specificity of ␦ ribozymes in both the cis-and transacting forms (2,(5)(6)(7)(8)(9)(10)(11)(12), most, if not all, experiments were carried out by randomly changing the base pairing combinations or by introducing mismatches which interfere with the Watson-Crick base pairing between the substrate and the ribozyme in the P1 stem (Fig. 1). It was demonstrated that cleavage activity was not destroyed by the interchanging of one to four nucleotide pairs between the substrate and the ␦ ribozyme (2,8,11,12). One or two nucleotide mismatches at any position of the P1 stem, except positions 5 and 11 (numbering according to Fig. 1), completely destroyed the activity (2,(5)(6)(7)(8)(9)(10)(11)(12). Although these are composite results from various versions of ␦ ribozymes, these findings could be interpreted as indicating that the positions located at both extremities of the base paired stem formed by the substrate and the ribozyme were more likely to tolerate a mismatch, resulting in distortion of the P1 stem, than the internal positions. There is no information on how each nucleotide of the substrate affects the cleavage activity and its kinetics since most investigations were carried out at only one or two positions at a time, and the findings generally reported in a plus/minus manner (e.g. cut or uncut). Therefore, the substrate specificity of ␦ ribozyme could not be deduced from previous reports. To determine how substrate sequences affect ␦ ribozyme cleavage activity, we performed kinetic studies using a collection of short oligonucleotide substrates (11 nt) with a trans-acting ␦ ribozyme. In this report, we demonstrate that each nucleotide of the P1 stem contributes differently to the cleavage activity. We compare the observed cleavage rate constants for cleavable substrates and the equilibrium dissociation constants for the uncleavable substrates with those of the wild type substrate. We present evidence that strongly suggests that the nucleotides located in the center of the P1 stem formed between substrate and ribozyme ( Fig. 1, positions 7 and 8) are important not only for substrate recognition but probably also for subsequent steps, for example a conformation change yielding a transition complex.
Plasmids Carrying ␦ Ribozymes
The antigenomic ribozyme sequence of the hepatitis ␦ virus described by Makino et al. (13) was used to generate a trans-acting ␦ ribozyme with some modifications as shown in Fig. 1. Briefly, the construction was performed as follows. Two pairs of complementary and overlapping oligonucleotides, representing the entire length of the ribozyme (57 nt), were synthesized and subjected to an annealing process prior to cloning into pUC19. The annealed oligonucleotides were ligated to HindIII and SmaI co-digested pUC19 to give rise to a plasmid harboring the ␦ ribozyme (referred to as p␦RzP1.1). A mutant ribozyme (␦RzP1.2) was then constructed by modifying the substrate recognition site of p␦RzP1.1 by ligation of an oligonucleotide containing the altered sequence flanked by restriction endonuclease sites to RsrII/SphI predigested p␦RzP1.1. The sequences of engineered ribozymes were confirmed by DNA sequencing. Plasmids containing wild type and mutant ribozymes were then prepared using Qiagen tip-100 (Qiagen Inc.), digested with SmaI, purified by phenol and chloroform extraction, and precipitated for further use as templates for in vitro transcription reactions.
RNA Synthesis
Ribozyme-In vitro transcription reactions contained 5 g of linearized recombinant plasmid DNA as template, 27 units RNAGuard RNase inhibitor (Amersham Pharmacia Biotech), 4 mM of each ribonucleotide (Amersham Pharmacia Biotech), 80 mM HEPES-KOH, pH 7.5, 24 mM MgCl 2 , 2 mM spermidine, 40 mM dithiothreitol, 0.01 unit of pyrophosphatase (Boehringer Mannheim) and 25 g of purified T7 RNA polymerase in a final volume of 50 l, and were incubated at 37°C for 4 h.
Substrates-Deoxyoligonucleotides (500 pmol) containing the substrate and T7 promoter sequence were denatured by heating at 95°C for 5 min in a 20-l mixture containing 10 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 50 mM KCl 2 , and allowed to cool slowly to 37°C. The in vitro transcription reactions were carried out using the resulting partial duplex formed as template under the same conditions as described for the production of the ribozyme.
After incubation, the reaction mixtures were fractionated by denaturing 20% polyacrylamide gel electrophoresis (19:1 ratio of acrylamide to bisacrylamide) containing 45 mM Tris borate, pH 7.5, 7 M urea, and 1 mM EDTA. The reaction products were visualized by UV shadowing. The bands corresponding to the correct sizes of either ribozymes or substrates were cut out, and the transcripts eluted overnight at 4°C in a solution containing 0.1% SDS and 0.5 M ammonium acetate. The transcripts were then precipitated by the addition of 0.1 volume of 3 M sodium acetate, pH 5.2, and 2.2 volumes of ethanol. Transcript yield was determined by spectrophotometry.
End-labeling of RNA with [␥-32 P]ATP
Purified transcripts (10 pmol) were dephosphorylated in a 20-l reaction mixture containing 200 mM Tris-HCl, pH 8.0, 10 units of RNAGuard ® , and 0.2 units of calf intestine alkaline phosphatase (Amersham Pharmacia Biotech). The mixture was incubated at 37°C for 30 min and then extracted twice with a same volume of phenol:chloroform (1:1). Dephosphorylated transcripts (1 pmol) were end-labeled in a mixture containing 1.6 pmol [␥-32 P]ATP, 10 mM, Tris-HCl, pH 7.5, 10 mM MgCl 2 , 50 mM KCl, and 3 units of T4 polynucleotide kinase (Amersham Pharmacia Biotech) at 37°C for 30 min. Excess [␥-32 P]ATP was removed by applying the reaction mixture onto a spin column packed with a G-50 Sephadex gel matrix (Amersham Pharmacia Biotech). The concentration of labeled transcripts was adjusted to 0.01 pmol/ml by the addition of water.
Cleavage Reactions
To initiate a cleavage reaction, we tested different procedures and chose the method that yielded the highest cleavage rate constant and the maximum cleavage product as described by Fauzi et al. (14). Various concentrations of ribozymes were mixed with trace amounts of substrate (final concentration Ͻ1 nM) in a 18-l reaction mixture containing 50 mM Tris-HCl, pH 7.5, and subjected to denaturation by heating at 95°C for 2 min. The mixtures were quickly placed on ice for 2 min and equilibrated to 37°C for 5 min prior to the initiation of the reaction. Unless stated otherwise, cleavage was initiated by the addition of MgCl 2 to 10 mM final concentration. The cleavage reactions were incubated at 37°C, and followed for 3.5 h or until the end point of cleavage was reached. The reaction mixtures were periodically sampled (2-3 l), and these samples were quenched by the addition of 5 l of stop solution containing 95% formamide, 10 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol. The resulting samples were analyzed by a 20% polyacrylamide gel electrophoresis as described above. Both the substrate (11 nt) and the reaction product (4 nt) bands were detected using a Molecular Dynamics radioanalytic scanner after exposition of the gels to a phosphorimaging screen.
Kinetic Analysis
Measurement of Pseudo First-order Rate Constant (k cat , K m and k cat / K m )-Kinetic analyses were performed under single turnover conditions as described by Hertel et al. (15) with some modifications. Briefly, trace amounts of end-labeled substrate (Ͻ1 nM) were cleaved by various ribozyme concentrations (5-500 nM). The fraction cleaved was determined, and the rate of cleavage (k obs ) obtained from fitting the data to the equation where A t is the percentage of cleavage at time t, A ϱ is the maximum percent cleavage (or the end point of cleavage), and k is the rate constant (k obs ). Each rate constant was calculated from at least two measurements. The values of k obs obtained were then plotted as a function of ribozyme concentrations for determination of the other kinetic parameters: k cat , K m and k cat /K m . Values obtained from independent experiments varied less than 15%. The requirement for Mg 2ϩ by both ribozymes was studied by incubating the reaction mixtures with various concentrations of MgCl 2 (1-500 mM) in the presence of an excess of ribozyme (500 nM) over substrate (Ͻ1 nM). The concentrations of Mg 2ϩ at the half-maximal velocity were determined for both ribozymes.
Determination of Equilibrium Dissociation Constants (K d )-For mismatched substrates that could not be cleaved by the ribozyme, the equilibrium dissociation constants were determined using a slight modification of the method described by Fedor and Uhlenbeck (16). Eleven different ribozyme concentrations, ranging from 5 to 600 nM, were individually mixed with trace amounts of end-labeled substrates (Ͻ1 nM) in a 9-l solution containing 50 mM Tris-HCl, pH 7.5, heated at 95°C for 2 min and cooled to 37°C for 5 min prior to the addition of MgCl 2 to a final concentration of 10 mM, in a manner similar to that of a regular cleavage reaction. The samples were incubated at 37°C for 1.5 h, at which time 2 l of sample loading solution (50% glycerol, 0.025% of each bromphenol blue and xylene cyanol) was added, and the resulting mixtures were electrophoresed through a nondenaturing polyacrylamide gel (20% acrylamide with a 19:1 ratio of acrylamide to bisacrylamide, 45 mM Tris borate buffer, pH 7.5 and 10 mM MgCl 2 ). Polyacrylamide gels were prerun at 20 W for 1 h prior to sample loading, and the migration was carried out at 15 W for 4.5 h at room temperature. Quantification of bound and free substrates was performed following an exposure of the gels to a phosphorimaging screen as described earlier.
RESULTS
The trans-acting ␦ ribozymes used in this report were derived from the antigenomic ␦ ribozyme of HDV (13). Some features of the antigenomic ␦ ribozyme were modified to improve its structural stability and to aid in transcript production. Based on a pseudo knot-like structure described by Perrotta and Been (2), Fig. 1 shows the structure of the ␦ ribozymes used with some modifications: (i) the singlestranded region between substrate and ribozyme (region J1/2) was eliminated to separate the substrate molecule from the ribozyme; (ii) the substrate contains only 11 nt and produces 7and 4-nt cleavage products, and the GGG at the 5Ј-end was added to increase the yield during in vitro transcription (17); (iii) three G-C base pairs were introduced in the P2 region to improve both the structural stability and transcript production; and (iv) the P4 stem was shortened to the minimum length reported to result in an active ribozyme (18). Prior to performing a cleavage reaction, native gel electrophoresis was used to test for the possible presence of aggregates or multimer forms of the transcripts. Various concentrations of ribozyme, ranging from 5 nM to 2 M, were mixed with trace amounts of end-labeled ribozyme (less than 0.5 nM) and fractionated under nondenaturing conditions as described under "Materials and Methods." We detected the presence of a slow migrating species of ribozyme in the mixture containing 2 M ribozyme (data not shown). The quantification of the slow migrating band showed that the band amounted to approximately 2% of the total radioactive material. However, a single band was detected at the concentrations used for kinetic analysis and under single turnover conditions (5-600 nM). Similar experiments were performed for each substrate. There was no substrate multimer detected at the concentrations used (data not shown). The equimolar mixture of end-labeled substrate and ribozyme was also fractionated under nondenaturing conditions, and it resulted a single band of ribozyme and substrate complex similar to those observed for the K d measurement shown in Fig. 4.
Cleavage Kinetics of Constructed Antigenomic ␦ Ribozymes
Two forms of trans-acting ␦ ribozymes (␦RzP1.1 and ␦RzP1.2) were used with their corresponding substrates (11 nt) for the kinetic studies. ␦RzP1.2 differs from ␦RzP1.1 in that ␦RzP1.2 has two nucleotides, at positions 22 and 24 of ␦RzP1.1, interchanged ( Fig. 1, 5Ј-CCCAGCU-3Ј). Time course experiments for cleavage reactions catalyzed by both ␦RzP1.1 and ␦RzP1.2 were monitored by the appearance of the 4 nt cleavage product. An example of a time course experiment for a cleavage reaction catalyzed by ␦RzP1.1 is shown in Fig. 2, panel A. In this particular experiment, 100 nM of ␦RzP1.1 was incubated with 1 nM end-labeled substrate, SP1.1. The newly formed product and the remaining substrate bands at each time point were quantified, and the percentage of cleavage was plotted as a function of time (Fig. 2, panel B). ␦RzP1.1 cleaved approximately 60% of the substrate within 10 min. The data were fitted to a single exponential equation as described under "Materials and Methods" so as to obtain the observed rate constant (k obs ϭ 0.21 min Ϫ1 ). We attempted to fit the data as biphasic reactions as described for the hairpin (19) and the hammerhead (20) ribozymes. We observed that the standard deviation ( 2 ) of data fitted to a double-exponential equation was higher ( 2 ϭ 0.01203) than that fitted to a single exponential equation ( 2 ϭ 0.000203). Although we could not exclude or dismiss completely the possibility that more than one conformation of the active ribozyme could be formed, the data were treated as if the reactions were monophasic in their kinetics for comparison purposes.
Similar experiments were performed using trace amounts of substrate (Ͻ1 nM) and various ribozyme concentrations to measure k obs at each ribozyme concentration. The values of k obs of both ␦RzP1.1 and ␦RzP1.2 increased with an increase in ribozyme concentration up to approximately 200 nM (Fig. 3, panel A). The concentration of ribozyme at which the reaction velocity reached half-maximal (apparent K m , K m Ј) is 17.9 Ϯ 5.6 nM for ␦RzP1.1 and 16.7 Ϯ 6.4 nM for ␦RzP1.2. Under the reaction conditions used, in which the increase in ribozyme concentration has no significant effect on the rate of cleavage, the cleavage rate (k obs ) is therefore represented by the catalytic rate constant (k cat ). The cleavage rate constants are 0.34 min Ϫ1 for ␦RzP1.1 and 0.13 min Ϫ1 for ␦RzP1.2. Apparent second-order rate constants (k cat /K m Ј) were calculated to be 1.89 ϫ 10 7 min Ϫ1 M Ϫ1 for ␦RzP1.1 and 0.81 ϫ 10 7 min Ϫ1 M Ϫ1 for ␦RzP1.2 (Table I).
Since we observed that the k cat of ␦RzP1.2 is about 3 times less than that of ␦RzP1.1, whereas the K m Ј is similar, we investigated whether an increased amount of Mg 2ϩ in the cleavage reaction would affect the k cat of ␦RzP1.2. Under single turnover conditions, in which the ribozyme and substrate concentrations were kept at 500 and 1 nm, respectively, we found that both ribozymes cleave their complementary substrates at Mg 2ϩ concentrations as low as 1 mM, which is the estimated physiological concentration of Mg 2ϩ (21). At this concentration, the k obs obtained were 0.11 Ϯ 0.01 and 0.04 Ϯ 0.01 min Ϫ1 , for ␦RzP1.1 and ␦RzP1.2, respectively (Fig. 3, panel B). A maxi-mum k obs for ␦RzP1.2 was observed when the concentration of Mg 2ϩ was 10 mM. Higher concentrations of Mg 2ϩ did not increase either the k obs or the extent of cleavage for both ribozymes. We did not observe a decrease in the cleavage rate when higher concentrations of Mg 2ϩ were used (e.g. 500 mM). The requirement for magnesium at half-maximal velocity (K Mg ) was 2 mM for both ␦RzP1.1 and ␦RzP1.2.
Substrate Specificity
To compare the specificity of the ␦ ribozyme with various substrates, ␦RzP1.1 was used under single turnover conditions as described above. The cleavage reactions were performed with a trace amount of each substrate (Ͻ1 nM) and 500 nM ␦RzP1.1. Under these conditions, the observed rates reflect the rates of cleavage without interference from either product dissociation or inhibition. For each substrate both the observed cleavage rate constants (k obs ) and the extent of cleavage were calculated and compared with those of the wild type substrate, as shown in Table II.
Shorter Substrates-Three shorter substrates containing 10, 9, and 8 nt were tested individually and compared with the 11-nt substrate (SP1.1) in which 7 nt base paired with ␦RzP1.1. The 10-, 9-, and 8-nt substrates contain 6, 5, and 4 nt regions complementary to ␦RzP1.1, respectively. We observed that the 10-nt substrate was cleaved with a k obs of 0.02 Ϯ 0.01 min Ϫ1 and a maximal cleavage of 28.8% (Table II). We could not detect the cleavage product formed when the 9-and 8-nt substrates were used, even after a 3.5-h incubation time. The cleavage reactions were also carried out in the presence of 100 mM Mg 2ϩ instead of the 10 mM concentration used in a regular cleavage reaction. We observed no improvement in the values of the k obs and the extent of cleavage for the 10-nt substrate and still detected no cleavage for both the 9-and 8-nt substrates.
Mismatched Substrates-We have generated a collection of substrates in which single mismatches were individually introduced into the P1 region of the substrate and then used in the cleavage reactions (Table II). Mutation at position 5 resulted in at least a 9-fold decrease in k obs as compared with that of SP1.1 (0.34 min Ϫ1 ). However, for SG5A, in which A was substituted for G at position 5 of SP1.1, the extent of cleavage was only reduced by half. When this nucleotide was changed to cytosine, the cleavage was reduced almost to nil (ca. 1.7%). ␦RzP1.1 cleaved approximately 4% of the SG6A and SG6U substrates, in which A or U were substituted for G at position 6. The alteration of either position 7 or 8, located in the middle of the P1 stem, yielded uncleavable substrates (SG7A, SG7U, SU8C, SU8G). The k obs was also drastically decreased when the C at position 9 was altered to A or U. The extent of cleavage was reduced to approximately 50%, when SC9U was used. The SG10U substrate, in which U was substituted for G at position 10, gave a similar result to SC9A. Finally, ␦RzP1.1 cleaved the substrate SG11U almost as well as SP1.1, although the k obs was considerably slower (0.01 min Ϫ1 ). The relative activity of each single mismatched substrate was calculated to obtain an apparent free energy of transition-state stabilization, ⌬⌬G ‡ (22,23). We found that the values of ⌬⌬G ‡ range between Ϫ0.96 to Ϫ2.25 kcal Ϫ1 mol Ϫ1 . This apparent difference in activation energy was also observed when substrates of leadzyme were altered and used in a cleavage assay (22).
Equilibrium Dissociation Constant (K d )
The four substrates containing a single mismatch either at position 7 or 8, which were not cleaved by ␦RzP1.1, were used to determine an equilibrium dissociation constant (K d ). Trace amounts of end-labeled substrates (SG7A, SG7U, SU8C, or SU8G) were individually incubated with various concentra- tions of ␦RzP1.1 for the gel shift analysis as described under "Materials and Methods." To ensure that the dissociation equilibrium was reached, we incubated the reaction mixtures at various intervals. We found that the equilibrium was reached within 5 min, and that a longer incubation of 28 h did not affect the measurement of K d . Since SP1.1 can be cleaved under native gel electrophoresis conditions, we therefore used its analog which has a deoxyribose at position 4 (SdC4) to obtain the estimated K d of the wild type substrate. This analog could not be cleaved by ␦RzP1.1 under the conditions used (2), and has been shown to be a competitive inhibitor of ␦RzP1.1 cleavage. 2 An example of a gel shift analysis carried out for the analog is shown in Fig. 4. In this particular analysis, trace amounts of SdC4 (Ͻ1 nM) were incubated with 11 concentrations of ␦RzP1.1 ranging from 5 to 600 nM. An autoradiogram of the resulting gel obtained by a Molecular Dynamics radioanalytic scanner is shown in Fig. 4, panel A. The bands of the bound SdC4 and the free SdC4 at each ␦RzP1.1 concentration were quantified, and the percentage of the bound SdC4 was plotted as shown in Fig. 4, ␦ ribozymes derived from the genome of HDV are of interest in the development of a gene regulation system in which the designed ribozymes would down-regulate the expression of a target gene. The facts that ␦ ribozymes are derived from HDV and that this pathogen naturally replicates in animal systems, suggest that this catalytic RNA could be used to control gene expression in human cells. Like other ribozymes, the designed ribozyme should specifically cleave its target substrates while leaving other cellular RNA molecules intact. We designed a trans-acting ␦ ribozyme harboring a recognition sequence similar to the HDV antigenomic ␦ self-cleaving motif so as to have a minimal system for the study of the specificity of the base pairing interaction between the ␦ ribozyme and its substrate.
Although a number of trans-acting ␦ ribozymes have been generated, they appear to have variable cleavage rate constants. The discrepancy of cis-acting ␦ ribozyme activities has been reviewed, and it was suggested that the variation of the cleavage activity, at least for cis-acting forms, may result from the nonribozyme flanking sequences used by each investigator (24). Our trans-acting ␦ ribozyme, ␦RzP1.1, exhibited an activity with a cleavage rate of 0.34 min Ϫ1 , or a t 1/2 of 2 min, under pseudo first-order conditions. These data are in good agreement with the observed rate constant (0.35 min Ϫ1 ) of a cisacting ␦ ribozyme derived from antigenomic HDV RNA (7). We found that the extent of cleavage is approximately 60%, regardless of the concentration of ribozyme used, suggesting possibilities that (i) a fraction of the substrate was bound to an inactive form of the ␦ ribozyme; (ii) substrate was bound to Cs of the 3Ј of the ribozyme, instead of to the P1 region of the ribozyme, causing a misfold or a nonactive substrate-ribozyme complex; or (iii) a portion of the ribozyme might adopt another conformation following substrate binding. Based on the latter hypothesis, the alternative form of ribozyme-substrate complex could undergo cleavage at a very low rate. We first investigated whether or not the presence of the alternative form could be a result of an infidelity of the T7 RNA polymerase transcription. Two batches of purified T7 RNA polymerase were tested using various amounts of enzyme and incubation times (data not shown). We found that the transcripts produced by both Bold letters represent the nucleotides of wild type substrate recognized by ␦RzP1.1. The numbers in subscript indicate the nucleotides of wild type substrate that were individually altered to generate shorter or mismatched substrates. k obs is the observed rate of cleavage calculated from at least two measurements. Cleavage extent (%) is obtained by fitting the data to the equation A t ϭ A ϱ (1 Ϫ e Ϫkt ), where A t is the percentage of cleavage at time t, A ϱ is the maximum percentage of cleavage, and k is the rate constant. k rel is the relative rate constant as compared to that of wild-type substrate. ⌬⌬G ‡ , the apparent free energy of transition-state stabilization, was calculated using the equation ⌬⌬G ‡ ϭ RT ln k rel , where T ϭ 310.15 K (37°C) and R ϭ 1.987 cal ⅐ K Ϫ1 mol Ϫ1 . batches of purified T7 RNA polymerase at the different incubating times exhibited a similar cleavage pattern and extent, suggesting that it is the nature of ribozyme transcripts to adopt an alternative form in the reaction mixtures, as previously reported for the hairpin ribozyme (19). A possible occurrence of misplaced or misfold substrate-substrate complex was dismissed since there is no evidence of other formed complexes detected under nondenaturing gel electrophoresis and also by RNase mapping. 3 Finally, the possible occurrence of a slow cleaving form of ␦ ribozyme was assessed following cleavage reactions. We attempted to fit the experimental data using a multiphasic kinetic equation. Since we could not clearly describe the kinetics of our trans-acting ␦ ribozyme as biphasic or multiphasic reactions, we measured initial rates of cleavage for comparative purposes.
To summarize the cleavage reactions catalyzed by ␦RzP1.1 and ␦RzP1.2, free energy diagrams of the reaction coordinates were constructed (Fig. 5). The diagrams relate the two states in the cleavage reactions using kinetic parameters obtained under single turnover conditions. ␦RzP1.1 and ␦RzP1.2 differ in that they have two base pairs in the middle of the P1 stem interchanged. As expected, the free energies of substrate binding are virtually identical (Ϫ11 kcal Ϫ1 mol Ϫ1 ). The base pair interchange in ␦RzP1.2 increased the value of ⌬G ‡ by approximately 0.5 kcal Ϫ1 mol Ϫ1 . It is interesting to note that the free energy of the transition state was affected by the changes in the base pairing of the P1 stem. Although several kinetic parameters were greatly different from those reported here, similar findings were previously reported when two nucleotides (positions 7 and 8) of the substrate were interchanged and complemented by the ␦ ribozyme (12). Since the kinetics of ␦ cleavage reactions appear to be affected by the particular combination of base pairs, it is very likely that in addition to P1 base pairing a tertiary interaction might also participate in substrate recognition. In this scenario the substrate-ribozyme complex would undergo a conformational transition, following forma- tion of P1 stem, which involves tertiary interaction(s). These interactions might result in the positioning of the scissile bond in the catalytic center, a key step in the reaction pathway. The substrate specificity of the ␦ ribozyme was studied using ␦RzP1.1. First, we found that the ␦ ribozyme can cleave a substrate having a minimum of 6 nucleotides adjacent to the cleavage site. This result is in an agreement with those previously reported for both the cis-acting form (5) and the transsystems (11) that a minimum of 6 base pairing is required for cleavage. The k obs of the 10-nt substrate is at least 10 times slower than that of SP1.1 (Table II). We also used shorter substrates generated by alkali hydrolysis as described by Perrotta and Been (11) to verify the cleavage reactions catalyzed by the two similar trans-acting ␦ ribozymes. Due to the slow cleavage rate, the detection of the disappearance of shorter substrates in the mixtures could not accurately be measured (data not shown).
Second, to estimate the contribution of base pairing interaction of the P1 stem to the cleavage reaction, a collection of single mismatched substrates was generated by introducing point mutations into the substrate sequence. Although there are a number of reports on the base pairing requirement of the P1 region (8, 10 -12, 14), no extensive investigation has been performed on each individual nucleotide of either cis-or transacting ␦ ribozymes. The determination of ribozyme specificity against various substrates was first attempted by comparing the apparent second-order rate constant (k cat /K m Ј) of each substrate to that of wild type substrate. We found that the ribozyme cleaved single mismatched substrates very slowly and gave a low percent cleavage (maximum of 2-20%) within the reaction time studied (3.5 h). As a consequence, the measurement of the apparent second-order rate constants as a function of ribozyme concentration yielded values with a high margin of error. We thus reported the cleavage activity of the ribozyme against various single mismatched substrates in terms of extent of cleavage and k obs , which at a high ribozyme concentration reflects the k cat of the cleavage reaction. In all cases, we observed the decrease in cleavage extent, which we suspected to be due mainly to the poor binding between the substrate and the ribozyme. The wobble base pair (G-U) at the cleavage site is required to maintain a high level of cleavage (10,11). Mismatches at this position, which create either an A-U or a C-U pairing, decreased the cleavage activity in a manner analogous to that reported in another version of trans-acting ␦ ribozymes (10). It is interesting to note that the extent of cleavage decreases proportionally to the mismatches introduced into the 3Ј and 5Ј positions of the middle of the P1 stem. The simultaneous alteration of two nucleotides in the middle of the P1 stem was reported to give rise to an uncleavable substrate in both the cisand trans-acting systems (11). However, in both cases the activity could be restored by the generation of a complementary ribozyme or a substrate.
The calculated free energy of transition-state stabilization (⌬⌬G ‡ ) for each substrate listed in Table II varies between Ϫ0.9 and Ϫ2.25 kcal Ϫ1 mol Ϫ1 . Each position of base pairing between the substrate and the ribozyme appears to affect the reaction pathway differently, at least with regard to transition state complex formation. If we assume that mismatched substrates yield the same level of ⌬G E⅐S , various end points of cleavage for mismatched substrates could be resolved depending upon the height of the energy barrier level to be overcome in the transition state. To address these questions precisely, more experiments on the equilibrium binding constant and the internal equilibrium of the reactions are required. We have determined the calculated K d of P1 duplex formation using the equation described by Serra and Turner (25) to be 28.5 nM. By using an analog, we have shown that the K d of the wild type substrate to its ribozyme is 31.9 nM. It is very interesting to note that the mismatch introduced at position U8 of the substrate has little effect on substrate binding affinity. However, the change completely eliminated cleavage activity. The mismatch introduced at position G7 of the substrate affected both the binding and chemical steps since it not only lowered the binding affinity of the substrate for the ribozyme, but also destroyed the cleavage activity. These findings suggest that some base pairs of the P1 stem have dual roles, participating in the substrate binding and subsequent steps leading a chemical cleavage, as was observed for the base pair interactions between the hammerhead ribozyme and its substrates (26). To address these findings more precisely some preliminary experiments have been carried out using the metal-ion induced cleavage method to study the tertiary structure of ␦ ribozyme 3. The data obtained to date suggests that positions U8 and G7 are likely involved in the formation of an essential metal-ion binding site. The mismatches introduced at either of the two positions destroyed the formation of this metal-ion binding site, a process which has been found to be highly associated with cleavage activity.
We present here evidence that aside from the base pairing between the substrate and the ribozyme, tertiary interactions, especially ones involving the P1 stem, appear to dictate the reaction pathway of ␦ ribozyme. To fully comprehend how the cleavage reactions are governed, the elucidation of these tertiary interactions is essential. | v3-fos-license |
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} | pes2o/s2orc | Structural Properties of Casein Micelles in Milk , the effect of salt , temperature , and ph Research Article Open Access
Milk is a complex liquid, which contains many different species, for example proteins, fat, minerals etc. It is the primary source of nutrition for young mammals before they are able to digest other types of food. The proteins can be divided into two groups: caseins and whey. Whey proteins are about 20 wt % of the total protein amount in milk, whereas the caseins corresponds 80 wt % of the total protein content in milk. The largest structures in the fluid portion of the milk are “casein micelles” which are aggregates of several thousands of protein molecules. The micelle is considered to be spherical and the diameter is in the micrometer size. The caseins can be divided in four types: αs1casein, αs2 casein, β casein, and k casein. ß casein is one of the most abundant caseins and it also self assembles to larger aggregates. In this thesis we have used a simple model to try to capture how electrostatic interactions affects the structure of β casein micelles in milk. The micelles have been modeled as hard spheres, with a central net charge of --140e, and a radius of 75 Å. These parameters have been taken from experimental data published in the literature. The structure of the solution has been studied by comparing the radial distribution functions for different solution conditions, such as the salt concentration and valency, pH, and the temperature. Popular scientific description Milk is a complex liquid, which contains many different species, for example proteins, fat, minerals etc. It is the primary source of nutrition for young mammals before they are able to digest other types of food. The proteins can be divided into two groups: caseins and whey. Whey proteins are about 20 wt % of the total protein amount in milk, whereas the caseins corresponds 80 wt % of the total protein content in milk. The largest structures in the fluid portion of the milk are “casein micelles” which are aggregates of several thousands of protein molecules. The micelle is considered to be spherical and the diameter is in the micrometer size. In this thesis we have used a simple model and computer simulations to try to capture how electrostatic interactions affects the structure of β casein micelles in milk. The micelles have been modeled as hard spheres, with a central net charge of 140e, and a radius of 75 Å. The volume fraction of was set to 5%, which is the actual volume fraction in the real product. The structure of the solution has been studied by comparing the radial distribution functions for different solution conditions, such as the salt concentration and valency, pH, and the temperature. It was noticed that due to the fact that the micellar charge is very large, the electrostatic repulsive interaction dominates, and the mean distance between the micelles are almost always obtained. Moreover, an increase of the temperature does not affect the structure at all i.e. the entropic contribution due to increased temperature can be neglected in comparison with electrostatic repulsion between the micelles. Also, there might also be an influence of the electric permittivity since it was kept constant during the simulations, When the salt concentration was increased to 80 mM, which corresponds to the ionic strength in milk, the structure of the β casein micelles resembles the structure of an ideal gas i.e. the electrostatic repulsive interactions are screened
Elsaid Younes (2017), Structural Properties of Casein Micelles in Milk, the effect of salt, temperature, and pH. Int J Biotech & Bioeng. 3:6, 204-220. DOI: 10.25141/2475-3432-2017 Introduction: Milk is as ancient as human beings themselves. Historians believe that humans started to drink milk over 10,000 years ago, along with the start of the domestication of animals. Milk is the normal product of the mammary glands of female mammals. Its purpose is primarily to meet the nutritional requirements of the neonate. It contains more than 100 substances, and it is the most complete and complex known natural food. Milk is perishable because it is a very good environment for the growth of the microorganisms that caused the FDA to warn people from drinking raw milk in March 2007. They wrote: Pasteurized milk does safe live! [41] The pasteurization of milk is heating the milk in a certain way to save the nutritional value of the milk while destroying the harmful bacteria.
The tropical countries are not considered consumers of milk, because of the high temperature and the lack of refrigeration, which makes the storage of milk difficult. The north part of the world, especially northern Europe and North America, is considered a big consumer of milk and milk products. Milk has traditionally been preserved by converting it to more stable products like making cheese and yogurt by milk fermentation. [33] Milk processing: Pasteurization: Because of the perishability of milk, milk storage has been an important process. The most widely used process, for preserving fresh milk, is pasteurization, which was named after the French microbiologist Louis Pasteur in 1864.
The most popular methods of pasteurization are: 1)HTST: high temperature short time pasteurization, which requires heating milk to 720 C for 15 seconds, and can extend the shelf life to a week.
2)UHT: ultra high temperature pasteurization, which requires heating the liquid to 1380 C for 2 seconds, and can extend the shelf life for weeks. It causes some changes of the structure of the milk protein. [2] Fermentation Other processes, that are popular for preserving milk, involve preserving milk components like fat and protein. These processes are modifying the environment of the milk and extend the lifetime of the products. Adding lactic acid bacteria to the milk converts lactose (milk sugar) to lactic acid, which leads to the dissociation of the casein micelles. After that, the viscous gel solution has the sour taste of yogurt. After adding the lactic acid bacteria to the milk, the lactose is converted to lactic acid, which decreases the pH of the milk. Rennet is added to convert the casein micelle into a stable protein that does not dissociate in water.
The content of the milk differs according the mammals it comes from as shown in table 1. The components also depend on the nutrition and the season. Cow's milk for example contains about 87% water. [34]
Milk components: Milk fat:
Fat represents about 3.5--6.5 % of milk, and it is the most variable component. It is secreted as a fat globule surrounded by a lipid bilayer membrane. When raw milk is centrifuged, the fat moves to the top and forms a cream layer, because the lipid is lower in buoyant density, see Figure 1. Elsaid Younes (2017), Structural Properties of Casein Micelles in Milk, the effect of salt, temperature, and pH. Int J Biotech & Bioeng. 3:6, 204-220. DOI: 10.25141/2475-3432-2017 Figure 1 shows the formation of two layers upon centrifugation of the milk. The upper layer is the lipid and it forms a creamy layer, and the layer at the bottom is formed from skim milk (milk without fat). This is because the lipid layer has a low buoyant density.
The cream contains some proteins (Mucin, Xanthine oxidase and butyrophilin), which are carried by the fat globules. It has been suggested that the fat globules are accumulated between the two halves of the milk lipid globule membrane (MLGM), which is part bilayer and part protein (see figure 2). Milk fat globules (MFG), whose diameters range from 0.2 -m to 20 -m, with a mean diameter of 4-m (Mulder and Walstra, 1974), are essentially from triglycerides, which are esters derived from glycerol combined with three fatty acids. It has been found that the triglyceride forms 98% of the fat content, and the phospholipid forms 1% of the fat content.
Figure 2.
The left image of the adipocytes ''lipid cells' shows the fat globules between the membrane, and the right image shows the membrane, which is composed of a combination of a phospholipid bilayer and the protein (blue ovals).
Over 400 fatty acids have been identified in milk fat. The most common type of fatty acids is triglycerol or triglyceride, see figure 3, which is composed of three fatty acids.
Figure 3
Three fatty acids form a triglyceride. Elsaid Younes (2017), Structural Properties of Casein Micelles in Milk, the effect of salt, temperature, and pH. Int J Biotech & Bioeng. 3:6, 204-220. DOI: 10.25141/2475-3432-2017 Milk fat melts over a wide range of temperatures from --400C to 400C, and it can be degraded by excess heat or light, and actions of some enzymes cause it to be inactivated.
Lactose
Lactose is the major carbohydrate in milk, and it is a disaccharide derived from galactose and glucose as seen in Figure 4. It is found dissolved in the milk serum in two forms: alpha--anomer and beta--anomer. The ratio between the two forms changes with temperature. The beta--anomer is sweeter and more soluble than the alpha--anomer. Short pasteurization time does not have a significant effect on the glucose, but (UHT pasteurization) motivates the Brownian reaction between the Lactose and the protein, leading to undesirable flavor and color. Figure 4 shows the Lactose structure.
Minerals:
The majority of minerals found in milk are Ca, P, Mg, K and Zn. Calcium and phosphorus combine together to form Calcium Phosphate salt and it is called Colloidal Calcium Phosphate (CCP). It was suggested to play an important rule in binding the casein micelle by immigrating in or out the micelle with changing temperature. Vitamins Milk is not considering a major source of vitamins in a diet. There are two groups of vitamins that exist in milk according to their solubility: •The water--soluble vitamins group, including the vitamins (B1, B2, B3, B5, B6, B12, C).
•The fat--soluble vitamins group, including the vitamins (A, D, E, K). HTST does not affect the vitamins, but UHT decreases the water--soluble vitamins. Exposure to light affects the vitamin A content.
Milk proteins
The proteins in milk are unique, and make milk an important source of human nutrition. Milk proteins are digestible by almost everyone in the intestine. Generally, there are many types of proteins according to the sequences of the amino acids. There are 20 amino acids, which form the different protein types where 9 of them are essential for diet and all of which are present in milk. The amino acids are bonded to each other by polypeptide bonds, see Figure 5. The N--terminal (terminus) is the amine part and the C--terminal (terminus) is the carboxylic acid part. They carry negative charge (the N terminal) and positive charge (C--terminal), and they neutralize each other. If the hydrocarbon side chain can be titrated, the charge of the protein depends on the pH, which will be discussed in detail later. A denatured protein is the protein unfolded from its native state. The denaturation can be useful in the industry or digestion as it gives the enzymes access to the protein chains. Figure 5 shows how the peptide bond binds the amino group to the carboxylic acid to connect the amino acids.
Bovine milk proteins are the most common proteins. Caseins resemble about 80% of all the protein and it is present in milk of all species. All other proteins are grouped together in a group called whey proteins. The major whey protein in cow milk is alpha-lactalbumin and beta--lactoglobulin. The only known milk allergy is caused by beta--lactoglobulin, because of its poor digestibility. The whey protein has also an important use in the industry as it is used in binding meat and water in meat and sau-sage products, and it will not form a gel upon denaturation and acidification.
There are four types of caseins in milk, each having appropriate amino acids that make the milk essential for growth and development of the young. They they are also phosphorylated to various degrees, which plays an important role in the stabilization of the casein itself. A casein micelle is composed of several similar proteins, forming a multi--molecular granular structure. The casein micelle contains water plus salts (mainly from Calcium and Phosphorus). The casein micelle plays an important role in many milk product industries such as in cheese making. The casein can be separated from whey by centrifugation where the casein is pelleting and the whey is in supernatant, or it can be precipitated by adding acid. The average diameter of a casein micelle is around 120 nm to 180 nm, and it is about 1/50 of the fat globule size. The micelle is composed of proteins with salt and the salt is primarily nano--clusters of Calcium and Phosphorus. Researchers think that the Calcium and Phosphorus play a role in connecting the proteins because by removing the salts, the micelles dissociate into small parts called sub--micelles. The micelles were found to have essentially 4 proteins; ---s1, ---s2, -and χ--casein in the ratio 4:1:4:1. [2] The isoelectric point of the casein is at pH 4.6 i.e. the pH at which the net charge is zero. At pH 6.6, the nano--clusters, which are mainly Calcium and Phosphorus salts, are dispersed as small particles with a radius of 1.72 nm--2.27 nm. There are many articles about the internal structure, and all of them agree that there is a micelle formed by the proteins and the colloidal calcium phosphate (CCP).
[27]- [30] Generally, the studies that use light and x--ray and neutron scattering show that the micelle is formed from sub--units (called sub--micelles) with the help of CCP. CCP acts like cement, as it connects the sub--micelles. The studies that use electron microscopes show that there are no sub--micelles, but there is a protein matrix and channels in the inside of the micelle. However, some recent studies, using high--resolution microscopes and carefully made samples show that the sub--micelle model probably exists. Horne (2006) has given an explanation, to the sub--micelle model, as is shown in Figure 6, and illustrates that the whole micelle consists of small spherical sub--units (sub-micelles) connected with the colloidal calcium phosphate. The sub--micelles on the outer surface of the micelle are rich with k--casein, having protruding hairs of negative charges which induce a steric repulsion between micelles, thus preventing them to coagulate and hence stabilizes the milk. The Holt model explains how the nano--cluster of the CCP acts like cement, and connect the proteins to form the big micelle as is shown in Figure 7. Both alpha and beta casein are attached to the cluster. The difference between the proteins enables them to make a bridge with the cluster. Old electron microscope studies say that there is no inhomogeneity in the micelle more than a few nanometers, which means that all the components form one part, and that there are no small molecules looking like the big micelle. However, the previous electron microscope photos contain artifacts, since the samples were supposed to be fixed by drying or freezing, with a possible loss of protein flexibility. Recent studies by more advanced electron microscopes, with carefully prepared samples, declare that the sub--micelle particles might exist, see Figure 8. In general all studies confirm that there is a casein micelle, composed of four types of protein, namely alpha s1, alpha s2, beta and kappa casein. The protein micelle is similar to the surfactant micelle with both a hydrophobic part and a hydrophilic one, motivating the micelle formation by their behavior towards the water phase. The polar part, which is presented by the N and C terminals, tends to face the water. The hydrophobic part is presented by two portions, the first of which consisting of non polar side chains and the second is the repeated sequence of P, F (Phenylalanine and Proline). It has been found that P, F sequences have less polarity than other sequences. Milk can be considered as a colloidal solution that contains: Solution from milk sugar in water. Fat emulsions (oil in water). Proteins suspended in water. Because of the great role played by the casein micelle, a simple model has been used to simulate the role of electrostatic interactions between ß--casein micellar structures in milk with emphasize on the following: 1)The effect of the electrostatic environment on the micelle--micelle interaction.
2)The effect of temperature of the micelle--micelle interaction.
3)The effect of changing pH on the protein charge on the micelle--micelle interaction.
Electrostatic interactions:
Milk contains approximately 87% percent water. It is extremely time consuming to consider the effects of all the individual water molecules in computer simulations, and therefore we will only consider the global effect of water, by letting the electrostatic interaction energy (uel) between charges, be screened by the dielectric permittivity of water, denoted -r.
The electrostatic energy (-!") depends on the electrostatic potential (Φ!"). The electrostatic potential depends on the charges found in the system. The electrostatic potential in vacuum at a point, separated by a distance rj from a point charge j, is given by: Elsaid Younes (2017), Structural Properties of Casein Micelles in Milk, the effect of salt, temperature, and pH. Int J Biotech & Bioeng. 3:6, 204-220. DOI: 10.25141/2475-3432-2017-6.0202 where -! is the dielectric permittivity of vacuum. The electrostatic interaction energy between two particles i and j (in vacuum) can be obtained by: This equation represents Coulomb's law. Coulomb's equation for electrostatic interaction in water is given by: As mentioned above, the introduction of the dielectric permittivity coefficient of water -!, accounts for the screening of the electrostatic interaction between charges when treating water molecules implicitly as a structureless continuum.
The effect of the concentration, the valence of the ions, and the temperature on the system, can be studied using the model (explained above). The Debye length can be considered as a resisting factor of the electrostatic force and is defined as length . The Debye length can be depicted as the radius of the circle with a point charge oriented in its origin, and the circumference of this circle is the farthest position that can be influenced by this point charge.
The Debye length affects the electrostatic potential, and the electrostatic potential from particle j, which affect particle i, becomes, which is the Debye Hückel expression for the electrostatic potential. Here Rj is the radius of the particle, and rij is the distance between i and j. It is obvious that the Debye screening length decrease by the increasing of the concentration of salt and the charge, but also while increasing temperature. In our model, the radial distribution function between the micelles has been calculated numerically to show the electrostatic behavior of the model when changing the environment.
It is possible afterward to express the total charge by mole fractions at constant pH by summing -!! over all the basic groups, substracting them from the sum of -!"! over all the acidic groups.
2.22
Here < z > is the net average charge of the protein.
Model and Method
3:1 Model A simple model system has been designed to study the micelle-micelle interactions in milk. The charge of the proteins which build up the micelle, can be estimated from the following steps: At a temperature of 250C and pH=6.7 (conditions of fresh milk) the net charge of a micelle protein is about --7 elementary charges.
In our model, we treated the micelles as hard spheres with a radius of 75 Å, and we neglected any titration effects during the coarse of the simulations. Practical experiments have been done to calculate the micellization number of the ß casein. [38] It is found to be around 20, i.e., there are 20 proteins/micelle, and the total charge would thus be around (--140) e/micelle. In order to make the system overall electroneutral, 140 counterions/micelle were added to the system. The ions were modeled in the same way as the micelles, i.e. as point charges, enclosed in impenetrable spheres, but with a diameter of 4 Å. The smallest investigated system consisted of 21 micelles and 2940 counterions, enclosed in a cubic simulation box with side lengths equal to 906 Å. The dimensions of the box were chosen as to give a micelle volume fraction of about 5 %, a value corresponding to the conditions in milk. The volume fraction was simply calculated as the total volume of the spherical model micelles, divided by the box volume. A snapshot of the model system is provided in Figure 8.
Method:
Fortunately, most of the computer simulations are based on the assumption that classical mechanics can be used to describe the motion of atoms and molecules. The fact that the quantum system can be found in different states is needed. Some important defini-tions with respect to computer simulations are: Ergodic system: when the time average of the sequence of events is equal to the ensemble average. The difference between analytical and numerical calculations: the analytical calculations are exact and can be done by using pen and paper and the solutions are exact. The numerical calculations are more complicated and can be done by using computer simulations, accompanied with an inevitable statistical noise. Markov chain: a system is said to undergo Markov chains when it undergoes a chain of transitions among finite possible states and the next state only depends on the current state and not the sequence of the state. Importance sampling: A technique used for estimating a particular distribution for a sample generated by random distribution. For example, two distributions as shown in Figure 10 where f(x) is the random distribution, and h(x) is the particular one ''certain one''. Importance weights: It is a function used to measure the error between the two distributions, = 3.1 hence the rejection rate depends on the importance weights. We need a new distribution related to h(x) only. By using the importance weight technique, approximation of the new distribution can be obtained.
where E is called the Monte Carlo estimator. The variance can be calculated.
Metropolis method Assume that we have two states (o) and (n), and these states are obeying Markov chain, where: • the N function denotes the probability density, • acc(initial state→final state) denotes the accepted move from the initial state to final one, • -(Initial state→final state) denotes the transition matrix from the initial state to the final one and -is the underlying matrix of the Markov matrix, chosen to be symmetric.
In the equilibrium condition to move between the states is equal so that the system does not change so: 3.4 The probability density of the certain state is the probability to find this state inside the system, The choice of Metropolis: or --< -(-), the accepted moves will be !(!) from the total moves !(!) Cluster moves technique The cluster moves technique is a sampling technique in order to speed up the simulation. It is an artificial move and instead of moving one particle from the cluster in an unfavorable environment, one moves the particle with surrounding ions and thereby one can speed up the simulation and so it increases the acceptance rate. From the previous explanation of Metropolis method, the acceptance of moving the cluster from the position (o) to a new position (n), 3.9 But this equation doesn't care about the equilibrium conditions for the micelle with the counterions (the cluster in our model). In order to modify the equation to serve the equilibrium conditions of the cluster, the equation should not accept moving more than one particle to or from the cluster. This term has been introduced p(k,l), where p is the probability of moving the particle, which is a part from the cluster between two positions k, l : p(k,l)= 1 when rk,l< rc and = 0 when rk,l> rc where rc is the radius of the cluster. The acceptance equation will be !"# acc(n→o)= min [1,exp(--ß∆U) ! !,! ] 3.10 !" !!"#(!,!) This equation guarantees the acceptance moves will obey the equilibrium of the cluster.
3.2.3
Ensembles One special class of ensemble is the ones that do not evolve over time. These ensembles are known as equilibrium ensembles and their condition is known as statistical equilibrium. Statistical equilibrium occurs if, for each state in the ensemble, the ensemble also contains all of its future and past states with probabilities equal to that state. The study of equilibrium ensembles of isolated systems is the focus of statistical thermodynamics. In this study the NVT ensemble has been used i.e. the number of molecules, volume and temperature are being constant.
3.3
Structural analysis A protein is a polymer formed naturally from a specific sequence of amino acids (monomers), and the amino acids are bonded together by peptide bonds, as shown in Figure 11. To calculate the size of the proteins precisely, the radius of gyration is recommended because it considers the distance between all the mono- Elsaid Younes (2017), Structural Properties of Casein Micelles in Milk, the effect of salt, temperature, and pH. Int J Biotech & Bioeng. 3:6, 204-220. DOI: 10.25141/2475-3432-2017 mers, not the average distance as in the hydrodynamic radius. The radius will be discussed later in this section. Figure 11 shows the structure of the protein.
The radial distribution function:
The radial distribution function (r.d.f or g(r)) is the variation of the number density (-= !, the unit of -is number/volume) of particle as a function of the ! distance from a certain particle, see schematic picture in Figure 12 (left). Or more simply spoken: it is the probability of finding a particle at a distance r far from a reference particle. The g(r) is calculated by using a Histogram technique, and it is a graphical representation of data distribution to estimate the probability distribution of a contentious variable. The radial distribution function is determined by calculating the distances between all the particles pairs, and put them all together in a histogram. Figure 12 shows the radial distribution function g(r).
[42] It is a schematic picture to the left and the histogram in a graphical representation to the right The graph of the radial distribution function can be understood from the following: The beginning of moving from zero in g(r) axis is the start of appearing a particles after the center particle, and it is equal to twice of the radius, if it is a pure hard sphere potential. By assuming that (rmax) is the distance (r) at the maximum g(r), means that at the (rmax) distance from the center particle is the most crowded position. Also the height of the peak refers to the number of the configuration. The g(r) gives a link between the microscopic determined from the intermolecular forces, like in this thesis ¨electrostatic interactions¨, and the other microscopic thermodynamic properties like energy (E) and entropy ( S).
End to end distance of the polymeric chain
The distance from one end to another. By assuming that the polymeric chain has N monomers, and bonds with length req bond the polymers to each other. The end--to--end distance Ree corresponds to the displacement Ld of a random walk with number of steps Nr -1 and step length req. Then ! ! ! ! ! < -!! > != < -! > != -! − 1 !-!" 3.11
3.12
where N is number of monomers and rij the distance between i and j monomers.
3.3.4
The radius of gyration Is the root mean square of the distances between the monomers and their centers of mass, and it can be expressed by the formula: ! ! 3.13 the ratio between the mean square of the end to end distance(Ree), and the radius of gyration (Rg), is related to the factor, and the shape factor is related to the polymer conformation.
Results and discussion:
The charges of the micelles have been calculated according to equation (2.22) at the pH of the normal fresh milk (6.7) and it has been found to be (--140e), which for 21 micelles gives rise to 2940 counterions. In the simulations, all the counterions have been explicitly taken into account, hence the systems are electroneutral. The existence of charged species in the solution gives rise to Columbic forces, i.e. attractive electrostatic interactions between particles of the opposite sign, and repulsive electrostatic interactions between particles of the same sign of charge. Electrostatic interactions have an effect on the distribution of micelles, and since the micelles repel each other, and attract the counter ions, a cluster move technique has been applied i.e. it has been possible to move a micelles with condensed counterions in one MC step. The effect of the electrostatic forces: The radial distribution function between the micelles is shown in Figure 13 (blue curve), and the corresponding g(r) for non-charged micelles are shown in the red curve. The blue curve shows the effect of the electrostatic interactions on the micellar structure. The g(r) is zero for r less than one molecular diameter due to hard sphere repulsive forces. The electrostatic interactions induce repulsion between the micelles and there is a prominent peak at around r = 328 Å, which is more than the double of the radius of the micelles (150 Å). The height of the peak reaches two, which means that it is two times more likely to find two micelles of this distance than in an ideal gas. Figure 13 shows the radial distribution between charged micelles (blue curve), and neutral micelles (red curve). Figure 14 shows the slight system size dependence. Elsaid Younes (2017), Structural Properties of Casein Micelles in Milk, the effect of salt, temperature, and pH. Int J Biotech & Bioeng. 3:6, 204-220. DOI: 10.25141/2475-3432-2017 The ideal system and the effect of counterion volume In Figure 15, the blue curve shows the "ideal system¨ with neutral micelles, and as expected, the closest approach distance between the micelles is twice the micelle radius due to hard sphere inter-actions. The red curve gives the structure for a system with again 21 neutral micelles, but now including 2940 neutral hard sphere "ions". From this simple test, we conclude that the excluded volume effect from the small ionic species is negligible.
Figure 15:
The radial distribution function versus the distance between the micelles for two different systems. The blue curve corresponds to a system with 21 neutral micelles, while the red curve corresponds to a system with 21 neutral micelles together with 2940 neutral "counterions".
The effect of salt concentration:
The monovalent salt concentration of 80 mM is interesting, since it corresponds to the real ionic strength in milk. The salt solution screens the electrostatic interactions and to be able to study the effect of salt, a screened electrostatic potential has been used. The g(r) in Figure 16 shows that when the salt concentration is increased, the electrostatic interaction is not as strong as in the reference system, and that it becomes more like the ideal system c.f. green curve (ideal system) with the red curve (80 mM). Figure 16 shows the response in the micelle--micelle structure with respect to the addition of 80 mM of monovalent salt.
The blue curve is for the reference system (explained above) and it is clear that the micelles are more separated than in the red curve, which is for the system with 80mM concentration (more screened electrostatic interaction). The third system is the ideal solution without any charges, where no long--ranged order is visible. Notice that the red and green curve deviate from each other at shorter interparticle distances, hence at these distances the micelles "feel" each other and repulsion is obtained. The screening length for 80 mM salt is ≈ 11 Å.
The effect of salt valence on milk:
The monovalent counterions in the reference system have been replaced by divalent and tetravalent ions where the aim was to study how the valency of the ions affects the structure, and if correlation effects can be obtained. Correlation effects means that two highly charged objects of the same sign can attract each other due to charge fluctuations, see schematic mechanism in Figure 17. This contra intuitively effect is only visible for higher ordered salts and most probably important in the aggregation process of proteins. The blue curve in Figure 18 corresponds to the reference system with monovalent counterions, the red curve corresponds to a system where the monovalent ions have been replaced by divalent ions, and the green curve corresponds to a system where the monovalent ions have been replaced by tetravalent ions. As clearly shown, when the valency is increasing the interparticle distance between the micelles is decreasing and the largest effect is visible for system with tetravalent ions. Here the micelles are more or less in molecular contact with each other, only separated by the counterions. Moreover, in the tetravalent system g(r) ≈ 13 at the peak position, which indicates that at r ≈ 160 Å, it is 13 times higher probability to find neighboring micelles in comparison with an ideal gas. Figure 18 shows the difference in the distribution of micelles with the variation of the valency of the counterions. In the green curve, the micelles look freer than in the red and the blue curve, and this behavior occurred because of the screening of the electrostatic force, which increases sharply when increasing the valence. Figure 19 shows micelles in monovalent and divalent and tetravalent respectively from right to left.
The effect of pasteurization:
Increasing the temperature is of special interest, because that is used in the pasteurization processes. Even without adding the values of the right permittivity coefficient, the graph still shows some variations of the behaviors of the micelles. These variations occur because of the effect of temperature on the Debye screening length, which is directly proportional to the root mean square of the -!!. Figure 20. The micellar structure has been studied as an effect of temperature to mimic the pasteurization process The radial distribution functions for the three different temperatures are given in Figure 20. As shown, they coincide and one reason might be because we kept the permittivity coefficient constant in the simulation. At 740C, the permittivity coefficient of the water falls to 63:61, and at 1380C it falls to 48:46.
[40] The entropy increases by increasing the temperature, but since the inter--micellar electrostatic repulsion is very strong, it does not have any influence on the structure.
The effect of fermentation
It is well--known that the charge of the protein depends on the pH as explained above. The protein charges have been calculated by the equation (1.15) for different pH as shown in Figure 21 Figure 21 shows how the protein charge is varying with the pH Figure 22 shows the g(r) for micelles in ordinary milk (blue curve) and in fermented milk (red curve) as well as test micelle where the charge is 1/3 of the micellar charge in milk. As visible there are no traceable effects on micellar structure between milk and fermented milk system (in which we increased the micelle charge to 420 e.) This is due to the fact that the screening of the counter decreases the Debye length to 21.5Å from 37.3Å for the reference system. Summary Milk is indeed a complex liquid, which contains many different components. In this study, we have used a simple model of hard spheres to mimic ---casein micelles in milk. The aim was to investigate how electrostatic interactions affect the micellar structure by varying the solution conditions. The structure of the solution has been analyzed by comparing the radial distribution function as a function of pH, salt concentration and valency, and temperature. It was noticed that due to the fact that the micellar charge is very large, the electrostatic repulsive interaction dominates, and the mean distance between the micelles are almost always obtained. The mean distance was calculated to be approximately 330 Å for a micellar volume fraction of five percent. Moreover, an increase of the temperature does not affect the structure at all i.e. the entropic contribution due to increased temperature can be neglected in comparison with electrostatic repulsion between the micelles. When the salt concentration was increased to 80 mM, which corresponds to the ionic strength in milk, the structure of the ---casein micelles resembles the structure of an ideal gas i.e. the electrostatic repulsive interactions are screened. 80 mM salt corresponds to a screening length of approximately 11 Å. 6.Suggestions for future work I.Verifying the model by simulation larger systems. II.Investigate how a temperature affects the dielectric constant, in our study; it was kept constant to 78.4. III.Investigated the effect of mixed micelles. IV.Investigate how the protein volume fraction affects the structure. V. It is of interest to get a general understanding of the micellar formation, and which theory that is applicable. | v3-fos-license |
2019-09-17T02:59:47.670Z | 2019-09-11T00:00:00.000 | 203404755 | {
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} | pes2o/s2orc | Ascorbic acid insufficiency impairs spatial memory formation in juvenile AKR1A-knockout mice
AKR1A, an aldo-keto reductase, is involved in the synthesis of ascorbic acid as well as the reduction of a variety of aldehyde compounds. AKR1A−/− mice produce considerably less ascorbic acid (about 10%) compared to AKR1A+/+ mice and require ascorbic acid supplementation in order to breed. To elucidate the roles played by AKR1A in spatial memory, AKR1A−/− male mice were weaned at 4 weeks of age and groups that received ascorbic acid supplementation and no supplementation were subjected to a Morris water maze test. Juvenile AKR1A−/− mice that received no supplementation showed impaired spatial memory formation, even though about 70% of the ascorbic acid remained in the brains of the AKR1A−/− mice at day 7 after weaning. To the contrary, the young adult AKR1A−/− mice at 13–15 weeks of age maintained only 15% of ascorbic acid but showed no significant difference in the spatial memory compared with the AKR1A+/+ mice or ascorbic acid-supplemented AKR1A−/− mice. It is conceivable that juvenile mice require more ascorbic acid for the appropriate level of formation of spatial memory and that maturation of the neural system renders the memory forming process less sensitive to an ascorbic acid insufficiency.
Introduction A scorbic acid (AsA), vitamin C, has pleiotropic roles in maintaining healthy conditions in the mammalian body. (1) A deficiency of AsA results in metabolic abnormalities and an imbalance of redox homeostasis, notably in the cardiovascular system, and in severe cases, fatality increases, as typically observed in scurvy. AsA is concentrated in central nervous system (CNS) and appears to be involved in a number of metabolic processes including dopamine b-hydroxylase activity in the biosynthesis of catecholamine. (2) AsA is released from astrocytes upon stimulation by glutamate and is then taken up by neurons. (3) While AsA exerts neuroprotective action by suppressing oxidative stress that is triggered by glutamate excitotoxicity, (4) it also promotes oligodendrocyte generation and remyelination. These results imply that AsA could have therapeutic potential for the treatment of demyelinating diseases, such as Multiple Sclerosis. (5) Thus AsA is beneficial in the CNS from standpoint of physiology and pathology.
Rodents are popular laboratory animals, but because they have the ability to synthesize AsA they cannot be used as model animals in investigations of the AsA functions. The enzyme Lgulono-g-lactone oxidase (GULO) catalyzes the final step in the biosynthesis of AsA synthesis, using molecular oxygen and releases AsA. (6) Primates are incapable of synthesizing AsA due to a mutation in the GULO gene, which likely occurred about 63,000,000 years ago. (7) The genetic ablation of GULO shows a total defect in AsA synthesis in mice. (8) In turn, gluconolactonase (GNL) catalyzes the dehydration of L-gulonate to L-gulono-glactone, the penultimate reaction in the AsA synthesis pathway. Mice with a genetic ablation of GNL, which is identical to the senescence marker protein 30, also show complete inability to biosynthesize AsA. (9) Aldehyde reductase (AKR1A) and aldose reductase (AKR1B), members of the aldo-keto reductase (AKR) superfamily, (10) have been reported to catalyze the NADPH-dependent reduction of D-glucuronic acid to L-gulonate, the reaction immediately before that catalyzed by GNL. (11,12) The ablation of these genes, therefore, result in impaired AsA synthesis, and the contributions of AKR1A and AKR1B to AsA synthesis in mice reduced to 85-90% and 10-15%, respectively. While AKR1A -/mice show pathological characteristics similar to scurvy and do not survive beyond one year in the absence of AsA supplementation, (13) neither the phenotypic abnormality associated with an AsA insufficiency nor an altered longevity has been reported for AKR1B -/mice. (11,14,15) AKR1A also plays roles in metabolic reactions in addition to AsA synthesis, which appears to confirm its existence in primates. (16)(17)(18)(19) Regarding the function of AsA in CNS, AKR1A -/mice are hypersensitive to pentobarbital anesthesia and this can be reversed by the administration of AsA. (20) On the other hand, AsA has no influence over the aggressive behavior caused by an AKR1A deficiency. (21) Thus, AKR1A appears to play differential roles in the CNS or other neuronal systems, but no rational explanation has been provided for this. In this study we subjected AKR1A -/mice to the Morris water maze test and evaluated the effects of an AsA insufficiency in the light of the spatial memory formation.
Materials and Methods
Animals. AKR1A -/mice with a C57BL/6 background that were generated using a gene-targeting technique, (12) were bred in our institution and used throughout the study. The male AKR1A +/ + and AKR1A -/mice were weaned at 30 days of age and fed a standard diet (Picolab 5053, LabDiet, St. Louis, MO) ad libitum with free access to either water or water containing 1.5 mg/ml AsA until they were used. The supplemented AsA concentration was sufficient to allow the AKR1A -/mice survive longer than one year. (13) Animal experiments were performed in accordance with the Declaration of Helsinki under the protocol approved by the Animal Research Committee at our institution.
A Morris water maze test. To evaluate spatial memory in the AKR1A -/mice, the Morris water maze test was performed. (22) A circular target platform (10 cm in diameter) was immersed in a pool (diameter 120 cm) 7 cm below the surface of the water, and four black-and-white drawings were attached to the inside wall of the pool above the water surface. The water temperature was maintained at 20 ± 1°C. The test was conducted on 4 consecutive days. Each mouse was examined four times per day, starting at a different position each time, in submerged platform trials in whitecolored water containing skim milk. The swimming was videotracked for 90 s. When the mouse reached the platform within 90 s, it was allowed to remain on the platform for 15 s and view the drawings. If the mouse did not reach the platform within 90 s, it was forced to view the drawing on the platform for 15 s. Escape latency, escape distance and swimming speed were measured in the quadrant where the platform was located using a video tracking system Compact VAS ver 3.0x (Muromachi Kikai, Tokyo, Japan).
Measurement of the reduced form of AsA. A fluorescent probe, 15-(Naphthalen-1-ylamino)-7-aza-3, 11-dioxadispiro [5.1.58.36]hexadecan-7-oxyl (Naph-DiPy), was synthesized (23) and used to measure the concentration of AsA. (18) Fresh blood plasma prepared from either the tail vein or the heart at autopsy was used for the AsA assay. In a typical run, a blood sample was collected in the presence of excess EDTA. The blood plasma was obtained by centrifugation of the sample at 800´g for 3 min at room temperature. Hippocampus tissue was dissected from mice, quickly frozen in liquid nitrogen, and stored at -80°C until used. After homogenizing the hippocampus tissue in 10 volumes of phosphate-buffered saline followed by centrifugation at 17,400´g for 15 min at 4°C, the supernatant was diluted with phosphate-buffered saline. The blood plasma or the diluted tissue extract were incubated with Naph-DiPy for 30 min at room temperature in the dark. The AsA concentration was calculated by measuring the fluorescence at an excitation wavelength of 310 nm and an emission wavelength of 430 nm using a microplate reader (Valioskan Flash, Thermo Fisher Scientific, Waltham, MA).
Measurement of choline, acetylcholine, glutathione and cysteine. LC-MS analyses of choline, acetylcholine, cysteine (Cys), and glutathione (GSH) in hippocampus extracts were performed as described in a previous report (24) with minor modifications. (25) 10 mg of tissue samples were homogenized in 100 ml buffer containing 20 mM N-ethylmaleimide (NEM) and 50 mM ammonium bicarbonate, pH 8.0, to block the sulfhydryl groups in Cys and GSH. The resulting homogenate was incubated for 10 min at room temperature. After adding a 200 ml portion of methanol containing 5 mM N-methylmaleimide (NMM)-derivatized GSH as an internal standard and another 200 ml of chloroform, the mixture was thoroughly stirred and centrifuged at 12,000´g for 15 min 4°C. The upper aqueous layer was filtered through a 0.45 mm filter (Millex ® -LH, Merck Millipore, Burlington, MA). A 90 ml aliquot of the filtrate was lyophilized, the residue dissolved in 30 ml of 50% acetonitrile, and subjected to liquid chromatography (LC)-mass spectrometry (MS) analysis. A Q Exactive Hybrid Quadruple-Orbitrap mass spectrometer (Thermo Fisher Scientific) equipped with a heated electrospray ionization source was operated in the positive ionization mode for this analysis. An Ultimate 3000 liquid chromatography system consisted of a WPS-3000 TRS autosampler, a TCC-3000 RS column oven, and a HPG-3400RS quaternary pump (Dionex, Sunnyvale, CA). A SeQuant ® ZIC ® -pHILIC column (2.1´150 mm, 5 mm particle size; Merck KGaA, Germany) was maintained at 30°C. The mobile phase A was 20 mM ammonium bicarbonate, pH 9.8, and the mobile phase B was 100% acetonitrile. System control, data acquisition and quantitative analysis were performed with the Xcalibur 2.2 software. Standard curves for choline, acetylcholine, GSH-NEM, and Cys-NEM showed linearity in concentration ranges examined.
Measurement of neuroactive amines in hippocampus
tissue. The levels of amines were measured by high performance liquid chromatography (HPLC), as previously described. (26) Hippocampus tissue was homogenized in 0.2 M perchloric acid (10 ml/mg tissue) containing 100 mM EDTA-2Na and isoproterenol (100 ng) was added as an internal standard. After centrifugation at 20,000´g for 15 min, the supernatants were transferred to another tube and the pH adjusted to 3 by adding 1 M sodium acetate. Samples were diluted to 1/20 and 1/400 with 0.2 M perchloric acid containing 100 mM EDTA-2Na. Samples (10 ml) were then analyzed by HPLC.
Histological analyses of brain. Histological analyses were performed at the Pathological Analysis Center, Institute for Promotion of Medical Science Research, Yamagata University. Dissected brains were fixed in 10% buffered formalin followed by embedding in paraffin. Brain sections (5 mm thick) were subjected to either hematoxylin and eosin (H&E) staining or Nissl staining. Photographs of the sections were taken with a BZ-X700 microscope (Keyence, Osaka, Japan).
Protein preparation. Brains were weighed and homogenized in 5 volumes of RIPA buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Roche) and centrifuged at 17,400´g in a microcentrifuge. The supernatant was used for protein determination using a Pierce ® BCA TM Protein Assay Kit (Thermo Fisher Scientific).
Statistical analysis. The results are expressed as the mean ± SEM. Statistical analysis was performed using the Student t test or one-way ANOVA, followed by the Tukey-Kramer test for multiple groups. A p value of less than 0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001.
Results
AKR1A knockout impairs spatial memory formation in juvenile mice but not young adult mice. During their period of lactation, the drinking water for the AKR1A -/mice was supplemented with AsA (1.5 g/L) for the purpose of breeding but was ceased at the time of weaning at 30 days after birth, while a second group of AKR1A -/mice continued to receive the AsA supplement to distinguish the effects of AsA from other functions of AKR1A. We subjected three groups of juvenile male mice at 4-weeks of age; AKR1A +/+ mice, AKR1A -/mice without AsA supplementation, and AKR1A -/mice with AsA supplementation, to the Morris water maze test after weaning. The findings indicated that a latency to reach the platform was significantly decreased in the AKR1A +/+ mice and the AKR1A -/mice with AsA supplementation during the trial period (Fig. 1A). On day 1 and day 2, no statistically significant differences were noted among the three groups, but on day 3 and day 4, the AKR1A -/mice without AsA supplementation showed a delay in escape latency compared with the other groups. The escape distance was not significantly shortened in the AKR1A -/mice during the session but gradually became shortened in the case of the AKR1A +/+ mice or the AKR1A -/mice with AsA supplementation (Fig. 1B). Swimming speed remained about the same during the session for all three groups (Fig. 1C). On the other hand, when the same experiment was conducted on young adult mice at 13-15weeks of age, no significant differences were observed in latency among three groups of mice (Fig. 1D). Body weights were about the same among the three groups of mice (data not shown).
AsA levels are preserved in the brain longer than other organs. We measured AsA levels in blood plasma and whole brains of juvenile mice at 5 weeks of age and young adult mice at 13-14 weeks of age. The plasma levels of AsA were about 15% in juvenile AKR1A -/mice and less than 10% in young adult AKR1A -/mice compared to corresponding AKR1A +/+ mice ( Fig. 2A). Supplementation with AsA increased these levels to 70% of the corresponding values for AKR1A +/+ mice in juvenile AKR1A -/mice and young adult AKR1A -/mice, respectively. To the contrary, the brain maintained higher levels of AsA; 70% in juvenile AKR1A -/mice and 15% in young adult AKR1A -/mice (Fig. 2B). It is also noteworthy that the AKR1A -/mice that received AsA-supplementation maintained the same levels of AsA as that for the AKR1A +/+ mice.
When AsA levels were measured in other organs, the liver and kidney, as well as blood plasma and brain of juvenile AKR1A -/mice at days 0, 3 and 7 after the cessation of AsA at 4 weeks of age, the levels declined rapidly in blood plasma, liver, and kidney ( Fig. 2C and D). However, the brains of AKR1A -/mice originally contained several-fold higher levels of AsA than the liver or kidney and more than 70% of the AsA was preserved on day 7. These findings suggest that there is a specific mechanism for preserving AsA in the brain compared to other organs.
Levels of neurotransmitters amines, acetylcholine, redox compounds in the hippocampus. We hypothesized that the production of neurotransmitters might be affected by the status of AsA and that this might influence the neuronal function of the juvenile mice. Because dopamine is released into the dorsal hippocampus, binds to D1/D5 receptors and promotes several responses, including spatial learning, (30) the dopamine levels, together with other neurotransmitter amines in the hippocampus were measured by HPLC. Essentially no significant differences were observed in their levels among three mice groups, except for finding that the norepinephrines were slightly increased in AKR1A -/mice with AsA supplementation compared with AKR1A +/+ mice (Table 1). Since the hippocampus is regarded as the site of action for the effects of nicotine on spatial learning, (31) we also measured the levels of acetylcholine and choline together with the antioxidative molecules, cysteine and GSH, in hippocampus area of three groups of juvenile mice by LC-MS. Again, there were no significant differences in their levels among the three groups of the mice.
No changes in brain histology or related proteins.
When histological analyses of the brain were performed on the three groups of mice at 5 weeks of age, no evident changes were observed in the brain sections that had been subjected to either H&E staining (Fig. 3A) or Nissl staining (Fig. 3B). To further explore the reason for the defect in the spatial memory formation in the AKR1A -/mice, the levels of AKR1A and AKR1B and antioxidative enzymes in the brains of these mice were assessed by immunoblot analysis. The absence of AKR1A was confirmed in the AKR1A -/mice, and no changes were observed in the levels of AKR1B among the three groups of mice ( Fig. 4A and B). The levels of major antioxidative enzymes, SOD1, SOD2, GPX1, and catalase remained unchanged. We also assessed possible variations in AKR1A and AKR1B levels in the AKR1A +/+ mice during aging ( Fig. 4C and D) and no changes in their levels were detected at 5, 13, and 31 weeks of age. Thus the AKR enzymes and antioxidative system remained unchanged during aging and appeared to not be the cause for the differential response in the neuronal function.
Isoflurane anesthesia had no affect on the spatial memory formation in young adult mice. Because a variety of stress conditions can exerts an oxidative insult and AsA suppresses it by virtue of its antioxidative function, it is possible that stress caused by anesthesia might affect the spatial memory formation in the AsA-insufficient adult AKR1A -/mice. We used isoflurane, an inhaled anesthetic, that reportedly affects spatial memory formation via oxidative stress or endoplasmic reticulum stress in juvenile rodents. (32,33) Male AKR1A +/+ mice or AKR1A -/mice that had grown to 12-13 weeks of age without AsA supplementation were treated with a relatively high dose of isoflurane (2%) for 2 h on day 0, and were then subjected to the water maze test in following days as shown in Fig. 1 (Fig. 5). The results indicated that the isoflurane treatment had no significant effects on spatial memory formation both in the AKR1A +/+ mice or in the AKR1A -/mice, suggesting that the young adult mice had a robust resistance against an AsA insufficiency.
Discussion
The findings reported herein show that AKR1A -/mice at the juvenile stage had a defect in spatial memory formation, as judged by the results of a water maze test (Fig. 1A). Although AsA levels were relatively well preserved in brains compared to other organs after the cessation of supplementation (Fig. 2D), spatial memory formation was impaired by a minor decline in the AsA contents in juvenile AKR1A -/mice. The impairment in the memory formation became less evident in adult AKR1A -/mice (Fig. 1D), implying that the maturation of the neuronal system rendered the CNS resistance to an AsA deficiency.
Roles of AsA in CNS have been extensively examined in GULO -/mice (34,35) and GNL -/mice. (9,36,37) However, there are substantial differences between our results and these studies. The ablation of AKR1A continues to enable the production AsA at a level of 10-15%, (11,12) while the ablation of either GULO or GNL resulted in a decrease in AsA production to negligible levels. (8,9) The complete absence of AsA for a long period would affect a variety of physiologic processes, and would show more profound phenotypic abnormalities in GULO -/or GNL -/mice than in the AKR1A -/mice. The advantage of using AKR1A -/mice would be that a certain amount of AsA would persist in the brain, which is similar to the actual pathological state of a human with an insufficient AsA dietary intake.
High levels of AsA were retained in the brains of these mice, while the AsA contents rapidly declined in other organs of the AKR1A -/mice after the cessation of AsA supplementation ( Fig. 2), which is consistent with the previous reports using other genetically modified mice. (34) An AsA deficiency during gestation causes developmental defects in the neonatal cerebellum and impairs its function in adult GULO -/mice via alteration in cellular composition at that location. (35) Impaired spatial memory has also been reported in juvenile guinea pigs, (38) which, like primates, are unable to synthesize AsA due to a hereditary defect in GULO. Neuron numbers in the brain are reduced in the guinea pig, implying a causal connection between the immature neuron development and impaired spatial memory. However, this is not the case for the juvenile AKR1A -/mice because no apparent difference was observed in the number of neurons, probably due to the recruitment of AsA via cord blood until the time of weaning and high preservation of AsA in brain (Fig. 3).
A deficiency in AsA synthesis is associated with an elevation in oxidative stress in the brains of GNL -/mice (9,36,37) and GULO -/mice. (39) While antioxidation is the putative function of AsA, AKR1A -/mice showed no changes in the levels of redox molecules, such as glutathione and cysteine, in the hippocampus compared to the AKR1A +/+ mice (Table 1), which suggests that robust oxidative damage in neuronal cells is not likely the cause for the impaired memory in the juvenile AKR1A -/mice. Treatment of the young adult AKR1A -/mice with isoflurane, which has been reported to affect spatial memory formation in juvenile mice by neurodegeneration, (33,40) showed no apparent difference in the spatial memory of the adult AKR1A -/mice that grew up without AsA supplementation (Fig. 5). This is consistent with anesthetic action of isoflurane, in that it does not affect duration of the loss of the righting reflex of young adult AKR1A -/mice, while pentobarbital anesthesia delays it compared to AKR1A +/+ mice. (20) Given these observations, clinical doses of isoflurane anesthesia would not be expected to cause any severe impairment in spatial memory formation in mice.
AsA reportedly supports the action of dopamine b-hydroxylase, an enzyme that catalyzes the conversion of dopamine to norepinephrine in vitro. (2) Adult GULO -/mice show a mild motor deficit, as observed by their slow swimming in water, which suggests that an AsA deficiency may affect neostriatal pathways via a dopamine-glutamate interaction process. (41) In our study, however, swimming speed was the same in all juvenile mice (Fig. 1C). There are no significant changes in norepinephrine content in adrenal glands between the AKR1A -/and AKR1A +/+ mice. (21) We also observed essentially the same results on neurotransmitters in the hippocampus, as evidenced by the fact that no difference was found in the levels of neurotransmitter amines or acetylcholine in the hippocampus of AKR1A -/and AKR1A +/+ mice (Table 1). We observed slight but significant increase in norepinephrine content in the AKR1A -/mice with AsA supplementation. AKR1A -/mice without AsA supplementation also showed a trend of high norepinephrine content. These findings collectively suggest involvement of other enzymatic characteristics of AKR1A than the AsA synthesis in either production or decomposition of norepinephrine in the hippocampus, although actual mechanism is not clear at present. Consistent with our results, it has been reported that an AsA treatment improves spatial memory in APP/PSEN1 mice, a mouse model for Alzheimer's disease, but does not alter monoamine levels in the nucleus accumbens. (42) In the meantime, AsA appears to play a protective role against the excessive action of glutamate receptor in dopaminergic neurons, (4) which suggests that the modulation of cellular signaling may be involved in spatial memory formation. Thus, precisely how AsA supports spatial memory formation remains unclear, and further studies will be required from the standpoint of the production and function of neuronal transmitters.
A recent study revealed that AKR1A has a novel role in energy metabolism of cells, (43) which may explain not only the function of AsA but also the phenotypic difference of AKR1A -/mice from GULO -/mice and GNL -/mice. AKR1A mice possess S-nitrosocoenzyme A reductase activity, (19) which protects sulfhydryl groups in proteins from S-nitrosylation, and hence the ablation of AKR1A increases the level of S-nitrosylation in proteins. A glycolytic enzyme pyruvate kinase M2 appears to be the main target of Snitrosylation via the S-nitroso-coenzyme A, which leads to the suppression of enzymatic activity and causes insufficient ATP production in AKR1A -/mice. (43) Because neurons are cells in which energy is mainly supplied from glycolysis, neuronal function would be suffered in the case of a short ATP supply. AsA stimulates the release of nitric oxide from the S-nitrosylated proteins (44) and, hence, supplemented AsA can cope with Snitrosylation reactions in the absence of AKR1A. Given this action of AsA, it is conceivable that supplemented AsA would protect pyruvate kinase from S-nitrosylation-mediated inactivation and rescue neuronal function in AKR1A -/mice. The influence of a short ATP supply would be evident notably in juvenile mice whose synaptic communication is premature and vulnerable. Thus, there are several possible mechanisms that could cause the impaired spatial memory formation in the juvenile AKR1A -/mice.
An AsA insufficiency causes a defect in spatial memory formation in juvenile mice but not in young adult mice. Although the status of AsA had no effect on production of neurotransmitters, AsA may support neuronal function either via direct action on neurotransmitter function, ameliorating stress conditions, or maintaining energy metabolism. In any event, these results, point to the importance of taking sufficient amounts of AsA for maintaining proper memory formation particularly at the juvenile stage of animals that are unable to synthesize AsA. 5. Effects of isoflurane on spatial memory formation. Young adult AKR1A +/+ mice (WT + Iso; A) and AKR1A −/− mice (KO + Iso; B) were treated with 2% isoflurane (Iso) for 2 h on day 0 and then subjected to Morris water maze test. Identical sequences of start locations were used each day. Data are latency (s) to reach the goal (n = 7 for each group). Data corresponding to the AKR1A +/+ and AKR1A −/− control mice were redrawn from the corresponding data in Fig. 1D for reference. Values are expressed as the mean ± SEM. No statistical difference was observed between control group and isoflurane treated group in both genetic mice. | v3-fos-license |
2020-04-30T09:11:43.437Z | 2020-01-01T00:00:00.000 | 218893729 | {
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} | pes2o/s2orc | T-DYNMOGA-Q w : Detecting Community From Dynamic Residue Interaction Energy Network and Its Application in Analyzing Lipase Thermostability
A new type of residue interaction network named residue interaction energy network (RINN) is built. Then, a multi-objective optimization dynamic network community discovery algorithm T-DYNMOGA-Q w has been proposed to detect communities from dynamic RINN. T-DYNMOGA-Q w sets a threshold during the initialization process and optimizes weighted modularity Q w as the objective function. Setting the threshold can better find the stable structure in the dynamic RINN. The resolution limit of modularization has been broken by using objective function Q w . After Community detection from dynamic RINN of wild type of lipase (WTL) and its mutant 6B from 300K to 400K, it is found that the communities in 6B network can still maintain a tight structure even at higher temperature. Stable community is benefit to the heat resistance of lipase 6B. The hydrogen bonds between mutated Ser15 and Ser17, and the Glu20 with other residues improved the structure stability. The mutated L114P, M134E, M137P, and S163P enhance the rigidity of the flexible region and tighten the secondary structure, which stabilize the protein structure.
I. INTRODUCTION
Protein function is closely related with its conformation. The change of protein conformation over time can be coded by a dynamic residue interaction network, whose node is residue and edge is interactions between residues at time scale. It is a powerful method to study the relationship between protein structure and function using complex network theory. In recent years, residue interaction networks have important applications in understanding molecular communication, exploring allosteric inhibitors and activators, and determining important residue sites related to activity, and so forth [1]- [7]. In addition, the analysis of residue interaction network is also available to study the thermostability of proteins [8].
It is very crucial to construct a network that can accurately describe protein three-dimensional structure. Unweighted networks are widely used in the early stage of using complex The associate editor coordinating the review of this manuscript and approving it for publication was Tossapon Boongoen . network theory to study the relationship between protein structure and function [9]- [11]. Later, it was found that weighted networks better described the relations between residues [12]. The total number of interactions between all atoms including the main chain and the side chain atoms is often taken as the weight of the edges [13], [14]. In addition, the interactions between all side chain atoms within the specified distance threshold are considered to be the edge weight [15]. As we know, the residue-residue interactions contain strong interactions and weak interactions, for instance, disulfide bridge is a kind of strong interaction (bond energy is 167 kJ/mol), while van der Waals interaction is one weak interaction (bond energy is only 6 kJ/mol). For the relationship between residues, considering both the interaction type and the strength can reflect the true relationship between the residues. The software gRINN [16] developed by Serçinoğlu Onur uses the network construction method proposed by Vijayabaskar [17], [18], which takes the average interaction energy between each residue pair as the edge weight.
The calculation of the interaction energy in this method is the sum of the short-distance Lennard-Jones and Coulomb interactions, averaged over the overall structure generated at a constant temperature (300 K). In fact, all forces and their energy need to be taken into account. In this paper, the sum of interaction energy of residues is used as the network weight to construct the residue interaction energy network (RINN).
For residue interaction network, mining and analyzing the community structure is an important method to find the functional related regions in the three-dimensional structure of protein. ZHANG et al. used ESPRA Algorithm and the Evolving Graph + Fast-Newman algorithm to detect the rigid community from xylanase dynamic weighted residue interaction networks [19]. Sun et al. combined the analysis of community structure with CRIP detection of 116 catalytic proteins and found that community structure and CRIP may be the structural basis of low-frequency motion [20]. Stetz et al. analyzed the network centrality and community, and proved that substrate binding may strengthen the connectivity of local interactions in communities, which leads to a dense interaction network that can promote an efficient allosteric communication [21]. Obviously, exploring the community structure and evolution is an important way to analyze the relation between protein structure and function.
Most methods for detecting communities from dynamic network include incremental clustering and evolutionary clustering. Compared to incremental clustering, evolutionary clustering [22] considers the characteristics of dynamic networks that change slowly over time. In the framework of evolutionary clustering, many novel algorithms have emerged, such as ESC algorithm [23], EMMC algorithm [24], PisCES algorithm [25], Kin-Han algorithm [26], ESPRA algorithm [27], DYNMOGA algorithm [28], and LDMGA algorithm [29]. Most of these algorithms obtain the community structure by maximizing the community modularity. Currently, it has been proved that the optimization of the modularity has a resolution limitation [30], with the result that important community structure on a small scale can not be found. Nandinee proposed weighted modularity Q w [31] to overcome the resolution limit of modularity function by incorporating a weight term in the modularity formulation. For the community detection algorithm based on GA (genetic algorithm), trajectory-based representation methods and graphics-based representation methods have a great impact on the algorithm performance. Different initialization strategies also have a great impact on the algorithm performance. The DYNMOGA algorithm uses a trajectory-based initialization method, so that the randomness of population production is very large. When the population numbers and the iteration times are increased to obtain the clustering quality, it will take more time. In order to reduce the randomness of population generation, Anwar calculated and screened the clustering coefficients of the initial population to obtain a highly modularity community [32]. Niu introduced the node degree-based label propagation algorithm [29] when initializing individuals. Community detection based on the characteristics of different networks helps to detect a more realistic community structure. Cheng [33] proposed a new method based on node vitality to detect overlapping communities in a dynamic network. Here, we consider the characteristic of RINN and introduce the threshold in the initialization stage to reduce the randomness of the population.
In this study, the randomness of population generation and the limitation of resolution for the optimization of modularity is considered at the same time, a community detection algorithm T-DYNMOGA-Q w is proposed for dynamic RINN based on DYNMOGA. The rest of the paper is organized as follows: Section 2 introduces the selection and process of data sets. The T-DYNMOGA-Q w is described in detail in Section 3 and Section 4. In Section 5, the reliability of T-DYNMOGA-Q w is tested, and the lipase network communities detected by TDYNMOGA-Qw is analyzed.
The conformations of lipase and xylanase are obtained by molecular dynamics simulation at 300K, 350K and 400K. The simulation time is 300ns and the conformation is saved 1 frame/ns. Then, each enzyme saved 300 three-dimensional structures in chronological order at each temperature. For every frame, RING2.0 [37] is used to calculate all types of interactions between residues. The distance thresholds are salt bridge 3.5 Å, disulfide bridge 4.0 Å, hydrogen bond 3.5 Å, van der Waals interaction 0.8Å, π-π stacking 7.0Å, and π-cationic 7.0Å. For RINN, the edge weight w ij is determined as follows.
if residue i and j exist interaction 0, others. (1) In the formula E a , E b , . . . , E f are the energy of different type of interaction.
Thus, a dynamic RINN contains 300 RINN in chronological order per temperature as follows:
A. EVOLUTIONARY CLUSTERING
Evolutionary clustering is a method of processing time series data to generate clustering sequences. It needs to consider two conflicting indicators at the same time: snapshot quality (ST) and temporal cost (TC) proposed by Chakrabarti et al [22]. The snapshot quality is used to represent the clustering result of the network G t (the network at time t) at current time. The temporal cost is used to represent the difference between the clustering result C t (the community of network at time t) at current time and the clustering result C t−1 at previous time. The optimal clustering result at each time is to maximize snapshot quality and minimize the temporal cost. The balance factor α is introduced to regulate the influence of the two indicators. The formula is as follows: A dynamic network with t networks in time can be described There are tk communities in the network at time point t, then the network G t can be described Definition 1: For a static network G t , the multi-objective clustering problem can be defined as: . . , f h (C t )) based on a vector of independent objective functions f i (i = 1, 2, . . . , h). f i is the function used to measure the quality of the cluster. The solution of the multi-objective optimization problem is C t where each of the partial functions in the above objective function takes a maximum value.
2) OPTIMAL SOLUTION FOR MULTI-OBJECTIVE PROBLEM
For the multi-objective optimization problem, the mutual constraints between the targets may make the improvement of the performance of one target often at the expense of the performance of other targets. Problem of the solution is usually a set of non-inferior solutions, that is, Pareto solution set.
Definition 2: Pareto optimal: if C * ∈ is Pareto optimal solution, then for ∀C ∈ , the following condition is satisfied: where I = {1,2,. . . ,h}, h is the number of individual objectives and at least there exists j∈i, such that The collection of Pareto solutions is the so-called Pareto Front. All solutions in the Pareto front are not dominated by solutions outside the Pareto Front (and other solutions within the Pareto Front curve).
IV. METHODOLOGY A. MULTI-OBJECTIVE OPTIMIZATION CLUSTERING ALGORITHM BASED ON THRESHOLD SELECTION AND WEIGHTED MODULARITY (T-DYNMOGA-Q w ) 1) FRAMEWORK OF CLUSTERING ALGORITHM BASED ON MULTI-OBJECTIVE OPTIMIZATION
T-DYNMOGA-Q w is developed from the DYNMOGA algorithm [28], which uses multi-objective genetic algorithm (NSGA-II). NSGA-II is a fast non-dominated sorting method. The algorithm constructs a population and each individual in the population represents a possibility which represents the community structure in the network in the algorithm. Individuals in a population are ranked according to non-dominated precedence, and individuals are constantly evolving in successive offspring. The algorithm optimizes two conflicting functions Q w and NMI. Q w is used to measure the clustering result of the network Gt (the network at time t) at current time t. NMI is used to measure the difference between the clustering result C t (the community of network at time t) at current time t and the clustering result C t−1 at previous time t-1. The algorithm process is shown in Fig 1. Nodes represent amino acid residues, edges represent the sum of the energy of different type of interactions between residues. (a) represents the initial input network; (b) represents the different divisions of community; (c) represents the population after calculating the objective function and assigning the rank; (d) represents optimal results after rounds of selection, crossover, and mutation. Examples are as follows in
2) ENCODING AND DECODING PROCEDURE
The genetic representation of the algorithm uses locus-based adjacency representation. An individual is composed of N genes g 1 , g 2 , . . . , g N . It is assumed that there exists an allele j in a gene. j is from 1 to N. Gene and allele represent the VOLUME 8, 2020 residue nodes of RINN. The value j assigned to the gene i indicates that residues i and j are in the same community. If there is no interaction between residues i and j, j will be replaced by a residue that interacts with residue i.
When decoding it, residues in the same group are assigned to a community. The main advantage of this representation is that the number of clusters is automatically determined by the number of components contained in the individual and determined by the decoding step. Examples are as follows in Fig 2.
3) INITIALIZATION BASED ON THRESHOLD SELECTION
The DYNMOGA algorithm has some limitations in community detection of RINN, including the population initialization. The random generation of the population usually results in a low adaptability of the population. It may eventually fall into a local optimum, and it takes longer to reach a global optimum. A better population initialization reduces the chance of falling into a local optimum and guarantees a fast approach to the global optimum. Algorithms that generate better populations may reach the global best state faster with fewer iterations. The RINN is constructed based on the number of inter-residue forces and the energy of various forces. The stability of the edges between network nodes is closely related to the strength of the interaction between residues. The energy of hydrogen bond, salt bridge, π-cation, π-π stack, disulfide bridge, and van der Waals interaction are as follows: 17 kJ/mol, 20 kJ/mol, 9.6 kJ/mol, 9.4 kJ/mol, 167 kJ/mol, 6 kJ/mol. The weak interactions between residues are relatively unstable, and the existence of the number of weak interactions makes the community structure of the network unclear, which increases the difficulty of community detection.
In order to detect dense and stable communities, we set thresholds during the population initialization phase to filter out the more unstable interaction forces between residues. The selection result of the threshold is shown in Supplementary Material.
4) CROSSOVER
The uniform crossover algorithm randomly generates a crossover mask with a length of N. One child is generated from the gene of the first parent corresponding to the mask of 0 and the gene of the second parent corresponding to the mask of 1. The uniform crossover can guarantee the maintenance of the effective connections of the nodes in the network in the child individual. Examples are as follows in Fig 3.
5) MUTATION BASED ON THRESHOLD SELECTION
The mutation operation is that the possible values that the allele can assume are limited to the neighborhood genes that meet the threshold, that is, the gene values at certain loci of individuals in the population are changed.
6) OBJECTIVE FUNCTION
The formula of weighted modularity Q w [31] is: Among them, a i = d i /2L is the fraction of edges with at least one end in community i, l i is the number of edges in the community i, d i is the sum of the degree of vertexes in community i, n i is the total number of nodes in the community i, e ii = l i /L is the fraction of the edges in the community i.
Normalized Mutual Information (NMI): A matrix is used to measure the similarity between the community structure at time t and the community structure at the previous moment. Assume a network with two partitions A = {A 1 , A 2 , . . . , A a } and B = {B 1 , B 2 , . . . , B b }. C is a matrix, where the element C ij is the number of nodes in the communities A i ∈ A and B i ∈ B simultaneously. The formula of NMI as follows: Among them, C A represents number of communities in division A, C B represents number of communities in division B, C i . represents the sum of the rows in matrix C, C .j represents the sum of the columns in matrix C, N is the number of nodes. if A = B, NMI (A, B) = 0. So the second objective function is to maximize NMI (C t , C t−1 ) at time t. 89442 VOLUME 8, 2020
B. T-DYNMOGA-Q w ALGORITHM
The algorithm is based on the framework of a multi-objective genetic algorithm (NSGA-II), it optimizes two complementary objective functions Q w and NMI which have been proven to detect the effectiveness of communities in complex networks. Refer to the parameter setting of the DYN-MOGA algorithm. The experimental setting parameters are: crossover probability 0.8, mutation probability 0.2, population number 300, iteration number 100. The algorithm process and parameter settings are as follows in Fig. 4.
C. COMMUNITY ANALYSIS INDICATORS
The graph G = (V, E) has n vertices and m edges. S is a cluster with n s nodes and m s edges. c s = {(u, v) |u ∈ S, v / ∈ S} is the number of edges in the boundary, {S 1 , S 2 , . . . , S k } are k clusters of G.
The following indicators are used to judge the quality of community structure.
Modularity Q [38]: The e ii and a i is the same as formula (6). Intra-Cluster Density [39]: It measures the inner edge density of a community. The formula is as follows: Inter-Cluster Density [39]: It measures the proportion of all possible edges leaving the community. The formula is as follows: When the modularity is greater than 0.3 [38], it is a reliable result of network community. The larger the intra-cluster density value and the smaller the inter-cluster density value, the better the quality of the community structure obtained.
V. RESULTS AND ANALYSIS A. COMPARISON OF T-DYNMOGA-Q w ALGORITHM AND DYNMOGA ALGORITHM ON COMMUNITY DETECTION
The T-DYNMOGA-Q w algorithm and the DYNMOGA algorithm were used in community detection 1-300 frame networks of xyna_strli, xyna_theau, WTL and 6B at 300K, 350K, 400K. The values of Q, community number, community size, inter-cluster density, intra-cluster density are shown in table 1: In table 1, the community modularity of the networks of xyna_strli, xyna_theau, WTL and 6B at 300K, 350K, and 400K are all around 0.6, and the communities detected by the T-DYNMOGA-Q w algorithm have higher modularity. The number of communities detected by the T-DYNMOGA-Q w algorithm is larger, the community size is smaller. And the detected communities have higher intra-cluster density and lower inter-cluster density. The T-DYNMOGA-Q w algorithm has better quality of the detected community structure.
Next, we analyze the key factors affecting the thermal stability of WTL and 6B in the rigid community detected by the T-DYNMOGA-Q w algorithm.
B. RIGID STRUCTURE ANALYSIS
Rigid structure is an important property of protein stability. After the communities of WTL and 6B networks at 300K, 350K and 400k were detected, we analyzed the residues and interactions in communities in the same frame and further identified the stable communities at each temperature. We set that there is a stable relationship between residues and residues that exist in the same community in 270 frames (total 300 frames). The results are shown in Fig. 5: As can be seen from Fig. 5, at 300K, the rigid structures in WTL and 6B are distributed in most of the α-helix, β-sheet and 3 10 -helix, and only a little part in the loop. As the temperature increases, the rigid structures in WTL and 6B gradually decrease, and the scale of rigid structures becomes smaller. When the temperature reaches 400K, the rigid structures in WTL are mainly distributed in αB, αC, αE, and a small part are distributed in β5, β6, and 3 10 -helix. The residues of the rigid structure in the loop only Asp118-Pro119-Asn120-Gln121. The reduction of rigid structures in 6B is less. Compared to WTL, αB, αF, 3 10 -helix and loop (between β4 and αB) in 6B have rigid structures. There are rigid structures in the loop (Asp43-Lys44-Thr45-Gly46-Thr47) between β4 and αB and the loop (Asp133-Met134-Ile135) between β7 and αE too. Rathi [40] et al. studied the lipase unfolding simulation of Bacillus subtilis based on the rigid theory and found that the loop region in the structure first disintegrated, followed by the α-helix and β-sheet. Comparing the changes in the rigid structure of WTL and 6B with increasing temperature, we found that increasing the stability of the α-helix and β-sheet structures contributes to the improvement of lipase thermostability. The left in Fig. 5 shows the rigid structure of WTL and 6B at 300K, 350K, and 400K, respectively. The circles in the figure represent residues, and the edges represent the sum of the forces and energies between the residues. The figure on the right shows the secondary structure corresponding to the rigid structure at each temperature.
By Analyzing the rigid communities of 300K, 350K, and 400K, we found the rigid structures that WTL and 6B are stable at all temperatures. The result is shown in Fig. 6: In Fig. 6, the number and scale of 6B rigid structures is much larger than that of WTL. Comparing the rigid structure in WTL and 6B, the rigid structure in WTL exist the corresponding structure among the rigid structures in 6B except Tyr86-Leu90. The corresponding three-dimensional structure of the rigid structure in the Fig. 6 is shown in Fig. 7 (blue): FIGURE 7. Rigid structure of WTL and 6B at all temperatures show in three-dimensional structure (a) shows the rigid structure of WTL at all temperatures, and (b) shows the rigid structure of 6B at all temperatures. Among them, the red structure represents α-helix, the yellow structure represents β-sheet, and the green structure represents loop region. The blue part indicates the rigid structure of WTL and 6B.
We found that most of the rigid structures in WTL also exist in 6B. Therefore, we analyze the unique rigid structure in 6B: (1) Residues Ser15-Ser17-Asn18 in a rigid structure are located in 3 10 -helix. The strenuous exercise of the 3 10 -helix will affect the stability of the structure [41]. The mutated Ser15 forms a strong hydrogen bond with the mutated Ser17, which stabilizes the 3 10 -helix structure in 6B. (2) Active site N18 and mutation site A20E locate near Gly21-Ile22-Tyr25-Leu26-Ser28-Gln29 in the rigid structure [34]. Ala20 is located at the N-terminus of αA. After mutation, the side chain of Glu20 interacts with water molecules, and water molecules interact with Ser24 (located on the αA helix). Glu20 not only forms hydrogen bond, but also forms salt bridge with Lys23, which contributes to the stability between Glu20 and Ser24. Glu20-Lys23 is also one of the unique rigid structures of 6B. Therefore, it can be speculated that the mutation of A20E contributes to the increase in the rigidity of the αA structure and the stability of the lipase structure. (3) The residues Ala113-Pro114-Pro115-Ile123-Leu124-Tyr125-Gln178 in the rigid structure are located in the loop and the partial structure of β7, and L114P is located in the longest loop [42], which is between β6 and β7. By analyzing the interactions related to L114P, we found that there is more VDW between the Pro114 residue and the Tyr125 residue after the mutation, and the Pro residue can stabilize the folded state of the protein. Therefore, the conversion of Leu to Pro helps to stabilize the structure of the protein, so that the relationship between this loop and β7 in 6B is closer than that in WTL. (4) The residues Asp133-Glu134-Ile135, Pro137-Tyr139-Leu140, Ser162-Pro163-Gln164-Val165 in the rigid structure, which are in the αE and αF terminal and loop regions. M134E, M137P and A163P residues are located at the positions where the end of the helix is connected to the loop region. Mutations at these positions of the WTL reduce the hydrophobic region on the surface of the protein molecule, which can prevent the protein from agglomeration at high temperatures and improve the thermostability [34], [43].
In summary, we find that site-directed mutation of residues and the increase of hydrogen bond between residues can improve the stability of lipase secondary structure. The loop, turn, and helix ends of the protein structure are very flexible regions. The introduction of Pro in these unstable regions can reduce structural flexibility and enhance rigidity. Improving the stability of the secondary structure and the rigidity of the unstable region can improve the heat resistance of the protein.
VI. DISCUSSION
In this study, we proposed an evolutionary multiobjective approach T-DYNMOGA-Q w based on DRINN for community discovery in dynamic networks. The threshold is set during the population initialization phase of the algorithm and the weighted modularity Q w is used as the objective function of the algorithm to optimize the resolution limitation of the modularity. Comparing the T-DYNMOGA-Q w algorithm with the properties of communities detected by DYNMOGA on the xylanase and lipase data sets, we find that the T-DYNMOGA-Q w algorithm can correctly divide the nodes and detect a stable community structure. However, it has the disadvantage of a large number of communities and a small community size. Community detection was performed on the lipase WTL and 6B networks at different temperatures. It was found that the communities detected from the 6B network could still maintain a tight structure when the temperature increased, indicating that the stability between the structures is the reason why 6B is thermophilic one. The mutated Ser15 forms a strong hydrogen bond with the mutated Ser17, and forms more force with other residues. Glu20 not only forms hydrogen bond, but also forms a salt bridge with Lys23. It can be speculated that the mutation of A20E can help increase the rigidity of the αA structure. VOLUME 8, 2020 The introduction of Pro in flexible regions such as the loop region, turns and the ends of the helix can reduce structural flexibility and increase rigidity. Therefore, the increase of hydrogen bond and the improvement of the rigidity of the flexible region contribute to the stability of the structure and have an important impact on the improvement of the protein thermostability.
[42] C. Magyar, M. M. Gromiha, Z. Sávoly, and I. Simon, ''The role of stabilization centers in protein thermal stability,'' Biochem. Biophys JINGSI JI is currently pursuing the master's degree with the Digital Media Institute, Jiangnan University, China. Her main research direction is bioinformatics. She has a strong interest in mining and analyzing biological data using complex networks and different algorithms.
YANRUI DING received the Ph.D. degree in bioinformatics from Jiangnan University, China. She is currently a Professor with the School of Science, Jiangnan University and a Visiting Scholar with New York University. She has published over 60 research articles. She has presided and finished several research projects including two projects supported by the National Natural Science Foundation. She applied two authorized patents and one soft-ware copyright. Her research interests include computing intelligence, data mining, and bioinformatics. VOLUME 8, 2020 | v3-fos-license |
2018-08-18T21:15:57.469Z | 2018-08-01T00:00:00.000 | 52022092 | {
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} | pes2o/s2orc | Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells
Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation.
Introduction
Over the years, many techniques leading to cardiomyocyte differentiation and isolation have been established. Unfortunately, by the use of these differentiation protocols, beating colonies of cardiomyocytes induced in vitro were not physiologically identical to cardiomyocytes isolated in vivo. Additionally, there have been several attempts to generate permanent, early cardiac cell lines. For example, H9c2-derived cells from embryonic rat heart [1], embryonal avian heart [2], transgenic mice with myocardial tumors [3], or neonatal rat myocardial cells transfected with SV-40 large T antigen [4] have been tested. In these cases, the established cell lines were not effective in studies of physiological processes leading to functional cardiomyocytes. The main reason for this result was that these cell lines were able to grow in culture for only a few passages because optimal cultivation conditions, including the composition of the cell culture media, for their growth in vitro had not been fully established [5]. From this view, Wobus et al. [6] studied the effect of retinoic acid on cardiomyocyte differentiation; these authors specifically analyzed cell induction into ventricle-like cardiomyocytes. In their protocol, the authors induced cardiomyocytes with Purkinje-and ventricle-like markers while a reduced number of pacemaker-and atrium-like cells were observed.
Cardiomyocyte differentiation has also been effectively studied in mouse and human embryonic stem cells (mESCs). These cells are characterized by pluripotency, self-renewal, broad differentiation plasticity, a relatively stable karyotype, and the ability to differentiate into cells from all three germ layers (ectoderm, mesoderm, and endoderm). Embryonic stem cells can be isolated from the inner cell mass (ICM) of blastocysts, and these cells are considered to be pluripotent. In this regard, Wobus et al. [7] explained that in mice, the fertilized oocyte and blastomeres of two-, four-, and eight-cell-stage embryos are totipotent, while cells from the ICM and the embryonic ectoderm and the primordial germ cells from fetal stages are only pluripotent. When transferred into an early embryo, these cells can physiologically mimic all cells of the embryo but not the placental tissue, and, therefore, these cells are not able to generate an organism.
For experimental studies, it is very useful to analyze the differentiation potential of threedimensional embryoid bodies (EBs) generated from ESCs in "hanging drops". EB development shows a multicellular arrangement like those of skeletal [8] cells, neuronal cells [9], blood vessels [10], and epithelial cells [11]. Moreover, EBs consist of various extracellular matrix components (collagen, laminin, nidogen, and fibronectin) [12,13]. In this complex multicellular structure, spontaneous beating cardiomyocytes appear between the epithelial layer and basal layer of mesenchymal cells [14]. In these highly specialized cells, it is generally accepted that beating attributes, such as beating frequency and an appearance of cardiomyocytes markers, including α-actinin, represent a tool for how to mimic cardiomyogenesis in vitro [15,16].
Cardiomyocytes are also characterized by specific epigenetic features. Epigenetics refers to heritable modifications of DNA, RNA, and histones. Epigenetic regulation, playing a role not only in cardiomyogenesis, includes DNA methylation and the function of non-coding RNAs or histone post-translational modifications (PTMs). Histone modifications are, for example, regulated by enzymes, such as kinases, histone acetyltransferases (HATs), histone deacetylases (HDACs), or histone methyltransferases/demethylases [17]. These epigenetic features can be affected by many factors, including pollution in the environment, diet (referred to as epi-diet), and disease progression or epigenetic therapy. In cardiomyocytes, for example, the p300 transcriptional co-activator, which is considered to be a HAT (responsible for GATA-4 acetylation) potentiates GATA-4 s DNA binding property [18]. Moreover, HDAC inhibitors (HDACi), including TSA, promote the differentiation of ESCs into cardiomyocytes; thus, it appears to be evident that HDACs in general play a role in cardiomyogenesis. As an example, a unique study of human hearts showed that HDAC4 inhibits the expression of pro-hypertrophic genes by recruiting a histone methyltransferase, SUV39H1, to regulatory elements for these loci that become methylated on histone H3 lysine-9 (H3K9) [19]. HDAC4 associates not only with SUV39H1 but also with the DNA binding transcription factor MEF2 (myocyte enhancer factor 2) and heterochromatin protein 1 (HP1). This nuclear event is essential for HDAC4-mediated downregulation of pro-hypertrophic genes. Conversely, from the view of hypertrophic gene activation, HDAC4 was found to be exported from the nucleus, and thus, the MEF2 factor was released to interact with histone acetyltransferases (HATs) that contribute to the upregulation of these genes. Cardiovascular diseases (CVD), including coronary artery diseases (CAD), such as angina and myocardial infarction, and other diseases, such as stroke, heart failure, rheumatic heart disease, hypertensive heart, and others, are one of the major reasons for death in the world. Unfortunately, the incidence has dangerously increased over the past 100 years. CVD is a group of multifactorial disorders linked to genetic risk factors [20]. These factors (mostly primary diseases) include hypertension, diabetes mellitus, familial hypercholesterolaemia, and familial hyperlipidaemia; all these diseases are characterized by a specific genetic background [21]. Thus, studies with experimental animals, focused not only on the physiology but also on the pathophysiology of cardiac tissue, enable a better understanding of the molecular mechanisms of CVD. From this view, it is well-known that epigenetic regulation plays a fundamental regulatory role on genes in which their expression is associated with CVD risk. Thus, an understanding of epigenetic events associated with CVD is essential from the view of potential therapy using epi-drugs. In this scenario, epigenetic approaches, especially the clinical application of HAT or HDAC inhibitors, are promising for new therapeutic strategies [22]. For example, an understanding of how histone acetylation affects cardiomyogenesis and how HATs and HDACs work during this process could initiate new and promising therapeutic approaches. The connection between changes in histone acetylation and cardiac hypertrophy was demonstrated by Gusterson and colleagues. These authors showed that overexpression of the transcriptional co-activators CREB-binding protein (CBP) and p300 HAT can induce cardiac hypertrophy due to the HAT activity of these proteins [23]. Antos et al. demonstrated that inhibition of HDACs by TSA shut down cardiac hypertrophy without affecting cell viability [24]. Cao et al. showed that autophagy processes in heart failure represent a target for therapy by HDACs inhibitors [25], and Liu et al. additionally documented the epigenetic background of the most common clinical cardiac arrhythmia: atrial fibrillation (AF). Mice overexpressing the HopX protein develop cardiac hypertrophy, which influences AF. Using a specific HDAC inhibitor (TSA), researchers have reduced atrial arrhythmogenesis as well as reversed atrial fibrosis [26]. Montogomery et al. highlighted the role of HDAC1 and HDAC2 in the control of cardiac function. Deletion of both genes (Hdac1 and Hdac2) in a mouse model resulted in early death caused by fetal arrhythmia or dilated cardiomyopathy [27]. Moreover, Montomery et al. showed that the phenotype of cardiac-specific Hdac3 gene deletion is distinct from those mutations in other Hdac genes [28]. This observation implies that the role of the Hdac3 gene is important for cardiac function. From this view, McKinsey, using a rodent model for heart failure, obtained promising data about the therapeutic potential of HDAC inhibitors [29]. According to the above-mentioned data, it appears likely that HDAC function, modified by HDAC inhibitors, is important for cardiovascular therapeutic applications. Thus, here we address the question of how HDAC1 depletion affects mESC differentiation into cardiomyocytes and how the histone signature, especially histone H3 and H4 acetylation or H3 methylation, is affected by HDACi treatment of HDAC1 wild-type (wt) and HDAC1 double knockout (dn) mESCs. Additionally, we analyzed embryonic heart epigenetic features. We tested clinically promising HDACi, including TSA (trichostatin A), SAHA (suberoylanilide hydroxamic acid, syn. vorinostat), and VPA (valproic Acid), on explanted mouse hearts. Our main hypothesis was that HDACi would modulate the epigenome of cardiac cells and HDAC1 depletion would affect the efficiency of cardiac differentiation.
The Beating of Cardiomyocytes Studied in wt and HDAC1 dn Cells
Cardiomyocyte differentiation was induced in the EBs of wt and HDAC1-depleted ESCs, and the beating was monitored every day. Cardiomyocytes beat from day 12 to day 25, with an average differentiation on the 20th day, but the beating period was affected by HDAC1 depletion (Figure 1A,B). Briefly, the beating period was not identical in the non-treated wt and the non-treated HDAC1 dn cells. HDAC1 wt mESCs started to beat at day 12-15 after differentiation, and beating lasted approximately 5 days ( Figure 1C). However, HDAC1 dn mESCs started to beat on the 10th day after differentiation, and when these cells began to beat in this early interval, the period of beating was 2.5 days ( Figure 1C). HDAC inhibitors, including TSA, SAHA, and VPA, had an ability to prolong the time of the beating irrespective of HDCA1 depletion ( Figure 1C). The beating time was 6-7 days in the case of HDCAi treatment ( Figure 1C). The size of the beating area was only reduced in the SAHA-treated HDAC1 dn cells ( Figure 1D). These data show that HDAC inhibitors, especially SAHA, have an effect on the beating of cardiomyocytes that were differentiated from mESCs.
Adherence of Embryonic Bodies Is Affected by HDAC1 Depletion
Embryonic bodies (EBs) represent an important cellular model for the study of embryonic development and are a useful tool for testing the role of pluripotency in vitro and the induction of the differentiation processes. The use of EBs is highly respected only when the cells have an ability to fully differentiate within these three-dimensional structures. In EBs, we observed the formation of the cavity. The cavitation should mimic the formation of the developing body cavity. The cavitation can represent a phenomenon linked to not only pericardial formation but also to the lateral plate mesoderm cavities, such as the pleural and peritoneal cavities. Interestingly, in EBs generated from wt mESCs, the cavity appears early, at day 6 (dd6) of differentiation. In HDAC1 dn mESCs, the appearance of cavitation started later than in wt cells (dd10), and here we show day 13 (dd13) that was additionally characterized by cavity malformations (Figure 2A).
Adherence of Embryonic Bodies Is Affected by HDAC1 Depletion
Embryonic bodies (EBs) represent an important cellular model for the study of embryonic development and are a useful tool for testing the role of pluripotency in vitro and the induction of the differentiation processes. The use of EBs is highly respected only when the cells have an ability to fully differentiate within these three-dimensional structures. In EBs, we observed the formation of the cavity. The cavitation should mimic the formation of the developing body cavity. The cavitation can represent a phenomenon linked to not only pericardial formation but also to the lateral plate mesoderm cavities, such as the pleural and peritoneal cavities. Interestingly, in EBs generated from wt mESCs, the cavity appears early, at day 6 (dd6) of differentiation. In HDAC1 dn mESCs, the appearance of cavitation started later than in wt cells (dd10), and here we show day 13 (dd13) that was additionally characterized by cavity malformations (Figure 2A).
Figure 2.
Studies on EB formation and adherence in HDAC1 wt and HDAC1 dn mESCs. Under transmitted light microscopy and through the use of bright-field microscopy, the formation of EBs was inspected at day 3 (dd3), 6 (dd6), and 13 (dd13) in (A) HDAC1 wt mESCs and (B) HDAC1 dn mESCs. Cavity formation in EBs is shown by white arrows. Scale bars show 500 µm. Adherence of EBs was reduced in HDAC1 dn mESCs as shown in panel (C). The asterisk shows a statistically significant difference at p ≤ 0.05 (*).
Here, we also study the adherence of EBs using wide-field microscopy, and the EBs were monitored on day dd3, dd6, and dd13 of cell cultivation and differentiation. The observations were performed using transmitted light microscopy (Figure 2A,B). We calculated the percentage of adherent EBs as shown in Figure 2C. In comparison to wt cells, we found a reduced number of adherent EBs that were generated from the HDAC1 dn mESCs. This result was statistically significant at p ≤ 0.05.
Differentiation-Specific Deacetylation of Histone H4 was Weakened in HDAC1-Depleted Cells.
Through the use of Western blots, we studied the level of selected histone markers in nondifferentiated wt and HDAC1 dn mESCs or these cells differentiated into cardiomyocytes. Terminally differentiated cardiomyocytes were additionally treated by HDAC inhibitors, including TSA, SAHA, or VPA. In wt cells, we observed the deacetylation of histone H4 that accompany cardiomyocyte differentiation. Interestingly, H4 deacetylation was weakened in the HDAC1-depleted cells ( Figure 3A,B and Ca,b).
We additionally studied H4K20ac, H3K9ac, pan-lysine acetylation, and H3K9me3 in wt and HDAC1 dn cells. In these histone markers, we observed a decrease in H4K20ac in differentiated wt Under transmitted light microscopy and through the use of bright-field microscopy, the formation of EBs was inspected at day 3 (dd3), 6 (dd6), and 13 (dd13) in (A) HDAC1 wt mESCs and (B) HDAC1 dn mESCs. Cavity formation in EBs is shown by white arrows. Scale bars show 500 µm. Adherence of EBs was reduced in HDAC1 dn mESCs as shown in panel (C). The asterisk shows a statistically significant difference at p ≤ 0.05 (*).
Here, we also study the adherence of EBs using wide-field microscopy, and the EBs were monitored on day dd3, dd6, and dd13 of cell cultivation and differentiation. The observations were performed using transmitted light microscopy (Figure 2A,B). We calculated the percentage of adherent EBs as shown in Figure 2C. In comparison to wt cells, we found a reduced number of adherent EBs that were generated from the HDAC1 dn mESCs. This result was statistically significant at p ≤ 0.05.
Differentiation-Specific Deacetylation of Histone H4 was Weakened in HDAC1-Depleted Cells
Through the use of Western blots, we studied the level of selected histone markers in non-differentiated wt and HDAC1 dn mESCs or these cells differentiated into cardiomyocytes. Terminally differentiated cardiomyocytes were additionally treated by HDAC inhibitors, including TSA, SAHA, or VPA. In wt cells, we observed the deacetylation of histone H4 that accompany cardiomyocyte differentiation. Interestingly, H4 deacetylation was weakened in the HDAC1-depleted cells (Figure 3(A,B,Ca,Cb)).
We additionally studied H4K20ac, H3K9ac, pan-lysine acetylation, and H3K9me3 in wt and HDAC1 dn cells. In these histone markers, we observed a decrease in H4K20ac in differentiated wt cells and HDAC1 dn cells. H3K9ac increased significantly in differentiated HDAC1 dn cells ( Figure 3A,B). The H3K9me3 level increased on day 7 (dd7) of differentiation and then maintained a relatively stable level in both the wt and HDAC1 mESCs induced to cardiac differentiation ( Figure 3A,B).
Here, we also show the results of the effect of HDACi on levels of H4ac, H4K20ac, and α-actinin (Figure 3(Ca-c)). We observed that, as opposed to SAHA and VPA, trichostatin A induced H4 and H4K20 hyperacetylation in wt cells on day 20 or 25 of cardiac differentiation, and in HDAC1 dn cells, this was observed on the 20th day of differentiation (Figure 3(Ca,b)). We found a significant increase in wt mESCs treated by TSA and when observed on day 25 (dd25) of differentiation (Figure 3(Cb)). Moreover, the α-actinin level, which is a marker of cardiomyogenesis, was elevated in the VPA-treated and differentiated wt cells (dd25), and the increased level of α-actinin was significant in the HDAC1 dn cells treated by TSA or SAHA on the 20th day (dd20) of cardiac differentiation (Figure 3(Cc)).
The differentiation-specific deacetylation effect, which was observed by Western blots ( Figure 3A,B), was also confirmed by immunofluorescence combined with confocal microscopy ( Figure 4A,B). From a cell morphology point of view, we observed that HDAC1-depleted cells were characterized by star-shaped α-actinin filaments (Figure 4(Ba), frame). Interestingly, the distribution pattern of the α-actinin filaments of the TSA-treated wt mESCs resembled tile-like contours (Figure 4(Bb)). This distribution pattern was observed in 70-80% of the α-actinin-positive wt cells. A similar distribution pattern was also observed in 40-50% of the SAHA-treated HDAC1 dn cells (Figure 4(Bc)). VPA changes only 5% of the wt cells to having a star-like shape (Figure 4(Bd), right bottom part in wt cells). Figure 3A,B). The H3K9me3 level increased on day 7 (dd7) of differentiation and then maintained a relatively stable level in both the wt and HDAC1 mESCs induced to cardiac differentiation ( Figure 3A,B).
Here, we also show the results of the effect of HDACi on levels of H4ac, H4K20ac, and α-actinin (Figure 3Ca-c). We observed that, as opposed to SAHA and VPA, trichostatin A induced H4 and H4K20 hyperacetylation in wt cells on day 20 or 25 of cardiac differentiation, and in HDAC1 dn cells, this was observed on the 20th day of differentiation (Figure 3Ca,b). We found a significant increase in wt mESCs treated by TSA and when observed on day 25 (dd25) of differentiation (Figure 3Cb). Moreover, the α-actinin level, which is a marker of cardiomyogenesis, was elevated in the VPAtreated and differentiated wt cells (dd25), and the increased level of α-actinin was significant in the HDAC1 dn cells treated by TSA or SAHA on the 20th day (dd20) of cardiac differentiation ( Figure 3Cc).
The differentiation-specific deacetylation effect, which was observed by Western blots ( Figure 3A,B), was also confirmed by immunofluorescence combined with confocal microscopy ( Figure 4A,B). From a cell morphology point of view, we observed that HDAC1-depleted cells were characterized by star-shaped α-actinin filaments (Figure 4Ba, frame). Interestingly, the distribution pattern of the α-actinin filaments of the TSA-treated wt mESCs resembled tile-like contours ( Figure 4Bb). This distribution pattern was observed in 70-80% of the α-actinin-positive wt cells. A similar distribution pattern was also observed in 40-50% of the SAHA-treated HDAC1 dn cells (Figure 4Bc). VPA changes only 5% of the wt cells to having a star-like shape (Figure 4Bd, right bottom part in wt cells). . Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, panacetylated lysines (K-ac), and α-actinin in (A) HDAC1 wt mESCs and (B) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH (C). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel (Ca) shows the levels of H4ac, (Cb) shows H4K20ac, and (Cc) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel (A,B), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel (C) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student's t-test; asterisks (*) in panel (Ca-c) show statistically significant differences at p ≤ 0.05. Note that the y-axis-scale in panel (Ca) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel (Ca), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells (Cb). In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH (C). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel (Ca) shows the levels of H4ac, (Cb) shows H4K20ac, and (Cc) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel (A,B), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel (C) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student's t-test; asterisks (*) in panel (Ca-c) show statistically significant differences at p ≤ 0.05. Note that the y-axis-scale in panel (Ca) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel (Ca), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells (Cb).
Histone Hyperacetylation Can Be Induced by HDACs Inhibitors in Hearts Explanted from Mouse Embryos at Stage e15
Explanted embryonic hearts (e15) also undergo histone hyperacetylation when treated with HDACi. We found that all tested HDACi (TSA, SAHA, VPA) increased the level of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines. TSA and SAHA were very strong hyperacetylating agents in terms of the total H4ac. When we focused on H4K20ac, the level of this epigenetic marker was also increased in e15 hearts ( Figure 5A,B). However, pronounced H3K9ac was observed only after the
Histone Hyperacetylation Can Be Induced by HDACs Inhibitors in Hearts Explanted from Mouse Embryos at Stage e15
Explanted embryonic hearts (e15) also undergo histone hyperacetylation when treated with HDACi. We found that all tested HDACi (TSA, SAHA, VPA) increased the level of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines. TSA and SAHA were very strong hyperacetylating agents in terms of the total H4ac. When we focused on H4K20ac, the level of this epigenetic marker was also increased in e15 hearts ( Figure 5A,B). However, pronounced H3K9ac was observed only after the treatment using TSA (Figure 5A,B). Using immunofluorescence and confocal microscopy, we additionally observed that H3K9ac is homogeneously distributed throughout e15 embryonic hearts, and this is additionally characterized by a nodal accumulation of H3K9ac in ventricular portions ( Figure 5C). treatment using TSA ( Figure 5A,B). Using immunofluorescence and confocal microscopy, we additionally observed that H3K9ac is homogeneously distributed throughout e15 embryonic hearts, and this is additionally characterized by a nodal accumulation of H3K9ac in ventricular portions ( Figure 5C). Additionally, we studied the level of α-actinin as a marker of cardiomyocytes, but the level of this marker (when normalized to the level of GAPDH) was not affected by HDACi treatment of explanted hearts ( Figure 5A,B). These data unambiguously show that even in explanted hearts, histones can be epigenetically modified using epi-drugs, which could be an important observation from the viewpoint of transplantation strategies. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. (B) Data from panel (A) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student's t-test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. (C) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions. Additionally, we studied the level of α-actinin as a marker of cardiomyocytes, but the level of this marker (when normalized to the level of GAPDH) was not affected by HDACi treatment of explanted hearts ( Figure 5A,B). These data unambiguously show that even in explanted hearts, histones can be epigenetically modified using epi-drugs, which could be an important observation from the viewpoint of transplantation strategies.
Discussion
It is well-known that the differentiation of ESCs into cardiomyocytes is regulated by the specific subset of genes that are furthermore regulated by epigenetic factors, including HATs and HDACs [30,31]. The present study shows that HDAC1 depletion, to some extent, prevents total H4 deacetylation, which is an epigenetic marker of cardiomyogenesis ( Figure 3A,B). Conversely, H3K9ac was relatively stable in differentiated wt mESCs, while HDAC1 dn mESCs that were induced to cardiac differentiation were characterized by a pronounced increase in H3K9ac ( Figure 3B). In parallel, the H3K9me3 level began to be elevated in both tested mES cell lines from dd7 of cardiac differentiation, and then the level of this histone mark remained stable. Interestingly, differentiated cardiomyocytes from both wt and HDAC1 dn cells were characterized by a lower level of α-actinin in comparison to e15 embryonic hearts ( Figure 3A,B). In detail, in wt mESCs, α-actinin was detectable at dd25 of cardiac differentiation, while in differentiated HDAC1 dn mESCs, the Western blot fragment showing α-actinin was visible at dd20 of differentiation and was even visible in the cells treated by HDACi ( Figure 3A,B). VPA treatment increased the level of α-actinin at dd25 in wt cells, but TSA and SAHA in HDAC1 dn cells elevated the α-actinin level earlier, at dd20 (Figure 3(Cc)).
Here, we document that HDAC1 depletion has an effect not only on the histone signature, especially H4ac, but also on beating areas in EB-containing cardiomyocytes ( Figure 1A,B and Figure 3(A-Ca)). From this view, the class I HDACs appear to be very important for cardiac differentiation and heart development. For example, HDAC2 promotes cardiac hypertrophy [32], and cardiac-specific deletion of HDAC3 in mice leads to cardiac hypertrophy and excessive myocardial lipid accumulation [28]. Moreover, HDAC6 regulates a structural and functional remodeling of atrial myocytes [33]. Here, we showed that HDAC1 depletion affects the timing related to the appearance of the cardiac marker α-actinin during in-vitro-induced cardiomyogenesis. Interestingly, the level of α-actinin was potentiated by HDACi treatment, likely due to hyperacetylation. This conclusion is in an agreement with Kawamura et al. [30] showing that TSA increased the acetylation of not only histones but also non-histone proteins, including the zinc finger protein GATA-4. This epigenetic event was induced during differentiation of ESCs into cardiomyocytes. These data document that non-histone proteins can also be affected by HDACs inhibitors. Similarly, Glozak et al. [34] showed that HATs and HDACs affect the function of non-histone proteins, including transcription factors, and their interaction potential. Moreover, HDACi are also functional, not only in cell lines cultivated in vitro but also in explanted mouse hearts in which we found that HDACi increased H4ac, H4K20ac, H3K9ac, and the acetylation of lysines (K-pan-acetylation) ( Figure 5A,B). This is a very interesting observation that shows that even explanted organs, at least 3 h after experimental surgery, underwent histone signature changes. Thus, from the viewpoint of tissue transplantation, HDACi might be considered as potential maintainers of tissue epigenome. Moreover, protein hyperacetylation could be beneficial from the viewpoint of enhanced expression of genes playing a role in graft acceptance and subsequent tissue regeneration. Thus, our claim fits well with Tao et al. [35] suggesting a post-transplantation role of HDACi. These authors showed that HDACi can affect regulatory T cells (Tregs), increase acetylation of Foxp3 protein, and cause chromatin remodeling. Thus, epigenetic regulation via epi-drugs could be taken into account from the viewpoint of therapeutic applications in transplantation medicine.
Cardio differentiation was initiated by seeding 400 cells per 30 µL into ES culture media without the addition of LIF factor using the "hanging drop" method. On the 3rd day of differentiation (dd3), the increased embryonic bodies (EBs) were plated onto non-adhesive (bacteriological) plastic dishes; on dd6, EBs were transferred to gelatin-coated culture dishes with DMEM/F12 (1:1) (#11320-033, Gibco, Paisley, UK) supplemented with insulin, transferrin, and selenium (ITS-100x, #41400-045, Gibco) (DMEM/F12-ITS). The adhesion efficiency was evaluated on dd7 and dd8. The serum-free DMEM/F12-ITS culture medium was changed every two days. On dd15, the HDACi (100 nM TSA, 8 µM SAHA, and 5 mM VPA) were applied for 3 h. The aim of treating the HDAC1-depleted cells using HDACs inhibitors was to potentiate the hyperacetylating effect of HDAC 1 depletion. Differentiation and cell monitoring were terminated on dd25.
Experimental Animals
To study epigenetic features of embryonic hearts, mouse strain C57Bl6 was used. Mice were housed in a specific pathogen-free (SPF) animal facility at the Institute of Biophysics of the Czech Academy of Sciences at a constant temperature of 21 • C and 60% humidity under a 12 h/12 h light/dark cycle with access to food and water ad libitum. All experiments with mice were performed according to the Agreement of the Ethics Commission of the Czech Academy of Sciences (document No.: 48/2016). After breading, embryos were explanted from female animals 15 days post conception (e15) and embryonic hearts were treated using HDAC inhibitors (200 nM TSA, 16 µM SAHA, and 15 mM VPA). HDACi were dissolved in DMEM supplemented by 10% of FBS. Explanted hearts were treated for 3 h in DMEM with 10% FBS, and the hearts were maintained at 37 • C in a humidified atmosphere containing 5% CO 2 . The concentrations of the HDACi were optimized in Večeřa et al. [39].
Immunostaining
After cell fixation with 4% paraformaldehyde, the interphase nuclei were permeabilized with 0.2% Triton X100 for 8 min, were treated with 0.1% saponin (Sigma-Aldrich) for 12 min, and then washed twice in PBS for 15 min. A solution of 1% bovine serum albumin in PBS was used for blocking of non-specific binding of antibodies. The procedure was performed at room temperature (RT) for 1 h. After washing with PBS for 15 min, samples were incubated overnight at 4 • C with the monoclonal antibodies of interest: α-actinin (#A-7811, Sigma-Aldrich), H3K9ac (#06-942, Merc Millipore, MA, USA) and H4ac (#382160, Merc Millipore). The next day, the cells were washed twice in PBS for 5 min and incubated for 1 h with the appropriate secondary antibody conjugated with the fluorochrome of interest (#A11032, Alexa Fluor 594 anti-mouse IgG, Life Technologies Corporation, Eugene, OR, USA; #ab150077, Alexa Fluor 488 anti-rabbit IgG, Abcam, Cambridge, UK). Immuno-stained preparations were washed three times in PBS for 5 min, and DAPI (4 ,6-diamidino-2-phenylindole dihydrochloride; #10236276001, Roche, Prague, CZ) was used for counterstaining the cell nuclei.
Confocal Microscopy
For analyses, a Leica TSC SP-5 X or SP-8 confocal microscope was used and was equipped with a white light laser (470-670 nm in 1 nm increments), an argon laser (488 nm), and UV lasers (355 nm and 405 nm). We used objectives with the following magnification: 20× HCX PL APO lambda blue (20.0× 0.7 IMM UV, Leica Microsystems, Mannhein, Germany) and an oil objective HCX PL APO 63× lambda blue with a numerical aperture (N.A. = 1.4). To prevent photo bleaching of fluorochromes, we used hybrid detectors (HyD) for time-lapse confocal microscopy; alternatively, fixed cells were monitored by photomultipliers (PMTs). The LEICA LAS AF software was used for data acquisition and analysis. The following microscope settings were used: 1024 × 1024 pixels, 400 Hz, and 8 × zoom [42].
The Tile-Scanning
Cryo-sections (7 µm) of whole embryonic hearts (at stage e15) were stained by an antibody against H3K9ac (#06-942, Merc Millipore) to visualize the distribution pattern of this histone marker. For image acquisition, we used the "tile-scanning" mode and the following objective: HCX PL APO lambda blue 20.0× 0.7 IMM UV (Leica Microsystems). Scanning was performed at a resolution of 512 × 512 pixels, and for image reconstruction, we used the auto-stitched tile-scanning mode involving the smooth-scanning mode with a slow/fine speed accuracy (set in the Leica LAS AF software connected to the Leica SP5 X microscope) [39].
Statistical Analysis, Image Analysis, and Image Processing
For statistical analyses, the following software was used: ImageJ, Sigma Plot 2000, and the Leica 3D software suite (LEICA LAS AF). Sigma Plot software was used in to analyze statistically significant differences using Student's t-test. The Leica 3D software suite (LEICA LAS AF) was used to measure the volume of the beating areas in EBs. Funding: This work was supported by the Czech Science Foundation (grant number: P302-12-G157). Work was also supported by a program of the Academy of Sciences of the Czech Republic, called Strategie AV21, Qualitas, the Center for Epigenetics (ICO: 68081707).
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
2019-11-14T14:12:34.935Z | 2019-11-01T00:00:00.000 | 207944939 | {
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} | pes2o/s2orc | The transcription factor FoxM1 activates Nurr1 to promote intestinal regeneration after ischemia/reperfusion injury
FoxM1 is involved in the regeneration of several organs after injury and expressed in the intestinal mucosa. The intrinsic mechanism of FoxM1 activity in the mucosa after intestinal ischemia/reperfusion (I/R) injury has not been reported. Therefore, we investigated the role of FoxM1 in mediating intestinal mucosa regeneration after I/R injury. Expression of FoxM1 and the proliferation of intestinal mucosa epithelial cells were examined in rats with intestinal I/R injury and an IEC-6 cell hypoxia/reperfusion (H/R) model. The effects of FoxM1 inhibition or activation on intestinal epithelial cell proliferation were measured. FoxM1 expression was consistent with the proliferation of intestinal epithelial cells in the intestinal mucosa after I/R injury. Inhibition of FoxM1 expression led to the downregulation of Ki-67 expression mediated by the inhibited expression of Nurr1, and FoxM1 overexpression promoted IEC-6 cell proliferation after H/R injury through activating Nurr1 expression. Furthermore, FoxM1 directly promoted the transcription of Nurr1 by directly binding the promoter of Nurr1. Further investigation showed low expression levels of FoxM1, Nurr1, and Ki-67 in the intestinal epithelium of patients with intestinal ischemic injury. FoxM1 acts as a critical regulator of intestinal regeneration after I/R injury by directly promoting the transcription of Nurr1. The FoxM1/Nurr1 signaling pathway represents a promising therapeutic target for intestinal I/R injury and related clinical diseases.
Introduction
Intestinal ischemia/reperfusion (I/R) injury is a common pathophysiological process in many clinical settings that includes small bowel transplantation, hemorrhagic shock, and necrotizing enterocolitis 1,2 . It can cause severe intestinal mucosa damage that provokes intestinal mucosal barrier dysfunction. Once the intestinal epithelium, one of the most rapidly proliferating tissues in the body, is damaged, it activates regeneration programs to restore its mucosal barrier function 3 . The intrinsic mechanism of intestinal mucosa regeneration is not always sufficient to restore mucosal barrier function damaged by I/R injury, which is associated with significant morbidity and mortality. The pathophysiology of intestinal regeneration after I/R injury is complex and involves many signaling pathways [4][5][6] . Several signaling pathways are involved in the proliferation of intestinal epithelial cells after I/R injury 7 . However, the intrinsic mechanisms of intestinal epithelial cell proliferation after I/R injury are still not known.
As a typical transcription factor, FoxM1 belongs to the family of Forkhead box (Fox) proteins and is associated with cell proliferation. It is expressed in several embryonic tissues and the testes, thymus and intestinal crypts in adult mice [8][9][10] . In addition, FoxM1 is a key regulator of cell cycle progression and critical for the replication of DNA and mitosis [11][12][13] . Studies have shown that FoxM1 expression is reactivated after organ injury and that FoxM1 has pleiotropic roles during mouse liver regeneration after partial hepatectomy injury 14 . Ackermann reported that FoxM1 is required for the proliferation of preexisting beta cells after 60% partial pancreatectomy 15 .
Ye et al. demonstrated that the expression of FoxM1 accelerates DNA replication and hepatocyte mitosis in the regenerating liver 16 . FoxM1, a key regulator of quiescence and self-renewal in hematopoietic stem cells, is mediated by control of Nurr1 expression 17 , and our previous research found that Nurr1 promotes intestinal mucosa epithelial cell proliferation after I/R injury by inhibiting p21 expression 18 . FoxM1, which is collectively considered a typical proliferation-associated transcription factor, is expressed in intestinal crypts. However, the effects of FoxM1 in regeneration of the intestinal mucosa after intestinal injury have not been examined.
Here, we propose that FoxM1 plays an important role in promoting intestinal mucosa regeneration after I/R injury. We determined that FoxM1 promotes intestinal mucosa epithelial cell proliferation via promoting the expression of Nurr1. Mechanistically, our findings demonstrate the direct transcriptional regulation of Nurr1 by FoxM1 in intestinal mucosa regeneration after I/R injury and that the FoxM1/Nurr1 pathway is involved in intestinal regeneration after I/R injury, providing new and potential therapies for intestinal I/R injury.
Intestinal I/R injury model and tissue analysis
Male wild-type Sprague-Dawley rats weighing between 180 and 220 g were purchased from the Animal Center of Dalian Medical University. The animal studies were performed at Dalian Medical University. The intestinal I/R injury model was described in a previous study in rats 19 . Briefly, after anesthetization of the rats with an intraperitoneal injection of pentobarbital (40 mg/kg), the superior mesenteric artery (SMA) and collateral vessels were interrupted with atraumatic clips. After 1 h of ischemia, the atraumatic clips were removed to initiate reperfusion for 3, 6, 12, or 24 h. Ileum tissue samples (1 cm) were collected for the various experimental evaluations required for this study. Rats in the sham group underwent laparotomy without SMA and collateral vessel occlusion. Rats in the sham group did not exhibit changes in FoxM1 expression, and pentobarbital anesthesia did not influence FoxM1 expression (supplementary material 1).
To test the roles of FoxM1 in intestinal mucosa regeneration after I/R injury, we used the FoxM1 inhibitor thiostrepton (TST) to inhibit the expression of FoxM1 20,21 . Rats were randomly divided into 4 groups: the sham, sham+TST, I/R, and I/R+TST groups. The sham and I/R groups were treated as described above, and 50 mg/kg TST was given daily by intraperitoneal injection for two days before I/R surgery. After 1 h of intestinal ischemia followed by 6 h of reperfusion, ileum tissue samples were collected.
To determine whether Nurr1-mediated FoxM1 promotes intestinal regeneration after I/R injury, we determined whether the Nurr1 activator 1,1-bis(39-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) 22,23 could abolish the inhibitory effects of TST on intestinal I/R injury. Rats were randomly divided into 4 groups: the sham, I/R, I/R+TST, and I/R+TST+C-DIM12 groups. Rats in the sham, I/R and I/R+TST groups were treated as described above. C-DIM12 (50 mg/kg) was given before surgery. After 1 h of intestinal ischemia followed by 6 h of reperfusion, ileum tissue samples were collected.
The protocols and experiments in this study were conducted according to the guidelines of Dalian Municipal Central Hospital Affiliated of Dalian Medical University and approved by the Institutional Ethics Committee of Dalian Municipal Central Hospital Affiliated of Dalian Medical University.
Western blot analysis
Protein expression in the intestinal samples or IEC-6 cells was analyzed by western blotting. The samples or cells were washed with ice-cold PBS and lysed in RIPA buffer (P0013, Beyotime, China) supplemented with phosphatase inhibitors and a protease inhibitor (P1048, Beyotime, China). Cells lysates were incubated for 2 h at 4°C and centrifuged at 13,000 rpm for 10 min at 4°C to remove cellular debris. The protein concentration was determined with a BCA protein assay kit (P0010, Beyotime, China). Whole cell lysates were loaded on a 12% SDS-polyacrylamide gel and transblotted to a nitrocellulose membrane after electrophoretic separation. Blocking was carried out in 5% PBS-milk, following which the membrane was incubated with anti-FoxM1 antibody (1:500, Santa Cruz Biotechnology, USA) and anti-Nurr1 antibody (1:500, Cell Signaling Technology, USA) overnight. The membrane was washed with 1× PBST and probed with HRP-conjugated anti-rabbit antibody (1:1000 dilution) for 2 h at 37°C, followed by enhanced ECL chemiluminescence (P0018, Beyotime, China) detection. As a loading control, membranes were incubated with anti-β-actin antibody (TA-09, ZSGB-BIO, China) for 2 h at 37°C, washed and probed with HRP-conjugated antimouse or anti-rabbit antibody. The results were calculated using the ratio of the density of the protein of interest corrected by the intensity of the protein used as the control (β-actin).
Histological and immunohistochemical staining
Histological and immunohistochemical staining was performed on paraffin-embedded intestinal tissue. Immunohistochemical staining was performed using primary antibody against Ki-67, a nuclear protein associated with cell proliferation (ab15580, 1:200 dilution, Abcam, USA). Then, a streptavidin-biotin-peroxidase kit (ZSGB-BIO, Beijing, China) was used according to the manufacturer's instructions. We measured the proliferation index, which was calculated as the average number of Ki-67-positive enterocytes per 100 enterocytes. At least 2000 enterocytes were assessed for these calculations 18 . Analysis and evaluation of the immunostaining results were independently carried out by two authors. Differences of opinion were reassessed together to reach a consensus.
Cell culture and hypoxia/reoxygenation (H/R) model IEC-6 cells were cultured, and an H/R model was developed as previously described 18 . IEC-6 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured at 37°C in a 5% CO 2 incubator. Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) was used as the cell medium. IEC-6 cells were incubated under hypoxic conditions (5% CO 2 , 1% O 2 , and 94% N 2 ).
Cell proliferation assay
The proliferation of IEC-6 cells was assessed using a Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Inc., Tokyo, Japan) according to the manufacturer's recommendations. A 96-well plate was seeded with IEC-6 cells at a total density of 2 × 10 3 cells/well in 100 μL of DMEM. Cells were allowed to attach and then subjected to H/R. At the indicated time points, the cells were washed with 100 μL of DMEM. The cells were incubated for 2 h in 10 μL of CCK-8 solution, and the absorbance at 450 nm was measured using an ELISA microplate reader (Thermo Scientific, USA).
Luciferase reporter assay
The wild-type rat Nurr1 promoter and mutated Nurr1 promoter, that harbored a mutation in the FoxM1binding motif were synthesized by GenePharma (Shanghai, China). The wild-type and mutated promoters were separately cloned into the pGL3-Basic luciferase vector between its NheI and HindIII sites. When IEC-6 cells reached 60-70% confluence, they were transfected with plasmid (2 µg/well) with Lipofectamine 2000. After transfection for 48 h, the cells were lysed with cell lysis buffer, and dual-luciferase reporter assay reagents (TransGen Biotech, Beijing, China) were then used according to the manufacturer's instruments. Luciferase activities were determined and normalized to Renilla luciferase activity.
Immunofluorescence
In vivo intestinal tissue samples were cryosectioned (10 µm thickness), and IEC-6 cells were postfixed in 4% paraformaldehyde in vitro. Then, tissues and cells were incubated with a primary antibody. The primary antibodies used were as follows: primary anti-Ki-67 (ab15580, 1:200 dilution, Abcam, USA) and primary anti-phosphorylated histone H3 (pH3, a mitosis marker) (1:50, Biogot Technology, Co., Ltd., Nanjing, China). Then, a secondary antibody (Invitrogen Life Technologies, Carlsbad, CA, USA) and DAPI (Beyotime, Shanghai, China) were added. A Leica DM 4000B microscope was used to examine staining. We also measured the proliferation index, which were the same as those of immunohistochemical staining, to analyze and evaluate proliferation of the intestinal mucosa.
Patients
Five ischemic intestinal samples were collected from 5 clinical patients who underwent intestinal ischemia at Dalian Municipal Central Hospital Affiliated of Dalian Medical University. Written informed consent was obtained from the families of patients. A total of 5 patients underwent an operation for acute mesenteric arterial embolism, strangulated intestinal obstruction or incarcerated hernia. We excluded patients with tumors or inflammatory bowel disease, patients who took immunosuppressants and patients who disagreed. The patients consisted of four males and one female with an average age of 66.6 years. The ischemia duration for the intestinal samples was greater than 6 h. After excision of ischemic intestinal tissue, small intestine tissue samples were harvested and stored immediately in liquid nitrogen for western blotting and PCR analysis. Tissues from another part of the intestine were fixed in 10% formalin for histological and immunohistochemical staining. Intestinal samples were obtained with the approval of the Institutional Ethical Committees of Dalian Municipal Central Hospital Affiliated of Dalian Medical University.
Statistical analysis
All values are expressed as the means ± SDs. Analysis of data between two groups was performed using two-tailed Student's t-tests. One-way analysis of variance and Student-Newman-Keuls tests were used to compare means among more than two groups. All data analyses were performed with Statistical Product and Service Solutions and GraphPad Prism 5.0. P < 0.05 indicated significance.
FoxM1 is induced during intestinal mucosa regeneration after I/R
We previously investigated intestinal epithelium regeneration after 1 h of ischemia and 3, 6, 12, or 24 h of reperfusion 18 . To determine the expression of FoxM1 in intestinal mucosa regeneration after I/R injury, we tested the expression of FoxM1 in intestinal tissues that underwent 1 h of ischemia followed by 3, 6, 12, or 24 h of reperfusion. The protein and mRNA expression levels of FoxM1 were tested after reperfusion for different lengths of time (Fig. 1a, b). The mRNA and protein expression levels of FoxM1 were consistent with proliferation of the Fig. 1 FoxM1 is induced during intestinal regeneration after I/R in vivo and in vitro. a, b Representative protein and mRNA levels of FoxM1 in rats subjected to ischemia for 1 h followed by 3, 6, 12, or 24 h of reperfusion (n = 6). c, d Representative protein and mRNA levels of FoxM1 in IEC-6 cells subjected to 6 h of hypoxia followed by 3, 6, 12, or 24 h of reoxygenation (n = 6). The values are presented as the means ± SDs. * P < 0.05 compared to the sham group or control group, ** P < 0.01 compared to the sham group or control group intestinal epithelium after I/R injury. These results indicated that FoxM1 is induced during intestinal mucosa regeneration after I/R injury in vivo.
We also examined FoxM1 expression in IEC-6 cells in vitro. After 6 h of hypoxia and 3, 6, 12, or 24 h of reoxygenation, the expression of FoxM1 was associated with IEC-6 cell proliferation after I/R injury (Fig. 1c, d).
Overall, these findings suggested that FoxM1 is induced during the proliferation of IEC-6 cells after H/R in vitro.
Reduced expression of FoxM1 inhibits intestinal epithelial proliferation after I/R in vivo
As FoxM1 was induced during mucosa regeneration after intestinal I/R injury, we used the FoxM1 inhibitor TST to test the effect of FoxM1 on intestinal epithelial proliferation 20,24 . First, the data suggested that TST inhibited the expression of FoxM1 (Fig. 2a, b). Then, we investigated the effects of TST on intestinal epithelial proliferation and histology after I/R injury. The I/R group showed impaired intestinal villi and reduced proliferation in the intestinal epithelium compared with the sham group (Fig. 2c, d). The group treated with TST following intestinal I/R injury showed worsened injury and an increased Chiu score compared to the I/R group. In addition, proliferation in the intestinal epithelium after I/R injury was further reduced (Fig. 2e, h). Collectively, these findings suggested that the reduced expression of FoxM1 inhibits intestinal epithelial proliferation induced by intestinal I/R.
FoxM1 promotes IEC-6 cell proliferation after H/R injury
As reduced expression of FoxM1 inhibited intestinal epithelial proliferation after I/R, we next tested the effect of FoxM1 knockdown and overexpression on IEC-6 cell proliferation after H/R injury. FoxM1 knockdown using siRNA (siFoxM1), FoxM1 overexpression using plasmid transfection (Fig. 3a, b) and IEC-6 cell proliferation after 12 h and 24 h of reoxygenation following 6 h of hypoxia were tested by the CCK-8 assay and determining the expression of Ki-67. FoxM1 knockdown dramatically decreased the proliferation of IEC-6 cells compared with that in the H/R group, whereas FoxM1 overexpression increased the proliferation of IEC-6 cells compared with that in the H/R group (Fig. 3c, d). These results showed that FoxM1 promotes the proliferation of IEC-6 cells after H/R injury.
Nurr1 is induced by FoxM1 during intestinal regeneration after I/R injury in vivo and in vitro
As a transcriptional factor, Nurr1 promotes intestinal mucosa regeneration after I/R injury 23 . Hou reported that FoxM1 can regulate Nurr1 expression in mouse and human leukemia cells 17 . Thus, we examined whether FoxM1-mediated intestinal epithelial proliferation is mediated by Nurr1. As expected, Nurr1 and FoxM1 expression levels were similarly inhibited in intestinal tissues after I/R injury (Fig. 4a, b). Similar results were demonstrated following the knockdown and overexpression of FoxM1 in IEC-6 cells after H/R injury (Fig. 4c, d). These results indicated that Nurr1 is induced by FoxM1 during intestinal regeneration after I/R injury in vivo and in vitro.
FoxM1 promotes intestinal regeneration after I/R injury in a Nurr1-dependent manner
As Nurr1 expression is closely associated with FoxM1 activation, we hypothesized that FoxM1 promotes intestinal regeneration after I/R injury in a Nurr1dependent manner. We used the Nurr1 activator C-DIM12 to examine the role of Nurr1 in the inhibition of FoxM1. Compared with the I/R group, the group treated with the FoxM1 inhibitor exhibited decreased FoxM1 expression (Fig. 5a, b). Furthermore, the FoxM1 inhibitor obviously inhibited proliferation of the intestinal epithelium after I/R injury. Nurr1 expression in the intestine was activated with the FoxM1 inhibitor TST, and TST no longer exerted any inhibitory effects of proliferation of the intestinal epithelium after I/R injury (Fig. 5c-f). These results showed that FoxM1 promotes intestinal regeneration after I/R injury in a Nurr1-dependent manner.
FoxM1 promotes IEC-6 cell proliferation after H/R injury in a Nurr1-dependent manner
Our results have showed that FoxM1 promotes intestinal regeneration after I/R injury in a Nurr1-dependent manner in vivo. We further detected the effect of Nurr1 overexpression in proliferation of IEC-6 cells with FoxM1 knockdown after H/R injury. The results showed that Nurr1 was overexpressed in IEC-6 cells with FoxM1 knockdown by plasmid transfection (Fig. 6a, b). Overexpression of Nurr1 reversed the inhibition of IEC-6 cell proliferation induced by FoxM1 knockdown (Fig. 6c, d). These results suggest that FoxM1 promotes IEC-6 cell proliferation after H/R injury in a Nurr1-dependent manner.
FoxM1 regulates Nurr1 expression by directly inhibiting Nurr1 expression
Our previous results showed that FoxM1 promotes intestinal mucosa regeneration after I/R injury in a Nurr1dependent manner. In addition, Hou reported that FoxM1 can directly regulate Nurr1 transcription in mice. To test the relationship between FoxM1 and Nurr1 in rats, we searched the proximal region of the Nurr1 promoter for the consensus FoxM1-binding site [T(G/A)TTT(G/A) TT] 25 . Two putative FoxM1-binding sites were identified upstream of the Nurr1 transcription start site (TSS) (Fig. 7a). In addition, a ChIP assay using IEC-6 cells showed that FoxM1 bound to site 1 of the upstream region of Nurr1 TSS directly, but not site 2 (Fig. 7b). Next, we performed dual-luciferase reporter assays to determine whether FoxM1 binds site 1 of the Nurr1 promoter. IEC-6 cells were cultured and transfected with plasmid containing wild-type or site 1 mutant Nurr1 promoter. Luciferase activity of cells expressing the wild-type Nurr1 promoter containing binding site 1 was activated by FoxM1 expression (Fig. 7c). These results revealed that the integrity of binding site 1 upstream of the Nurr1 TSS is required for FoxM1-mediated activation of Nurr1 expression.
Downregulation of FoxM1/Nurr1 in the ischemic intestine of clinical patients
To determine the function of FoxM1 in the proliferation of intestinal epithelial cells of the human ischemic intestine, we investigated proliferation in the human ischemic intestine by HE (Fig. 8a, b) and immunohistochemical (Fig. 8c, d) staining and the expression of FoxM1 and Nurr1 in clinical patients with intestinal ischemia (Fig. 8e, f). We observed the decreased expression of FoxM1 and Nurr1 and the expression of Ki-67 in the human ischemic intestine. These results were similar to those in the rat model of intestinal I/R injury. . e, f Immunofluorescence staining for pH3 in the intestinal tissues of different groups (n = 5) (bar = 100 µm). g, h Immunohistochemical staining for Ki-67 (n = 5) (bar = 100 µm). The values are presented as the means ± SDs. **P < 0.01 compared to the sham group, # P < 0.05 compared to the I/R group, ## P < 0.01 compared to the I/R group
Discussion
Intestinal I/R injury, a serious condition in intensive care units and among vascular surgery patients, is characterized by mucosal barrier damage. Once intestinal mucosal damage occurs, new intestinal crypt epithelial cells proliferate and migrate to the villus and subsequently restore proper mucosal barrier function 26,27 . Previous studies have shown that several genes are activated during the proliferation of intestinal epithelial cells after injury 28,29 . However, the intrinsic mechanism of intestinal epithelial proliferation remains unknown. Our previous study showed that Nurr1 is involved in intestinal regeneration after I/R injury by directly inhibiting p21 expression 23 . Our study is the first to find that (1) FoxM1 can be induced during intestinal mucosa regeneration after I/R injury, (2) FoxM1 promotes intestinal epithelial cell proliferation by promoting Nurr1 gene expression, and (3) Nurr1 is a novel downstream effector of FoxM1 that can be directly activated by FoxM1 and mediate the ability of FoxM1 to promote intestinal regeneration after I/R injury.
Intestinal epithelial cells proliferate to rebuild the proper structure of the epithelium after I/R injury, and nuclear factors are involved in intestinal mucosa regeneration after injury [30][31][32] . FoxM1 is involved in the regeneration of several organs after injury 33,34 . As a nuclear factor, FoxM1 promotes organ regeneration after injury via its transcriptional regulation of downstream genes 35,36 . FoxM1 is highly expressed in intestinal crypts, where it stimulates proliferation by promoting cell cycle entry into S phase and M phase, and may be a key target for regeneration after injury to some organs. In addition, inflammation can promote tissue regeneration after injury through poorly understood mechanisms 37 . As a transcription factor, FoxM1 acts as a critical mediator of the inflammatory response. Zeng et al. revealed that the inhibition of FoxM1 suppresses the production of inflammatory factors including tumor necrosis factor-α and IL-6 in osteoarthritis. The inflammatory mediators nitric oxide, prostaglandin E2 and cyclooxygenase-2 were also repressed by FoxM1 knockdown 38 . It has been reported that inflammatory diseases drive tissue hypoxia 39,40 . FoxM1 is involved in tissue regeneration after injury 33,34 . In our study, FoxM1 was induced in intestinal regeneration after I/R injury. In addition, the 6). The values are presented as the means ± SDs. *P < 0.05 compared to the control group, **P < 0.01 compared to the control group, # P < 0.05 compared to the I/R group, ## P < 0.01 compared to the I/R group expression of FoxM1 was consistent with that of Ki-67 and pH3 in the intestinal mucosa after I/R injury. These results revealed that the expression of FoxM1 is closely associated with intestinal epithelial cell proliferation. Furthermore, inhibition of the expression of FoxM1 resulted in the reduced proliferation of intestinal epithelial cells, and the activation of FoxM1 promoted increased cell proliferation. These data suggested that FoxM1 promotes intestinal mucosa regeneration after I/R injury.
Previous studies have shown that FoxM1 induces the expression of proliferation-associated genes that regulate cell cycle progression and promote cell proliferation in various tissues. Zhao et al. reported that overexpression of FoxM1 induces the expression of cell cycle-associated genes and promotes the proliferation of lung endothelial cells and epithelial cells in a different model of inflammatory lung injury 41,42 . Hou et al. reported that E2F1 target genes and other genes that promote S phase transition were increased when FoxM1 was absent in hematopoietic stem cells, providing molecular evidence of the promotion of S phase progression in hematopoietic stem cells and increased proliferation of hematopoietic stem cells. Moreover, downregulation of FoxM1 led to the loss of Nurr1 17 . Interestingly, our previous studies showed that the loss of Nurr1 markedly impairs intestinal epithelial cell proliferation and is associated with the upregulation of p21 18 . In this study, we found that the ectopic expression of Nurr1 could reverse the inhibition of intestinal epithelial cell proliferation induced by FoxM1 deletion, indicating that these roles are likely mediated by Nurr1. Furthermore, for the first time, our results demonstrated the physiological significance of the overexpression of FoxM1 and resultant epithelial regeneration in the mechanism of intestinal epithelial cell repair following I/R injury. FoxM1 activates Nurr1 expression, and the forced expression of Nurr1 reverses the inhibitory effect of FoxM1 deficiency on the proliferation of intestinal epithelial cells. Taken together, these results suggest that FoxM1 regulates intestinal epithelial cell proliferation through activating Nurr1-mediated pathways.
Next, we examined the intrinsic molecular mechanism by which FoxM1 inhibits the expression of Nurr1. FoxM1 6). The values are presented as the means ± SDs. * P < 0.05 compared to the sham group, ** P < 0.01 compared to the sham group, # P < 0.05 compared to the I/R group, ## P < 0.01 compared to the I/R group can bind to and transactivate target promoters by recognizing and binding to a specific FoxM1-binding site. Hou et al. identified two putative FoxM1-binding sites upstream of the Nurr1 TSS (-5585 to -5576 and -167 to -159) and showed that consensus site 1 (-5585 to -5576) in the promoter of Nurr1 was required for FoxM1mediated activation of Nurr1 expression (-5585 to -5576) in a mouse model 17 . Using bioinformatic analysis, we found a consensus FoxM1-binding site in the Nurr1 promoter that interacted with FoxM1. The results of subsequent ChIP and luciferase assays showed that the binding of FoxM1 to this site activated Nurr1 transcription. This evidence revealed the direct promotion of Nurr1 expression at the transcriptional level by FoxM1.
However, other transcriptional factors that involve additional regulatory mechanisms cannot be excluded.
The role of FoxM1 in intestinal I/R injury is unknown. By analyzing the expression of FoxM1 in clinical patients with superior mesenteric artery embolus, we found that FoxM1 was significantly downregulated in the ischemic mucosa of intestinal I/R patients. In our previous study, we tested Nurr1 expression was downregulated in the intestinal mucosa after ischemic injury 18 . More importantly, the expression of FoxM1 and Nurr1 was reduced along with the expression of Ki-67 in the ischemic intestinal mucosa. Thus, our data implicated the critical role of FoxM1 in promoting human intestinal mucosa epithelial cells and suggested that the upregulation of . e, f Immunofluorescence staining for pH3 in the intestinal tissues of different groups (n = 5) (bar = 50 µm). The values are presented as the means ± SDs. *P < 0.05 compared to the sham group, **P < 0.01 compared to the sham group, # P < 0.05 compared to the I/R group, & P < 0.05 compared to the I/R+TST group Nurr1 expression contributes to the proliferation of epithelial cells of the human intestinal mucosa.
In summary, we show that FoxM1 acts as a critical regulator of intestinal mucosa regeneration after I/R injury by promoting the expression of Nurr1. Moreover, we revealed that FoxM1 promotes intestinal mucosa epithelial cell proliferation by directly binding the promoter of Nurr1 and promoting the transcription of Nurr1. . c Immunofluorescence staining for Ki-67 in IEC-6 cells in different groups (n = 5) (bar = 100 µm). d The CCK-8 assay was used to examine the cell proliferation of IEC-6 cells in different groups (n = 6). The values are presented as the means ± SDs. *P < 0.05 compared to the H/R group, # P < 0.05 compared to the H/R + siFoxM1 group Endogenous binding of FoxM1 to the upstream region of Nurr1 in IEC-6 cells after H/R injury as determined by ChIP assay. IgG was used as a negative control. c The TGTTTATTT core motif was mutated into CGCCCACCC in the mutant Nurr1 promoter luciferase construct. Luciferase reporter assays: IEC-6 cells were transfected with wild-type or mutated Nurr1 promoter luciferase constructs and either FoxM1 or pEX-3 vectors Together, the results of our study reveal the important role of the FoxM1/Nurr1 signaling pathway in promoting intestinal regeneration after I/R injury. Therefore, we believe that the FoxM1/Nurr1 signaling pathway is a novel therapeutic target for intestinal I/R injury and related clinical diseases. | v3-fos-license |
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} | pes2o/s2orc | An Amperometric Immunosensor Based on a Polyelectrolyte/ Gold Magnetic Nanoparticle Supramolecular Assembly—Modified Electrode for the Determination of HIV p24 in Serum
A novel supramolecular amperometric immunosensor for the determination of Human Immunodeficiency Virus antigen p24 (HIV p24) was built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode with HIV p24 antibody (anti-p24) being immobilized on polyelectrolyte/gold nanoparticle multilayer films. The multilayer films were composed of poly(L-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized Fe3O4(core)/gold(shell) nano particles (GMPs).The immunosensor preparation steps were monitored by X-ray fluorescence spectrometry (XRFS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In pH 6.5 PBS, after the immunosensor was incubated with HIV p24 solution at 25 °C for 5 min, the electron transfer access of FeCN is partially inhibited, which leads to a linear decrease of peak current. In addition, the performance of the immunosensor was studied in detail. It offers high-sensitivity for the detection of p24 and has good correlation for the detection of p24 in the range of 0.1 to 100.0 ng/mL with a detection limit of 0.05 ng/mL estimated at a signal-to-noise ratio of 3. The proposed immunosensor was used to analyze p24 in human serum specimens and the results showed the developed immunosensor provides a promising alternative approach for detecting p24 in the early diagnosis of AIDS patients.
Introduction
There are currently an estimated 700,000 people living with HIV in China, including about 75,000 AIDS patients [1]. There is currently no curative therapy available for acquired immunodeficiency syndrome (AIDs), so detecting HIV at an early stage is the best hope of decreasing the mortality rate [2]. Some procedures for the detection of HIV relying on immunoassay recognition of HIV antibody (anti HIV) are available, but in the early stages of HIV infection when anti HIV has not yet appeared in the sufferers' serum (the window period), the patient is highly infectious [3]. If detection during this period is attempted, it is bound to miss patients suffering from HIV. Nevertheless, one of the diagnostic markers of HIV glycoprotein antigen (p24) has been found in human serum in the "window period", although its content is very low (<5 ng/mL) in the early one week after infection [4]. If p24 is found at this time, HIV can be diagnosed, which will effectively shorten the "window period ". At present, enzyme-linked immunoassay assay (ELISA) relying on a change in color detected by fluorescence, luminescence or radioactive emission is the main method to detect trace amounts of p24 [5]. However, these conventional immunoassay methods need enzyme or fluorescent-labeled antibody/antigen, not to mention its lengthy analysis that requires highly skilled personnel, specially equipped laboratories, and expensive chemicals [6]. Therefore, would be highly desirable for disease diagnosis to develop new methods for fast and convenient monitoring of p24.
The electrochemical immunosensor is one of the more attractive analytical tools due to several advantages such as specificity, simplicity, direct detection and time savings of the analyses compared with conventional immunoassay techniques [7,8]. As for the construction of an electrochemical immunosensor, the crucial step is the immobilization of an immune probe such as an antibody or antigen onto the electrode surface. One of the traditional shortcomings in the practical applications of electrochemical immunosensor has been the lack of simple methods to fix probes on the electrode surface and ensure their long-term biological activity. Furthermore, the main disadvantages of many reported amperometric immunosensor are the necessity to separate free from bound label which requires washing and separation steps, which increase the complexity of the assays [9]. Furthermore usually an electrochemical transducer molecular (eT) must be added to the reaction system to accelerate electron transfer between the electrode and immunoassay system [10]. In recent years, nanometal particles have been used as a basic interface to construct antibody protein monolayer [11]. Especially, nonmaterial composed of two-and three-dimensional assemblies of nanoparticles (NPs) with narrow size distribution are becoming increasingly important in analytical and materials chemistry, due to their practical applications in nanoelectronic and optoelectronic devices and biosensors [12]. Using the electrostatic and covalent interactions of bifunctional groups on the substrates, the assembly of individual NPs into three-dimensional structures has become an important and widespread research subject [13][14][15][16][17][18][19][20][21][22][23]. Electrodes modified with gold nanoparticles (AuNPs) are usually fabricated by assembling AuNPs on the electrode surface using organic linker molecules such as thiols and polymers which can provide antibody proteins with a stable environment similar to the native one and thus help retain their bioactivity [14], however the immobilization of AuNPs is usually difficult to achieve. Recently, increasing research has been focused on layer-by-layer (LBL) selfassembly methods using sequential adsorptions of ionized polyelectrolyte and oppositely charged materials in aqueous solutions which has many advantages, such as a being a simple, low temperature deposition, with no limit of thickness and thickness control on the nanoscale [15,[18][19][20]. Fe 3 O 4 (core)/gold(shell) composite nanoparticles (GMPs) has been investigated intensively in recent years because of their remarkable electrical and magnetic properties which can be employed for the development of biosensors via magnetic control with an external magnet [16,17]. Anti-p24 can be easily absorbed on GMPs from the solution mixtures with the aid of Au colloids in the outside shell of GMPs to form GMPs-anti-p24 composite probes. Such probes can then be easily separated from free anti-p24 by adding a magnetic field for the super-paramagnetic Fe 3 O 4 . Moreover in GMPs, there are several AuNPs coated on one Fe 3 O 4 particle, which means that there would be more antibodies immobilized sites than in separate AuNPs.
The focus of this report is the fabrication of a magnetic-controlled electrochemical immunoassay system based on a home-made detection cell for the measurement of p24 with switching and controlling of electrochemical signals by means of an external magnet. The immunosensor electrode was prepared on layer-by-layer self-assembled films which were composed of positively charged poly (L-lysine) (pLys) [19] and negatively charged mercaptosuccinic acid stabilized GMPs(MSA-pLys-GMPs) with an average diameter of 100 nm. We employed GMPs to stabilize HIV p24 antibodies because it has both advantages in magnetic separation and the ability to immobilize antibody proteins with Au colloid. Thus the immunosensor electrode modified with GMPs-anti-p24 would be expected to have a wider detection range than AuNPs modified electrodes. We immobilized MSA stabilized GMPs-anti-p24 in the pLys (pLys/GMPs-anti-p24) membrane layer by layer on the surface of Au|MSA electrode to prepare a p24 biosensor (Au|MSA/{pLys/GMPs-anti-p24} n ), which was then successfully employed in the detection of trace amounts of p24 in human serum.
Characterization of the HIV immunosensor
Electrochemical impedance spectroscopy (EIS) has been proven to be one of the most powerful tools for probing the features of surface-modified electrodes [8,20]. The equivalent circuit in Figure 1 has been shown to account satisfactorily for the impedance spectra used here to analyze the pLys/GMPs multilayer. In Figure 1, the solution resistance is represented as Rs, the double-layer capacitance as C DL and the semicircular part at higher frequencies corresponds to the electron transfer resistance (R et ). Figure 2 shows the electrochemical impedance of the bare Au electrode(a) and the layer by layer immunosensor(b~e) in the presence 5.0 mmol/L K 3 Fe(CN) 6 +K 4 Fe(CN) 6 ([Fe(CN) 6 ] 3-/4-). It can be seen that the bare gold electrode exhibited an almost straight line (Figure 2a), which was characteristic of a diffusion limiting step of the electrochemical process [23] can be seen that the change of electrochemical impedance occurred after MSA was modified on the surface of gold electrode (Figure 2b), indicating that the MSA film obstructed electron-transfer of the electrochemical probe. When the positively charged pLys/GMPs-anti-p24 was absorbed on negatively charged Au|MSA, the interfacial resistance increased highly (Figure 2c). It can also be seen that as the number of layers (pLys/GMPs-anti-p24) reached to 2 and 3, the resistance increased (Figure 2d,e) which implies that the deposited film obstructs the electron transfer of the electrochemical probe. pLys is positively charged in pH 6.5 PBS buffer and can be easily absorbed by the negatively charged GMPs-anti-p24 as a result of the adsorption of MSA in the fabrication process to obtain the desired number of { pLys/GMPs-anti-p24} n multilayer films. The electrode surfaces of (Au|MSA/{pLys/GMPs} 2 and Au|MSA/{pLys/ GMPs-anti-p24 } 2 ) were characterized using SEM ( Figure 3). From Figure 3a, one can see that there are many white bright spot on the electrode surface which may be caused by gold colloids on the surface of GMPs that reflect light [16]. The dimension of most GMPs particles was about 100~120 nm. The GMP film can be found evenly on the Au electrode modified with pLys which has a larger surface compared with the ordinary bare electrode. When the anti-p24 was coated on GMPs with aid of pLys to form pLys/ GMPs-anti-p24 composite and immobilized on a Au electrode by MSA, the electrode surface morphology had changed markedly. There were a lot of island-based spheres and presumably this was the morphology after the antibody was fixed on the electrode surface ( Figure 3b). TEM is an effective method to provide information on particle size and shape. Here, TEM was used to characterize the microstructure of pLys/GMPs (Figure 3c), and pLys/ GMPs-anti-p24 composite ( Figure 3d). The average size of pLys/GMPs was about 120 nm, which is slight larger than bare GMPs. As shown in Figure 3d, the pLys/GMPs-anti-p24 composite shows a uniform structure with an average size about 300 nm, which proved that GMPs is an excellent material to immobilize p24 antibodyies because of the virtues of the nano-Au surface to immobilize antibody proteins.
To further investigate the synthesis of the bionanoparticles, FT-IR was used (data not shown). The absorption bands of anti p24 were observed at 1,655 and 1,534 cm -1 , which were attributed to the protein amide I and amide II infrared absorbance bands. When anti-p24 molecules were modified on the GMP nanoparticle surface, the absorption bands for amide I and amide II were located at 1,674 and 1,560 cm -1 , respectively, which indicated that anti-p24 immobilized on the surface of the GMP nanoparticles retained their native structure. Moreover, the slight change in absorption wave numbers suggested an interaction between the GMPs and anti-p24.
Cyclic voltammetric behaviors of the immunosensor
The cyclic voltammetry (CV) curves of the immunosensor in pH 6.5 PBS are shown in Figure 4. A pair of stable and well-defined redox peak can be observed in Figure 4a. The anodic and cathodic peak potentials were at 215 and 135 mV at 100 mV/s scan rate, respectively, in correspondence with the redox of FeCN. Figure 4b shows the CV for the redox couple [Fe(CN) 6 ] 3-/4of Au|MSA, the redox peaks of [Fe(CN) 6 ] 3-/4diminished and peak-to-peak separation increased when the pLys/GMPs-anti-p24 layers were modified on Au|MSA to form the immunosensor. In pH 6.5 PBS, CVs of Au|MSA/{pLys/GMPs-anti-p24} 2 immunosensor, the experiment results indicated that nano GMPs, pLys, MSA, anti-p24 had no electrical activity, therefore the oxidation and reduction peaks correspond to the reversibility of the oxidation-reduction of FeCN. When the number of multilayers (n) increased, the immunosensor exhibits increased peak currents (see Figures 4c-e). The electrode response increased because the outer layer of pLys is positively charged and can easily absorb [Fe(CN) 6 ] 3-/4and GMPs have good conductivity, which can increase the electron transmission of [Fe(CN) 6 ] 3-/4-. Literature reports state that nano Au [21] and Fe 3 O 4 particles [22] not only strengthen the electron transfer function of Au surfaces, but also supply active points for adsorbing antibodies,and our research finds that when nano Au and Fe 3 O 4 form GMPs composites, these enhanced effects can be superimposed. These CVs are similar to those observed in poly-toluidine blue/nano-Aus electrode [20]. When more layers are added, the peak current increases. A comparison of the current responses of immunosensors with different numbers of layers (n) is shown in Figure 5. For n ≥ 3, no increase of current was obvious. Because the preparation time for one layer is very time-consuming (7 h), two layers were chosen for fabricating the immunosensor. When the immunosensor was incubated in 10 ng/mL p24, the CV current diminished because the immuoreactant of anti-p24 and p24 was formed in the surface of the electrode, which is nonconductive and prevents the electron transformation of FeCN to the electrode. The cyclic voltammetry (CV) curves of the immunosensor at scan rates from 50 to 300 mV/s showed the potential divergence (ΔE p ) between anodic and cathodic current peak increased with the change of scan rates, so we can obtain the average rate of transferred electron is 1.57 ± 0.12 s -1 . The surface coverage on the surface of MSA was estimated to be 9.02 ± 1.13 × 10 -10 mol/ cm -2 , which is same as the sub-monomolecular value [13]. The number of electron transferred was obtained as n = 1.07 ≈ 1, corresponding to the redox of FeCN(Fe(IV)CN→Fe(III)CN). CVs of the immunosensor in pH 4.0-8.0 PBS showed the redox peak potential shifts negatively with the increase of pH. ∂E pa /∂pH = -52 mV/pH; ∂E pc /∂pH = -54 mV/pH. Due to the number of electrons transferred (z) of FeCN was 1, the number of H + involved in the reaction was 1.
Electrochemical behaviors of the immunosensor and determination of p24
Differential pulse voltammetry (DPV) was employed for the determination of p24 due to its higher sensitivity than CVs. It can be seen that the peak current of FeCN by DPV decreased obviously when the immunosensor was incubated in p24 solution with different concentration (0~150 ng/mL) for 30 min (Figure 6a-t). And the ratio of decreased current value (ΔI) and I H (the highest I without p24) was proportional to the p24 concentration in the range from 0.1 to 100.0 ng/mL (Figures 6-insert).
The optimum conditions of the immunoassay
The effect of the concentration of GMPs-anti-p24 used for fabrication of the immunosensor was also investigated when different concentration of anti GMPs-p24 was immersed in the solution containing 100 ng/mL p24 antibody at 25 ºC from 0 to 7 h. The current declined, then essentially reached a stable value, representing the adsorption of antibody on GMPs-anti-p24 have reached saturation. Consequently the soaking time of 100 ng/mL GMPs-anti-p24 was 6 h. The performance of the immunosensor is usually related to the incubation temperature, the time, and the pH value of the detection solution. The dependence of the electrochemical behavior of the immunosensor on the pH of the working solution containing 1.0 mmol/L FeCN was studied. The peak current of the immunosensor increased with an increase of pH values ranged from 3.51 to 7.0, and the peaks became weak when the pH reached 6.6, thus the optimal pH value was 6.5. The amperometric response increased linearly with the FeCN concentration over the range from 0.1 to 1.0 mmol/L, and when the FeCN concentration was higher than 1.0 mmol/L, the ∆I tended to a constant value. Therefore, 1.0 mmol/L FeCN was chosen for the whole experiment. The effect of incubation time on the amperometric responses of the immunosensor was investigated. The value of ∆I 0 increased with increasing incubation time and tended to a maximum value at 30 min, and at this time immune response on the electrode surface was completely finished. The average incubation time by the sensor was 20 min or so which shortened 3fold compared with that of ELISA method which was more than 60 min [3]. This is because detection by the proposed method is based on a one-step immunoassay which reduced the need for addition of secondary antibody in the sandwich ELISA method.
Interference experiments, regeneration of the electrode surface and durability
Interference experiments were performed to assess whether the immunosensor could respond selectively to p24. The immunosensor was used to detect two 10 ng/mL p24 incubation solutions -one solution with an interferent [400 ng/mL α-fetoprotein (AFP); 2 µg/mL BSA, ascrobic acid (AA), uric acid (UA), dopamine (DA), L-lysine] and the other without. The results shown that the peak current responses in the two solutions showed less then 3.2% difference, which means that the immunosensor in this study could respond to p24 specifically.
A small amount of serum protein could be adsorbed on the surface of electrode as the determination times increase, so that the regeneration of immunosensor is very important for their practical use. The prepared immunosensor could be regenerated by simply immersing it in a 0.1 mol/L guanidine hydrochloride-PBS solution for 12 h and removed to wash with water after each determination. A relative standard deviation (R.S.D.) of 2.6% was obtained when the electrode was used for eight consecutive measurements. The stability of the successive assays was studied. After 100 CV measurements in working buffer, an R.S.D. of 2.9% was obtained The long-time stability of the immunosensor was also investigated over a 75 day period. The immunosensor had acceptable storage stability with 94.3% of initial activity remaining after the 75 days storage periods at 4 ºC. This result indicates that the immunosensor had acceptable storage stability and the present immunoassay method is suitable for the determination of p24 in human serum in routine clinical diagnosis.
Determination of p24 in real serum samples
We added 0.1~150 ng/mL p24 standards to normal human serum for a simulation in which the immunosensor was used to determine p24. The results are shown in Figure 7. The added p24 led to a linear decrease of the FeCN response current in p24 concentration ranges from 0.1~100 ng/mL with a detection limit of 0.05 ng/mL (3σ). Three real patient serum samples were determined by the method and the results compared with those obtained from a standard ELISA method, which were very close to standard results. The recovery was in the range from 94% to 103%, indicating that the immunosensor is suitable for the determination of HIV p24 in serum. The results are shown in Table 1.
Apparatus
Cyclic voltammetric measurements (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were carried out with a CHI 600 B electrochemistry workstation (Shanghai CH Instruments Co., China). A three-compartment electrochemical cell contained a platinum wire auxiliary electrode, a saturated calomel reference electrode (SCE) and the modified electrode as working electrode. The size of the GMP film was estimated from scanning electron microscopy (SEM; Hitachi S-3400N spectrometer, Japan). The sizes of GMPs, GMP-anti-p24 and pLys/GMPs-anti-p24 membrane were characterized by transmission electron microscopy (TEM; TECNAI 10, Philips, Holland). The elemental composition of GMPs was characterized by X-ray fluorescence spectrometry (S2 X-RANGER, Bruker Co., Germany)
Preparation of GMPs-anti-p24
The purified GMPs with diameter of 100 nm were dried under vacuum for 24 h at 50 ºC. Afterwards, GMP nanoparticles (10 mg) were treated in toluene solution for 6 h at room temperature with slightly stirring, and then purified by centrifugation and redispersion in pH 6.5 PBS. The purified GMPs were incubated in anti-p24 antibody solution (100 ng/mL) at 4 ºC for 12 h with light stirring. The excess anti-p24 antibody in supernatant was removed using a magnetic field and rinsed with PBS (pH 6.5). Following that, the anti-p24 antibody-modified GMPs were treated with 3.0 mg/mL BSA-PBS at 37 ºC for 1 h to block the unreacted and nonspecific sites of GMPs. Finally, the synthesized bionanoparticles were stored at 4ºC when not in use.
Fabrication of the immunosensor of Au|MSA/{pLys/GMPs-anti-p24}n
This procedure was performed according to the literature [19]. Freshly cleaned gold electrodes were immersed in a toluene solution (10 mol/L) of MSA for 18 h, and then the carboxylate groups of the acid adsorbed on the surface were activated to show negative charge by immersion in basic solution (0.2 mol/L NaOH) for 10 min, followed by washing with Millipore water. The modified electrode (Au|MSA) was then immersed in PBS solution of pLys (1 mg/mL) for 1 h. The positively charged polymer was then adsorbed on the negatively charged MSA membrane of Au|MSA with electrostatic force to form the Au|MSA/pLys electrode. Then Au|MSA/pLys was immersed in GMPs-anti-p24 anions for 6 h. The concentration of the GMPs solution was 0.22 mg/mL. After every layer deposition, the electrodes were washed with Millipore water to remove unbound polymer or GMPs. Alternating layers (Scheme 1) of cationic pLys and anionic GMPs were deposited up to maximum n = 2 layers on the electrode surface. The number of layers deposited is limited by the electrode response. The buildup of multilayers for the spectroscopic measurements was performed on quartz slides coated with 3aminopropyltriethoxysilane (3APTS) proceeding as described in the literature [18,19]. The procedures used for construction of the immunosensor were shown in Figure 1. The experiments were always carried out under an atmosphere of N 2.
Experimental measurements
The analytical procedure for the immunoassay was based on the inhibition of immunocomplex formation by electron transfer between FeCN and the electrode. According to the literature [7] we determine the amperometric response I H of the immunosensor electrolyzed for 120 s at a potential of 200 mV in 5 mL anaerobic pH 6.5 PBS in 0.1 mol/L FeCN, then the immunosensor was incubated with p24 solution at 37 ºC for 30 min, and amperometric response I in the same buffer with FeCN determined under the same conditions. The percentage decrease of the amperometric response of the immunosensor after incubation is given by the following expression: (I H -I)/ I H × 100%. The ratio is proportional to p24 concentration in a certain range. The ELISA tests were performed as follows: 1) each well of antibody coated plate is washed twice with 20-fold diluted wash buffer (350 μL); 2) diluted antigen standard (0, 7.5, 15, 30, 60 ng/mL) or specimen solutions (200 μL) are added to the washed wells, and incubated at 37 ºC for 30 min; 3) the solutions in the wells are removed by aspiration and the wells washed three times with 20-fold diluted wash buffer (350 μL); 4) diluted biotinyl antibody (100 μL) is added to the washed wells and incubated at 37 ºC for 30 min; 5) the solutions in the wells are removed by aspiration and the wells washed three times with 20-fold diluted wash buffer (350 μL); 6) diluted Enzyme-Labeled p24 antibody (100 μL) was added into the washed wells, and incubated at 37 ºC for 30 min; 7) the solutions in the wells are removed by aspiration and the wells washed three times with 20-fold diluted wash buffer (350 μL); 8) substrate solution (100 μL) was added into the washed wells and incubated at room temperature for 30 min; 9) after washing four times in 0.1mol/L (pH6.5) phosphate buffer saluting (PBS), the color was developed with tetramethylbenzidine (TMB) for exactly 5 min and the optical density (OD) was read at 630 nm. The assays conducted during the development phase were performed in triplicate and the assays performed to test the patient samples were performed in duplicate. The average OD 630 of duplicate or triplicate wells was plotted against the dilution factor for each test specimen on the same graphs with positive and negative serum specimens.
Electrochemical impedance tests: The modification procedures were monitored using electrochemical impedance spectroscopy (EIS) in the solution of 0.1 mol/L KCl and 5.0 mmol/L Fe(CN) 6 3/4at room temperature. A three-electrode system was used for recording the impedance spectra. Au|MSA/{pLys/GMPs-anti-p24} n served as the working electrode. A platinum electrode and saturated calomel electrode (SCE) were used as the auxiliary and the reference electrode, respectively.
Conclusions
A stable, sensitive and separation-free amperometric immunosensor for rapid determination of HIV p24 in human serum has been successfully fabricated based on the electrostatic interaction between oppositely charged molecules and nanometer-sized Fe 3 O 4 (core)/Au colloidal(shell) particles (GMPs). Due to the strong electrostatic force of attraction between negatively charged GMPs and positively charged pLys, the p24 antibody coated nano GMPs anions can be easily absorbed on cationic polymers-pLys to form multilayer films on the electrode by a layer-by-layer assembly process. GMPs were used to provide a larger electrode surface area and an easier antibody immobilization; the mediator attached on the electrode surface simplified the experimental design and reduced the consumption of expensive reagents. The modified process was simple, and the results have good reproducibility. This amperometric immunosensor is very suitable for the detection of p24 in the "window period", and has potential application value for the early diagnosis of HIV. The immunosensor shows high sensitivity, low detection limit, and satisfactory storage stability which should be useful for the practical diagnosis of HIV. | v3-fos-license |
2018-12-14T15:02:08.565Z | 2018-12-01T00:00:00.000 | 54901004 | {
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} | pes2o/s2orc | Crystal structure and mutation analysis revealed that DREP2 CIDE forms a filament-like structure with features differing from those of DREP4 CIDE
Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although our recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. In this study, we present the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE.
Scientific RepoRts | (2018) 8:17810 | DOI: 10.1038/s41598-018-36253-y proteins assemble into highly oligomeric forms in solution. Based on the structural study of DREP4 CIDE, which showed a helical filament-like structure even in the crystallographic asymmetric unit, we found that the CIDE domains of both DREP4 and DREP2 form filament-like structures in solution. In this study, the details of the helical assembly of the CIDE domain were determined and the filament-like helical oligomeric complex of DREP2 CIDE was confirmed by structural analysis and mutagenesis studies. Additionally, the differences in the characteristics of filament-like structures between DREP2 and DREP4 were determined.
CIDE domain of DREP2 forms a highly oligomeric state in solution.
Apoptotic DNA fragmentation, which is mainly mediated by the DFF40/DFF45 heterocomplex, is the hallmark of apoptotic cell death and conserved in fly. Four CIDE domain-containing proteins have been identified in fly: DREP1, DREP2, DREP3, and DREP4. Unlike DFF45, which contains only conserved acidic residues, the DREP2, DREP4, DFF40, and FSP27 CIDE domains contain two patches, acidic and basic, in one CIDE domain (Fig. 1A). Although the pattern of charge distribution of each CIDE domain is similar in that they contain two oppositely charged patches, their behaviours differed in solution by forming various oligomeric states based on the size-exclusion chromatography results (Fig. 1B). The DREP4 CIDE domain showed the greatest oligomerization in solution (Fig. 1B). In size-exclusion chromatography, the DREP2 CIDE domain was eluted at approximately 11-14 mL, corresponding to a molecular weight of 100-300 kDa, indicating formation of a highly oligomeric complex (Fig. 1B). We also performed native-PAGE with purified DREP2, DREP4, DFF40, and FSP27 proteins to check the behaviour of the CIDE domain in native state. As shown by the Fig. 1C, we found that DREP2 and DFF40 were stuck on the loading wall and did not migrate well on the native-gel, which might be due to large size of the homo-oligomeric structure. DREP4 migrated a little on the gel. This is also due to the large size of the DREP4 CIDE as indicated by size-exclusion chromatography. Interestingly, however, majority of the FSP27 CIDE, which was eluted as 18 mL on the size-exclusion chromatography, did not migrated well on the gel, although some low molecular weight Scientific RepoRts | (2018) 8:17810 | DOI:10.1038/s41598-018-36253-y particle was shown on the gel. This might be because FSP27 also formed higher oligomeric filament structure during the concentration process. Oligomerization of the CIDE domain of DREP4 was highly dependent on the concentration of salt 30 . To analyse the salt dependency of complex formation of DREP2, we conducted multi-angle light scattering (MALS), which reveals the absolute molecular weight of a particle. The molecular weight of DREP2 CIDE in 50 mM NaCl and 1 M NaCl were calculated by MALS. The theoretical molecular weight of DREP2 CIDE was 10.99 kDa, while the experimental weights determined by MALS were 281.6 kDa (2.04% fitting error) in 50 mM NaCl and 215.3 kDa (0.77% fitting error) in 1 M NaCl, indicating that complex formation of DREP2 CIDE is not dependent on the salt concentration (Fig. 1D,E). Oligomerization of the CIDE domain was expected, as several studies showed that CIDE domain-containing proteins formed highly oligomeric complexes via this domain 31 .
Structure of DREP2 CIDE. The 2.3 Å high resolution crystal structure of the DREP2 CIDE domain was solved using the molecular replacement (MR) method followed by refining to an R work of 22.0% and R free of 25.6%. The structure of the DREP2 CIDE domain exhibited an atypical CIDE domain fold, which is composed of an α/β roll fold with two α helices and five β strands, containing the α2 helix, but not β4 strand (Fig. 1F). This case is similar with the structure of ICAD CIDE and CIDE-A. The two helices comprised of residues 30-41 and 64-68 are indicated as α1 and α2, respectively. The four strands comprised of residues 10-15, 22-27, 50-53, and 74-78 are indicated as β1, β2, β3, and β5, respectively (Fig. 1F). Unlike DREP4 CIDE, in which one turn of the helical complex is formed by 10 molecules of DREP4 CIDE in the crystallographic asymmetric unit, there were four molecules in the asymmetric unit of DREP2 CIDE, which were referred to as chains A-D (Fig. 1G). A model of chains A and D was built from residues 7-84, while that of chains B and C was built from residues 8-84. There were extra residues of Leu, Glu, and His at the C-terminus, which were part of the vector construct. Based on the Ramachandran plot, 95% of the residues were in the most favourable region, whereas 5% were in the allowed regions. The collection and processing of data and refinement statistics are summarized in Table 1. Interestingly, there was no apparent symmetry between the four chains (Fig. 1G). Each monomer was nearly identical, as indicated by superimposition with a root mean square deviation (R.M.S.D.) of approximately 0.7-0.9 Å (Fig. 1H).
Four interfaces are formed in the oligomeric CIDE domain of DREP2. The four CIDE domains in the asymmetric unit are arranged to form a square-shaped complex through non-symmetric interactions between molecules, resulting in mediation of the tetrameric arrangement by four unique interfaces involving different parts of the surface and residues of the DREP2 CIDE domain. The interface between chains C and D (hereafter, interface C) showed the most extensive interactions burying the 500-Å 2 accessible surface area. Inspection of
Data collection
Wild-type R36E interface C revealed that the main interactions are mediated by polar residues involving K9, K13, W15, R22, K23, and N72 from chain D and D56, T58, Q59, E61, E64, and Y65 from chain C. These residues form three salts bridges and four hydrogen bonds, as well as contribute to van der Waals interactions ( Fig. 2A). The second largest interface, which is formed by chains A and B (hereafter, interface A), buries the 495-Å 2 accessible surface area and is mediated by the same residues observed in interface C, except for residues K9 (chain D) and E64 (chain C) (Fig. 2B). Interestingly, these two interfaces (A and C) are highly similar to those observed in the homo-dimeric and hetero-dimeric CIDE domain complex 23,26,28,29 . These two CIDE homo-dimers further assembled into a tetrameric complex by forming two unique interfaces between chains B and C (hereafter, interface B) and between chains A and C (hereafter, interface D). Interface B buries the 368-Å 2 accessible surface area, and the interaction is mediated by residues E31, T29, and G61 from chain B and E31, K38, and A45 from chain C (Fig. 2C). The last interface, D, buries the 134-Å 2 accessible surface area via an interaction mediated by residues E61, D62, and R67 from chain C and residues E36, D39, and K40 from chain A (Fig. 2D). In the tetrameric assembly, chain C forms three unique interfaces with chains A, B, and D involving a surface area of 1002 Å 2 which accounts for 20% of the total surface area of 4951 Å 2 of chain C, while chain D only forms an interface with chain C. Therefore, the chain C molecule is important in the tetrameric arrangement of the DREP2 CIDE domain in the asymmetric unit.
Putative oligomeric structure of CIDE domain of DREP2. The DREP2 CIDE domain exists as a highly oligomeric complex in solution, containing 16-18 molecules in the complex as determined by size-exclusion chromatography and MALS. Because the current tetrameric structure of the DREP2 CIDE domain is smaller than the calculated size, we examined the entire molecular packing of the CIDE domains in the crystal (Fig. 3A). Interestingly, each tetrameric complex of the DREP2 CIDE domain, which was detected in the asymmetric unit, was linked to the next tetrameric unit using interfaces previously detected in the hetero-dimeric and homo-dimeric complexes of CIDE domains ( Fig. 3B) 26,29 . The solution structure of the hetero-dimeric complex between DFF40 CIDE and DFF45 CIDE showed that the interaction is mediated by a basic patch (K9, K18, K32, and R36) on DFF40 CIDE and acidic patch (D66, D71, D72, and D74) on DFF45 CIDE 27 (Fig. 3C). Another structural study of the homo-dimer of the FSP27 CIDE domain revealed that homo-dimerization of the CIDE domain is mediated by basic patch formed by R46, R55, and K56 of one FSP27 CIDE and acidic patch formed by E87, D88, and E93 of the second CIDE (Fig. 3C). This interaction strategy may be the same as that used by neighbouring molecules in the structure of DREP2 CIDE. Superposition of the structures showed that the hetero-dimeric (Fig. 3D). These findings indicate that the orientation of the interface formed by crystallographic packing was similar to that formed by the hetero-dimeric complex and homo-dimeric complex of the CIDE domains. This structural analysis and the newly solved filament-like helical structure of DREP4 CIDE suggest that DREP2 CIDE forms a similar filament-like helical structure in solution. Therefore, we modelled the helical complex of DREP2 by identifying the symmetrical molecules responsible for forming the helical complex in the crystal lattice (Fig. 3A,E). Our previous structural study of the filament-like CIDE domain showed that 10 molecules of DREP4 CIDE form one turn of the helical assembly in the crystallographic asymmetric unit 30 . In the crystal lattice, the helical structure is continuous and stacks along the a-axis of the unit cell with a 56.5 Å rise/turn and ~105 Å diameter 30 . The modelled helical structure of DREP2 based on the crystal contains four molecules in the crystallographic asymmetric unit arranged into a filament assembly with eight subunits per turn, rise of 50.3 Å, and diameter of ∼90 Å (Fig. 3E).
Confirmation of helical complex of DREP2 by mutagenesis.
To determine the importance of the interface formed by crystallographic packing in the oligomeric complex followed by formation of further filament-like structures, we conducted a mutagenesis study. Based on interface analysis of the tetrameric DREP2 and previous analysis of the interface formed by the hetero-dimeric DFF40/DFF45 CIDE complex showing that the main forces generated in the interface were salt bridges formed by K9, K18, K32, and R36 on DFF40 and D66, D71, D72, and D74 on DFF45, we introduced a mutation at K13 on the DREP2 CIDE domain to D (hereafter, K13D), which is aligned with K9 in DFF40, to disrupt the interfaces A and B in the tetrameric DREP2 structure. We also introduced a mutation at R36 in the DREP2 CIDE domain to E (hereafter, R36E), which is critical for the formation of interface D in the tetrameric DREP2 structure (Fig. 2D). While wild-type DREP2 CIDE and R36E mutants formed a highly oligomeric homo-complex in solution that was eluted from the gel-filtration column at approximately 11 mL, the K13D mutant produced a monomeric peak in solution that was eluted from the gel-filtration column at approximately 18 mL, indicating that only K13D (disrupting interfaces A and B) disrupted complex formation (Fig. 4A). The molecular weight of the disrupted K13D mutant was confirmed by MALS. The theoretical molecular weight of the K13D mutant was 10.85 kDa and the experimental molecular weight determined by MALS was 12.14 kDa (8.59% fitting error), with a polydispersity of 1.000 (Fig. 4B). MALS showed that R36E still formed a ~217.4 kDa complex, with a similar size as the wild-type (Fig. 4C). To exclude the possibility that dissociation of the self-complex resulted from structural distortion caused by mutations, we conducted far UV circular dichroism (CD) analysis. As shown in Fig. 4D, the wild-type and two mutants showed similar CD spectrum patterns, with two pronounced minima at 208 and 222 nm and a maximum at 215 nm. These findings indicate that the mutations did not affect the DREP2 CIDE domain structure. Accordingly, the real form of DREP2 CIDE in solution is a helical filament-like complex rather than a tetrameric complex which is packed and detected in the asymmetric unit. The R36E mutant was crystallized under similar conditions as those used to produce the wild-type crystal. The structure of R36E was solved and refined to an R work of 18.0% and R free of 23.4%. Four molecules in the asymmetric unit were the same as those in the wild-type (Fig. 5). Mutated E36 formed a salt bridge with R67 from neighbouring molecule chain C (Fig. 5). This interface was formed as a crystallographic artefact, which was confirmed by mutagenesis analysis.
Characterization of DREP2 CIDE and comparison with DREP4 CIDE. Previous structural analysis
showed that the CIDE domain forms helical oligomers through repetitive head-to-tail polymerization via charged interfaces, which are disrupted by a high concentration of NaCl 30 . As the salt concentration was increased, the DREP4 CIDE particle sizes decreased. Because the particle size of the CIDE domain varied depending on the buffer condition, protein concentration-dependent oligomerization, which is another important factor affecting filament formation, was also examined by size-exclusion chromatography and electron microscopy. The size of the DREP2 CIDE particle did not change as protein concentration was decreased from 30 to 1 mg/mL (Fig. 6A). Electron microscopy of negatively stained samples also showed that both DREP2 and the R36E mutant formed similar-sized rings and filaments in solution and that the filament size was not affected by protein concentration (Fig. 6B). In contrast, filament formation of DREP4 CIDE was dependent on the protein concentration based on the results of size-exclusion chromatography (Fig. 6C) and electron microscopy (Fig. 6D). Smaller particles eluted at 15 mL in the size-exclusion chromatography (Fig. 6C) and smaller filaments were detected by electron microscopy (Fig. 6D) when low concentrations of DREP4 CIDE were used, strongly indicating that the filament formation of DREP4 CIDE depends on the protein concentration, whereas the DREP2 CIDE filament is not sensitive to protein concentration. This indicates that each CIDE domain exhibits unique features in filament formation, although the structure and surface features are identical.
Discussion
Apoptotic DNA fragmentation, which is a hallmark of apoptosis, is mediated by apoptotic nuclease DFF40. DFF40 contains a CIDE domain, which is a protein interaction domain. The CIDE domain-mediated interaction of DFF40 with DFF45, an inhibitor of DFF40 that contains a CIDE domain, is critical for controlling the activity of DFF40. Filament-like assembly of DFF40 via CIDE domain after removing DFF45 upon activation of apoptotic signalling is necessary for DNA fragmentation. In addition to its function in DNA fragmentation, the CIDE domain-containing proteins CIDEA, CIDEB, and FSP27 play important roles in lipid homeostasis. It has been reported that CIDEA, CIDEB, and FSP27 localize at lipid droplet contact sites, promoting lipid transfer and lipid droplet fusion in adipocytes and hepatocytes. Because of this involvement, CIDE-containing proteins were emerged targets for therapeutic intervention of metabolic disorders. The high resolution structure of the DREP2 CIDE showed that the structure exhibited an atypical CIDE structure that contains two helices, α1 and α2, and four strands, β1, β2, β3, and β5, by replacing β4 with a loop. A structural homology search conducted using DALI 32 revealed that the DREP2 CIDE domain is highly similar to ubiquitin-like domains and other CIDE domains (Table 2). DALI server picked six structures, including CIDE-A (PDBid: 2EEL), CAD (PDBid: 1F2R-I), ICAD (PDBid: 1F2R-C), ubiquitin (PDBid: 4NQK-E), SUMO-3 (PDBid: 1U4A-A), and HUB-1 (PDBid: 3PLU-A), as top matches. Pair-wise structural alignments of the DREP2 CIDE domain and structural homologues showed that the length and orientation of the α2 helices in the DREP2 CIDE domain differed slightly from those of in the other structures (Fig. 7A-F). A district β4 was only detected in the structure of CAD (Fig. 7B). No ubiquitin-related proteins contained β4 (Fig. 7D-F). One of the distinct features of ubiquitin-related proteins, including ubiquitin and Sumo, is that β2 is longer than the CIDE domain (Fig. 7A-F). Based on the structural similarity between the CIDE domain and ubiquitin, it would be interesting to functionally characterize and compare these domains. Although the structure of DREP2 CIDE is similar to those of other CIDEs in that it contains a typical α/β roll fold with two α helices and five (or four) β strands, the high-resolution structure revealed a possible biologically important higher oligomerization mechanism of the CIDE domain that functions through several novel dimeric interfaces formed between homo-dimers. Before solving helical filament-like oligomeric structure of DREP4, determining the DREP2 structure, which formed an unusual tetramer without any symmetry in the crystallographic asymmetry unit, was difficult. The current study showed that DREP2 CIDE also formed a filament-like structure with features differing from those of DREP4 CIDE in solution. The filament-like oligomeric structure of DREP2 was confirmed by mutagenesis analysis.
Proteins and accession numbers Z-score RMSD (Å) Identity (%) References
Although the function of CIDE domain-mediated filament-like structure in apoptotic DNA fragmentation was established in the current structural study, CIDE domain-mediated lipid metabolism and learning/memory in brain synapses require further investigation. The characterization and elucidation of the oligomeric forms of the CIDE domain and CIDE domain-containing proteins will provide important information regarding the function of various CIDE domain-containing proteins in apoptosis, lipid metabolism, and particularly lipid droplet (LD) growth and learning/memory in the brain synapses.
Methods
Sequence alignment. Clustal W has been used for analysing the amino acid sequences of CIDEs (http:// www.ebi.ac.kr/Tools/clustalw2/index.html).
Protein expression and purification. The expression and purification methods used in this study have been described in detail elsewhere 21,29,30 . Briefly, the DREP2 CIDE (amino acids 1-84), the DREP4 CIDE (amino acids , and FSP27 (amino acids 38-119) were expressed in Escherichia coli BL21 (DE 3) under overnight induction at 20 °C. The protein contained a carboxyl terminal His-tag and was purified by nickel affinity and size-exclusion chromatography with a S200 gel filtration column 10/30 (GE Healthcare, Little Chalfont, UK) that had been pre-equilibrated with 20 mM Tris-HCl at pH 8.0 and 150 mM or 500 mM NaCl. The protein was then concentrated for further use.
Native PAGE shift assay. Oligomeric forms of each CIDE domains were monitored by native (non-denaturing) PAGE on a PhastSystem (GE Healthcare) with pre-made 8-25% acrylamide gradient gels (GE Healthcare). Separately purified proteins were directly loaded onto the gel. Coomassie Brilliant Blue was used for staining and detection of the shifted bands. The uncropped scan of gel is provided at Supplementary Fig. 1. Crystallization and data collection. The crystallization conditions were initially screened at 20 °C by the hanging drop vapor-diffusion method using various screening kits. Initial crystals were grown on the plates by equilibrating a mixture containing 1 μL of protein solution (7-8 mg/mL protein in 20 mM Tris-HCl at pH 8.0, 500 mM NaCl) and 1 μL of a reservoir solution containing 300 mM magnesium formate dihydrate and 100 mM Bis-Tris pH 6.2 against 0.4 mL of reservoir solution. The best crystal was obtained by further optimization searching over a range of concentrations of protein and precipitant and pH ranges. The diffraction data set was collected at beamline BL-4A of the Pohang Accelerator Laboratory, Republic of Korea. Data processing and scaling were carried out using the HKL2000 package 33 . The mutant crystal was obtained in the similar crystallization condition. 2.3 Å and 3.3 Å data were collected for wildtype and mutant, respectively.
Structure determination and analysis. The initial molecular-replacement (MR) method was carried out using Phaser 34 with the solution structure of the CIDE-N domain of human cell death activator CIDE-A (PDB code 2EEL), which has 37% amino-acid sequence identity, as a search model. The MR solution gave rotation-function and translation function Z-scores of 5.4 and 18.9, respectively. Model building and refinement were performed using COOT 35 and Refmac5 36 , respectively. Model quality was evaluated using PROCHECK 37 . Pymol was used to generate all the cartoon figures 38 . Mutagenesis. Site-directed mutagenesis was conducted using a Quick-change kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's protocols. Mutagenesis was then confirmed by sequencing. Mutant proteins were prepared as described above.
Oligomerization assay by size-exclusion chromatography. For gel filtration analysis to detect oligomerization formation, the target protein was applied to a gel-filtration column (Superdex 200 HR 10/30, GE Healthcare) that had been pre-equilibrated with 20 mM Tris-HCl 8.0 and 500 mM NaCl. The peak fractions were collected and subjected to SDS-PAGE.
Multi-angle light scattering (MALS).
The molar mass of the highly oligomerized CIDE domain of DREP2 was determined by MALS. The target protein was injected onto a Superdex 200 HR 10/30 gel filtration column (GE Healthcare). The chromatography system was coupled to a three-angle light scattering detector (mini-DAWN EOS) and refractive index detector (Optilab DSP) (Wyatt Technology, Santa Barbara, CA, USA). Data were collected every 0.5 s at a flow rate of 0.2 mL/min and analysed using the ASTRA program, which gave the molar mass and mass distribution (polydispersity) of the sample. cuvette at a bandwidth of 1.0 nm, rate of 50 mm/min, and 5-s response time. The protein samples in buffer containing 20 mM Tris-HCl at pH 8.0 and 150 mM NaCl were diluted to 0.1 mg/mL prior to use. Four scans were accumulated and averaged, after which the α-helical content was calculated from the molar ellipticity at 222 nm 39 .
Electron microscopy. Wild-type DREP2 CIDE and R36E mutant samples after affinity chromatography purification were diluted to 0.8 mg/mL to prepare the high concentration sample and 0.1 mg/mL to prepare the low concentration sample. DREP4 CIDE samples were also diluted to 1 mg/mL as the high concentration sample and 0.1 mg/mL as the low concentration sample. For negative staining, 10 μL of each protein sample was placed onto a glow discharged copper grid and stained with 1% uranyl formate at pH 4.5 for 30 s and air-dried. The grids were imaged using a Tecnai G² Spirit BioTWIN Transmission Electron Microscope (FEI Company, Hillsboro, OR, USA) and recorded with an AMT 2k CCD camera (Thermo Fisher Scientific, Waltham, MA, USA).
Protein Data Bank accession codes.
Coordinates and structural factors have been deposited in the Protein Data Bank. | v3-fos-license |
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} | pes2o/s2orc | Activation of the complement system in an osteosarcoma cell line promotes angiogenesis through enhanced production of growth factors
There is increasing evidence that the complement system is activated in various cancer tissues. Besides being involved in innate immunity against pathogens, the complement system also participates in inflammation and the modulation of tumor microenvironment. Recent studies suggest that complement activation promotes tumor progression in various ways. Among some cancer cell lines, we found that human bone osteosarcoma epithelial cells (U2-OS) can activate the alternative pathway of the complement system by pooled normal human serum. Interestingly, U2-OS cells showed less expression of complement regulatory proteins, compared to other cancer cell lines. Furthermore, the activated complement system enhanced the production of growth factors, which promoted angiogenesis of human endothelial cells. Our results demonstrated a direct linkage between the complement system and angiogenesis using the in vitro model, which suggest the complement system and related mechanisms might be potential targets for cancer treatment.
enhanced tube formation activity of human endothelial cells. Additionally, we found that this tube formation is mediated by the upregulation of secreted growth factors including FGF1 and VEGF-A through ERK phosphorylation. In this study, we demonstrate for the first time activation of the complement system in osteosarcoma cells using NHS, and the complement system's impact on angiogenesis.
Activation of complement system in U2-OS osteosarcoma cancer cells. Previously, we established
the cell-based enzyme-linked immunosorbent assay (ELISA) technique to quantify the complement activation in eukaryotic cell surface 13 . With this method, we screened some cell lines for complement activation. Interestingly, the osteosarcoma cell line, U2-OS, activated the complement system through the addition of NHS (Fig. 1A). To confirm if U2-OS cells can activate the complement system, the deposition of MAC and C3b on cells were analyzed by an immunofluorescence assay (IFA) and flow cytometry, respectively (Fig. 1B,C). To exclude the possibility of complement activation by mycoplasma contamination, detection of mycoplasma was tested by PCR and the results indicated no contamination (Fig. 1D). After complement activation, cell viability was analyzed.
Only few apoptosis and cell death was observed both in NHS-and HHS-treated cells (Fig. 1E), suggesting that the activated complement system does not induce cell death in U2-OS cells. These results indicate that U2-OS cells have a potential to be used for complement activation with sublytic level of MAC. To investigate the deposition of MAC on osteosarcoma human tissue, bone and cartilage cancer tissue microarray slide was stained with the anti-MAC antibody (Fig. 1F). In osteosarcoma tissues, obvious staining of MAC was observed on the tumor cells. A non-immune rabbit serum, which was used as a negative control, did not induce any positive signal in the osteosarcoma lesions. Very weak or no MAC staining was observed in the osteoclastoma and chondrosarcoma tissues in the same microarray slide, suggesting MAC does not deposit on all kinds of cancer cells in bone or cartilage. Detection of MAC deposition on the osteosarcoma tissues indicates the complement system is activated in human osteosarcoma.
Alternative pathway of complement system was activated in U2-OS cells.
To investigate which pathway of the complement system was involved in U2-OS cells, we preincubated the cells with NHS containing 10 mM ethylenediaminetetraacetic acid (EDTA), which inhibits the activation of all complement pathways or 10 mM ethyleneglycotetraacetic acid (EGTA) with 2 mM MgCl 2 , which inhibit the antibody-dependent classical pathway. While EDTA-treated cells no longer had C5b-9 deposition, cells treated with EGTA together with MgCl 2 continue to have C5b-9 deposition ( Fig. 2A,B), indicating that C5b-9 deposition on U2-OS cells was mediated by the complement pathway, most likely through the alternative complement pathway. To confirm whether alternative complement pathway was activated, we examined C5b-9 deposition after depleting factor B from NHS. As expected, human serum with depletion of factor B failed to induce C5b-9 deposition; however, addition of factor B to the depleted human serum rescued the C5b-9 deposition (Fig. 2C). Together, the above results indicated that the alternative complement pathway was activated in U2-OS cell.
Negative regulatory proteins of the complement system were suppressed in U2-OS cells.
There are various mechanisms controlling the activation of the complement system. Some complement regulatory proteins including CD46, CD55, and CD59 on cell surfaces inhibit complement activation. We investigated whether the expression of these proteins has correlation with complement activation. Interestingly, the expression of CD46, CD55, and CD59 was suppressed in U2-OS cells compared with human endothelial cells or other cancer cells which did not activate the complement system (Fig. 3A). Endogenous expression of properdin or C3 is also related with complement activation 14 , we examined their expression on U2-OS cells. Because C3b is deposited on the cell surface by complement activation and properdin is incorporated into C3 convertase during activation of the alternative pathways, NHS-treated U2-OS cells were used as a positive control. Except positive control, no evidence for the endogenous expression of C3 or properdin was observed in U2-OS cells (Fig. 3B). Together, activation of the complement system on U2-OS cells would be mediated by suppression of the negative regulatory proteins of the complement system. Recent studies suggested that microRNA expression would be associated with the expression of complement regulatory proteins 15,16 . Therefore, we analyzed the previously reported microRNAs related with CD46 and CD55 in U2-OS cells. Interestingly, most analyzed microRNAs were significantly upregulated in U2-OS cells compared to other cancer cell lines, suggesting microRNAs might be one of the mechanisms for the regulation of these proteins (Fig. S1).
Complement activation of U2-OS cells increased angiogenesis of human endothelial cell through secreted factors.
To investigate if complement activation affects angiogenic activity in endothelial cells through the secretion of specific factors, cancer cells were treated with NHS or HHS for 1 h followed by changing culture media without human serum conditioned media. After 48 h, both conditioned media were collected and applied to human endothelial cells. While the supernatant from NHS-treated U2-OS cells enhanced in vitro tube formation of human endothelial cells, conditioned media from HeLa and T24 cells did not increase angiogenic effects (Fig. 4), suggesting that complement activation of U2-OS is associated with enhanced production of angiogenesis-related secreted factors.
Increased production of VEGF-A and FGF1 in complement activated U2-OS cells.
To determine which secreted factors from NHS-treated U2-OS cells enhance angiogenesis in vitro, various angiogenesis-related growth factors were analyzed by RT-qPCR (Fig. 5A). mRNAs of several growth factors were upregulated in NHS-treated U2-OS cells as compared to HHS-treated cells. Since VEGF-A and FGF1 showed the most significant difference between NHS-and HHS-treated cells, these growth factors were quantified by ELISA using the supernatant from NHS-or HHS-treated U2-OS cells. VEGF-A and FGF1 expression in complement activated cells was significantly higher than in HHS-treated cells (Fig. 5B,C). To confirm the association of the production
The increase of in vitro angiogenesis in U2-OS cells is regulated by the phospho-ERK signaling
pathway. The ERK1/2 and AKT pathways are known to be activated through complement activation by sublytic MAC or C3a 17 . Both signaling pathways are also implicated in angiogenesis and the production of VEGF/ FGF1 [18][19][20] . Therefore, we investigated if the AKT or ERK pathways are activated in NHS-treated U2-OS cells. Interestingly, the phosphorylation of ERK was higher in NHS-treated U2-OS cells compared to HHS-treated U2-OS cells, which may represent a mechanism for the enhanced angiogenesis of complement activated U2-OS cells (Fig. 6A,B). To investigate the association between VEGF-A/FGF1 production and phosphorylation of ERK, U0126 inhibitor was applied to HHS-or NHS-treated U2-OS cells and the supernatant was isolated after 48 h of incubation with varying concentrations of U0126. Western blot analysis of the cell lysates and ELISA for each supernatant showed that the phosphorylation of ERK and VEGF-A/FGF1 decreased in a U0126 dose-dependent manner, respectively (Figs S2 and 6C). Additionally, angiogenic activity of the supernatant from NHS-treated U2-OS cells was analyzed to find if there is a link between U0126 suppressed growth factors and in vitro tube formation activity. The analysis showed that angiogenic activity was significantly decreased by treatment of U0126 ( Fig. 6D-F). To confirm the association of ERK and induction of VEGF-A/FGF1 by the complement system, ERK-1/2 was suppressed by siRNAs (Fig. S3). Knockdown of ERK-1/2 with siRNAs also significantly suppressed the expression of VEGF-A/FGF1 in NHS-treated U2-OS cells (Fig. 6G). Together, our results suggest that the production of angiogenesis-related growth factors (VEGF-A and FGF1) in U2-OS cells through complement activation is mediated by ERK signaling pathway.
Discussion
Although there is increasing research in uncovering the biological responses of the complement system, its association with cancer progression remains controversial. Complement activation is considered damaging to cancer cells through complement-dependent cytotoxicity, which is recruited by anti-tumor monoclonal antibodies 21 . On the other hand, several studies have demonstrated the pro-tumor effects of the complement system in different experimental conditions 3 . Since the complement system has diverse roles depending on the microenvironment, it is not easy to elucidate the exact role of the complement system in vivo. Previous studies have suggested a role for complement proteins in angiogenesis, whereby C3a and C5a stimulate the secretion of VEGF in adjacent retinal pigmented epithelium and choroid cells in a dose-dependent manner 22 . C3 and MAC are deposited in laser-induced choroidal neovascularization, subsequently causing increases in VEGF and other angiogenic growth factors 23 . Using a novel in vitro model with sublytic levels of complement activation in cancer cells, we demonstrated that complement activation in cancer cells can lead to increased production of angiogenic growth factors. Furthermore, phosphorylation of ERK is associated with the complement-mediated production of VEGF-A and FGF1.
Complement activation is associated with cell death through MAC; however, in our previous and current studies, we have not seen any evidence for cell death of human endothelial cells, human mesenchymal stem cells, and various cancer cells through MAC deposition 13,24,25 . A possible explanation for the reason is that our in vitro model of complement activation was not mediated by antigen-antibody complex but through alternative pathway, which would only induce sublytic levels of C5b-9 deposition.
When NHS was applied to cancer cells, complement activation was observed in some cancer cell lines 13 . We found that the expression of complement inhibitory proteins was suppressed in U2-OS cells, which could be a reason for complement activation in eukaryotic cells 24,26,27 . A recent study suggested that microparticles released from cells cause activation of the alternative pathway of the complement system 28 .
Increasing evidence supports the nonimmunological function of the complement system, in which complement activation in the tumor microenvironment enhances tumor growth and increases metastasis 2,3 . MAC can upregulate oncogenic growth factors and cytokines to sustain tumorigenesis and angiogenesis, whose autocrine and paracrine actions promote tumor invasiveness and metastasis 2 . Anaphylatoxins, C3a and C5a, mediated by the activation of M2 macrophages, can also regulate angiogenesis 29 . However, more research is needed to elucidate the exact effects and mechanisms of the sublytic level of complement activation on cancer.
In our present study, we demonstrated the activation of the complement system in an osteosarcoma cell line, U2-OS, through NHS treatment. This activation enhanced angiogenic activity through the secretion of growth factors. Since the relationship between the complement system and tumors remains unclear, a complete theoretical framework has not emerged. This study presents a direct linkage of the complement system and angiogenesis in an in vitro cancer cell model, which could be useful in elucidating the relationship between the complement system and tumors and the underlying mechanisms.
Methods
Cell culture and reagents. U2-OS, HeLa, and T24 cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare, Little Chalfont, UK) supplemented with 10% fetal bovine serum (FBS; Welgene, Seoul, South Korea) and 1% antibiotics (Lonza, Allendale, NJ). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured in endothelial cell growth medium-2 (EGM-2; Lonza) bullet kit. The cells were maintained in a humidified atmosphere of 5% CO 2 at 37 °C. Pooled complement human serum was purchased from Innovative Research, Inc (Novi, MI) and used as normal human serum (NHS) in all experiments. Heat-inactivation was performed using this serum at 56 °C for 30 min and used as heat-inactivated human serum (HHS).
C5b-9 cell-ELISA. The cell-ELISA was performed as described with modifications 13 . Briefly, Cells were seeded at 10,000 cells/well in 96-well culture plates and incubated overnight at 37 °C in 5% CO 2 . Cells were then cultured in media containing 10% pooled human serum (Innovative Research, Novi, MI) for 1 h to activate the complement system. Plates were washed with phosphate-buffered saline (PBS) followed by fixing with 3% paraformaldehyde (PFA) for 15 min. The cells were incubated in blocking buffer (5% skim milk in Tris-buffered saline; TBS) for 1 h at 37 °C. A rabbit polyclonal C5b-9 antibody (Abcam, Cambridge, MA) diluted in blocking buffer (1:4,000) was added to the plate, and incubated with the cells for 2 h. The plate was washed three times with TBS/T for 15 min, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (GE Health Care, Buckinghamshire, UK) was added. After incubation at room temperature for 1 h, the substrate 3,3′,5,5′-tetramethylbenzidine (TMB, KPL, Gaithersburg, MD) was used. The absorbance at 450 nm was measured by a microplate reader (Molecular Devices, Silicon Valley, CA).
Immunofluorescence assay. Cells were seeded onto microscope cover glass. After culturing overnight, culture media containing 10% pooled human serum or heat-inactivated human serum was treated for 1 h. The cells were fixed with 4% PFA in PBS and blocked with 3% bovine serum albumin. Cells were incubated with rabbit polyclonal anti-C5b-9 (1:500, Abcam) overnight at 4 °C, followed by incubating with Alexa Fluor 588-labeled secondary antibodies (Invitrogen, Carlsbad, CA). After washing, nuclei were stained using In vitro endothelial cell tube formation assay. Matrigel (BD biosciences, San Jose, CA) was coated on μ-slide Angiogenesis plates (ibidi, GmbH, Germany). HUVECs (10,000 cells/well) were placed on prepared Matrigel matrix with culture media. The plate was incubated at 37 °C with 5% CO 2 and angiogenic activity was analyzed in three random fields of wells using the WimTube software (Onimagin Technologies SCA, Cordoba, Spain) 30 .
VEGF-A and FGF1 ELISA.
A total of 1 × 10 6 cells was seeded in 75 cm 2 cell culture flasks with DMEM containing 10% FBS. To activate the complement system, the culture media was replaced by DMEM containing 10% pooled normal human serum (NHS) and the cells were incubated for 1 h. After complement activation, cells were washed with PBS twice. Then the media was replaced again with FBS-free DMEM. The conditioned medium was collected after 48 h of incubation. Concentrations of VEGF-A or FGF1 in conditioned medium were measured by using the human VEGF-A or FGF1 ELISA kit (Elabscience, Houston, TX) according to the manufacturer's instructions.
Mycoplasma detecting PCR.
Mycoplasma in culture media were detected by the previously described mycoplasma detecting PCR 31 , using the following primers: Mycoplasma Universal s; 5′-ACACCATGGGAGCTGGTAAT -3′ and Mycoplasma Universal as; 5′-CTTCWTCGACTTYCAGACCCAAGGCA -3′. The 16 S ribosomal DNA region of the strain with which the cell lines were infected was amplified by PCR. The cycling conditions were as follows: initial denaturation at 95 °C for 2 min, 40 cycles consisting of denaturation at 95 °C for 30 sec, annealing at 58 °C for 30 sec, and extension at 72 °C for 60 sec, followed by a final extension at 72 °C for 5 min. The PCR products were analyzed using 1.5% agarose gel. DNA fragments were visualized with a Gel Doc XR system (Bio-Rad) after being staining with ethidium bromide.
Tissue microarray. Human paraffin embedded tissue array slide for bone and cartilage cancer tissue (BO241) was purchased from US Biomax, Inc (Derwood, MD). Tissue sections were deparaffinized in xylene followed by a graded series of alcohol washes prior to staining. Subsequently, all sections were treated with 3% H 2 O 2 for 10 minutes to block endogenous peroxidase activity, and then slides were blocked with normal goat serum (Vector laboratories, Burlingame, CA) for 1 h at room temperature (RT). After blocking, slides were incubated with the anti-C5b-9 antibody (1:100, Abcam Inc., Cambridge, MA) overnight at 4 °C. Then sections incubated with biotinylated goat anti-rabbit secondary antibodies (1:100, Vector Laboratories, Burlingame, CA) for 1 h at RT, followed Scientific RePoRTS | (2018) 8:5415 | DOI:10.1038/s41598-018-23851-z by 30 min incubation with Vectastain avidin-biotin complex reagents (Vectastain-Elite kit, Vector Laboratories, Burlingame, CA). Then color was developed with 3,3′-diaminobenzidine(DAB) and counterstained with Mayer's hematoxylin. Finally, Slides were observed under an Eclipse E400 microscope (Nikon Instruments Inc., USA) and images were captured with a Nikon Digital Sight DS-U2 camera.
Statistical analysis. Each experiment is performed at least three times independently, and the representative result has shown. The number of replicates was indicated in each figure legend as "N". Results are shown as means ± standard deviations. The one-tailed Student's t test was used to assess the significance of difference between groups. Statistical significance at P values of <0.05 and <0.01 is indicated by * and **, respectively. Data availability. The dataset analyzed during the current study are available from the corresponding author on reasonable request. | v3-fos-license |
2019-11-22T00:43:23.833Z | 2019-11-01T00:00:00.000 | 208299144 | {
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} | pes2o/s2orc | Oxidative Stress Increases Endogenous Complement-Dependent Inflammatory and Angiogenic Responses in Retinal Pigment Epithelial Cells Independently of Exogenous Complement Sources
Oxidative stress-induced damage of the retinal pigment epithelium (RPE) and chronic inflammation have been suggested as major contributors to a range of retinal diseases. Here, we examined the effects of oxidative stress on endogenous complement components and proinflammatory and angiogenic responses in RPE cells. ARPE-19 cells exposed for 1–48 h to H2O2 had reduced cell–cell contact and increased markers for epithelial–mesenchymal transition but showed insignificant cell death. Stressed ARPE-19 cells increased the expression of complement receptors CR3 (subunit CD11b) and C5aR1. CD11b was colocalized with cell-derived complement protein C3, which was present in its activated form in ARPE-19 cells. C3, as well as its regulators complement factor H (CFH) and properdin, accumulated in the ARPE-19 cells after oxidative stress independently of external complement sources. This cell-associated complement accumulation was accompanied by increased nlrp3 and foxp3 expression and the subsequently enhanced secretion of proinflammatory and proangiogenic factors. The complement-associated ARPE-19 reaction to oxidative stress, which was independent of exogenous complement sources, was further augmented by the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib. Our results indicate that ARPE-19 cell-derived complement proteins and receptors are involved in ARPE-19 cell homeostasis following oxidative stress and should be considered as targets for treatment development for retinal degeneration.
Introduction
One of the most oxidative environments in the body is the retinal pigment epithelium (RPE) [1], which is in close contact with the photoreceptors and maintains visual function [2]. Low levels of for 4 weeks with apical and basal media exchanges (first-day medium with 10% FCS), remaining time medium with 5% FCS). Before treatment, the FCS concentration was reduced to 0% within 3 days (5%-2.5%-1.25%). ARPE-19 cells were treated with either 0.5 mM H 2 O 2 for 1, 4, 24, and 48 h or with 0.5 mM H 2 O 2 and 0.01 mM olaparib (Biomol, Hamburg, Germany) for 4 h.
Transepithelial Resistance (TER) and Cellular Capacitance
TER and cell layer capacitance were recorded online using the established cellZscope device (nanoAnalytics, Münster, Germany), as previously described [36]. The dielectric properties of empty filter inserts were determined independently and were included in the equivalent circuit used for analysis. Fitting the parameters of the equivalent circuit to the experimental data was achieved via nonlinear least-squares optimization according to the Levenberg-Marquardt algorithm.
Real-Time, Quantitative Polymerase Chain Reaction (RT-qPCR)
Here, mRNA was isolated using a NucleoSpin ® RNA/Protein kit (Macherey-Nagel, Düren, Germany). Purified mRNA was transcribed into cDNA with a QuantiTect ® Reverse Transcription Kit (Qiagen, Hilden, Germany). Transcripts of complement components, receptors, and inflammation-associated markers were analyzed using a Rotor-Gene SYBR ® Green PCR Kit either with QuantiTect Primer Assays (Supplementary Materials, Table S2) or in-house-designed primer pairs (Metabion, Planegg, Germany) (described in the Supplementary Materials, Table S3) in a Rotor Gene Q 2plex cycler (Qiagen, Hilden, Germany). Data were analyzed using the delta delta Ct (ddCt) method. Values were depicted on a linear scale using log-transformed scores to equally visualize increases and decreases in expression levels.
Statistics
Expression statistical analyses for the mRNA were performed using a nonparametric, unpaired Kruskal-Wallis test with Dunn's multiple comparison correction, and protein secretion was statistically analyzed using an ordinary two-way analysis of variance ANOVA with Bonferroni's multiple comparisons test (GraphPad Prism 7; GraphPad Software Inc., San Diego, CA, USA).
Stressed, In Vivo-Like Cultivation of ARPE-19 Cells
We investigated cellular stress response and cell-specific complement expression in a cell line of human RPE cells, the ARPE-19 cell line. Aged ARPE-19 cells of passage 39 were cultivated under in vivo-like, unstressed conditions. This was visualized by staining zonula occludens 1 (ZO-1), an important protein for cell-cell contact, and this showed the formation of stable tight junctions and mainly mononuclear, polarized cell growth on transwell filters ( Figure 1A,D). Stable transepithelial resistance (TER), a measure of the cell layer's barrier function, and the cell layer's capacitance, which is indicative of the expression of membrane protrusions such as microvilli and other membrane folding, were characteristics of the in vivo-like cultivated ARPE-19 cells (Supplementary Materials, Figure S1A,B). H 2 O 2 treatment resulted in cellular stress, which was indicated by reduced cell-cell contact after 4 h ( Figure 1B) and a time-dependent translocation of ZO-1 from the cell membrane to the cytoplasm after 24 h ( Figure 1E). Evidence of induced cellular stress by H 2 O 2 was also observed in the increased mRNA expression of vimentin (vim) and α-smooth muscle actin (α-sma), a typical mesenchymal marker indicating an epithelial-mesenchymal transition (Supplementary Materials, Figure S1C,D) [38][39][40]. However, the majority of the ARPE-19 cells did not undergo apoptosis under these nonlethal oxidative stress conditions, as shown by a low number of TUNEL-positive cells ( Figure 1C
ARPE-19 Cells Increased Complement Receptor Expression under Oxidative Stress
ARPE-19 cells express cellular receptors, sense the cellular environment, and can react to complement activation products. Complement receptor 3 (CR3) is a heterodimer integrin consisting of two noncovalently linked subunits (CD11b and CD18) on leukocytes/microglia, and it is activated by C3 cleavage products (iC3b, C3d, and C3dg). We detected CD11b with low expression in mRNA and low protein levels in ARPE-19 cells (Figure 2A,B). Oxidative stress increased cd11b mRNA expression after 4 h, which was also shown in protein levels with immunostaining ( Figure 2A,C).
The activation of complement protein C5 was detected by complement receptor C5aR1, which was expressed by ARPE-19 cells ( Figure 2D). H2O2 treatment increased c5ar1 expression comparably to cd11b expression ( Figure 2D-F). C5aR1 protein accumulation was observed after 4 h in the cell nuclei ( Figure 2F), which was more distributed in/on the cell after 24 h ( Figure 2G). Increased C5aR1 protein levels were also confirmed in Western blots ( Figure 2H,I).
The transcription levels of complement receptor c3aR were not significantly changed in H2O2treated ARPE-19 cells (Supplementary Materials, Figure S2A).
ARPE-19 Cells Increased Complement Receptor Expression under Oxidative Stress
ARPE-19 cells express cellular receptors, sense the cellular environment, and can react to complement activation products. Complement receptor 3 (CR3) is a heterodimer integrin consisting of two noncovalently linked subunits (CD11b and CD18) on leukocytes/microglia, and it is activated by C3 cleavage products (iC3b, C3d, and C3dg). We detected CD11b with low expression in mRNA and low protein levels in ARPE-19 cells (Figure 2A,B). Oxidative stress increased cd11b mRNA expression after 4 h, which was also shown in protein levels with immunostaining ( Figure 2A,C).
The activation of complement protein C5 was detected by complement receptor C5aR1, which was expressed by ARPE-19 cells ( Figure 2D). H 2 O 2 treatment increased c5ar1 expression comparably to cd11b expression ( Figure 2D-F). C5aR1 protein accumulation was observed after 4 h in the cell nuclei ( Figure 2F), which was more distributed in/on the cell after 24 h ( Figure 2G). Increased C5aR1 protein levels were also confirmed in Western blots ( Figure 2H,I).
The transcription levels of complement receptor c3aR were not significantly changed in H 2 O 2 -treated ARPE-19 cells (Supplementary Materials, Figure S2A).
Complement Proteins Accumulated in ARPE-19 Cells under Oxidative Stress
Complement proteins, which can modulate the activity of complement receptors at the RPE, are locally produced in the retina [26,41] and by ARPE-19 cells (Figure 3; Supplementary Materials, Figure S2B-I). The mRNA expression and cellular protein levels of the stabilizing complement regulator, properdin, were increased after 24 h of H2O2 treatment ( Figure 3A,C-E), but properdin
Complement Proteins Accumulated in ARPE-19 Cells under Oxidative Stress
Complement proteins, which can modulate the activity of complement receptors at the RPE, are locally produced in the retina [26,41] and by ARPE-19 cells ( Figure S4I). However, cfh mRNA expression was not changed under oxidative stress ( Figure 3K). Figure S4I). However, cfh mRNA expression was not changed under oxidative stress ( Figure 3K).
Autocrine Complement Receptor Activation Following Oxidative Stress Was Correlated with the Release of Proinflammatory and Proangiogenic Factors
Intracellular complement proteins and cellular complement receptors have previously been associated with the autocrine regulation of cell differentiation and cell physiology in T-cells as well as lung epithelial cells [20,42]. In line with this, we found a colocalization of CD11b and C3 in ARPE-19 cells ( Figure 4A,B) and activated C3 fragments (C3b α', C3d) in the ARPE-19 cells ( Figure 4C), without adding any exogenous complement source. Intracellular complement proteins and cellular complement receptors have previously been associated with the autocrine regulation of cell differentiation and cell physiology in T-cells as well as lung epithelial cells [20,42]. In line with this, we found a colocalization of CD11b and C3 in ARPE-19 cells (Figure 4A,B) and activated C3 fragments (C3b α', C3d) in the ARPE-19 cells ( Figure 4C), without adding any exogenous complement source. The intracellular cleavage of complement proteins into active fragments (independently of the systemic complement cascade) can be mediated by intracellular proteases such as cathepsin B (CTSB) and cathepsin L (CTSL) [17,18]. Both proteases were expressed by ARPE-19 cells, and they were upregulated following oxidative stress ( Figure 5). The mRNA expression of ctsb and ctsl was increased after 24 h of H2O2 treatment ( Figure 5A,B). We confirmed a higher concentration of CTSL in ARPE-19 cells under stress conditions also on the protein level ( Figure 5C,D). The activation of complement receptor signaling regulates the pro-and anti-inflammatory response in T-and RPE cells [24,43]. This can induce inflammasome activation and regulate the mammalian target of rapamycin (mTOR)-pathway, involving the FOXP3 transcription factor [24,25,44]. After the detection of H2O2-dependent regulation of complement receptors (Figure 2), cellular complement protein accumulation (Figure 3), cell-derived C3 colocalized with CD11b, and C3 activation products C3b and C3d in ARPE-19 cells (Figure 4), we hypothesized that the NLRP3 The intracellular cleavage of complement proteins into active fragments (independently of the systemic complement cascade) can be mediated by intracellular proteases such as cathepsin B (CTSB) and cathepsin L (CTSL) [17,18]. Both proteases were expressed by ARPE-19 cells, and they were upregulated following oxidative stress ( Figure 5). The mRNA expression of ctsb and ctsl was increased after 24 h of H 2 O 2 treatment ( Figure 5A,B). We confirmed a higher concentration of CTSL in ARPE-19 cells under stress conditions also on the protein level ( Figure 5C,D). Intracellular complement proteins and cellular complement receptors have previously been associated with the autocrine regulation of cell differentiation and cell physiology in T-cells as well as lung epithelial cells [20,42]. In line with this, we found a colocalization of CD11b and C3 in ARPE-19 cells (Figure 4A,B) and activated C3 fragments (C3b α', C3d) in the ARPE-19 cells (Figure 4C), without adding any exogenous complement source. The intracellular cleavage of complement proteins into active fragments (independently of the systemic complement cascade) can be mediated by intracellular proteases such as cathepsin B (CTSB) and cathepsin L (CTSL) [17,18]. Both proteases were expressed by ARPE-19 cells, and they were upregulated following oxidative stress ( Figure 5). The mRNA expression of ctsb and ctsl was increased after 24 h of H2O2 treatment ( Figure 5A,B). We confirmed a higher concentration of CTSL in ARPE-19 cells under stress conditions also on the protein level ( Figure 5C,D). The activation of complement receptor signaling regulates the pro-and anti-inflammatory response in T-and RPE cells [24,43]. This can induce inflammasome activation and regulate the mammalian target of rapamycin (mTOR)-pathway, involving the FOXP3 transcription factor [24,25,44]. After the detection of H2O2-dependent regulation of complement receptors (Figure 2), cellular complement protein accumulation (Figure 3), cell-derived C3 colocalized with CD11b, and C3 activation products C3b and C3d in ARPE-19 cells (Figure 4), we hypothesized that the NLRP3 The activation of complement receptor signaling regulates the pro-and anti-inflammatory response in T-and RPE cells [24,43]. This can induce inflammasome activation and regulate the mammalian target of rapamycin (mTOR)-pathway, involving the FOXP3 transcription factor [24,25,44]. After the detection of H 2 O 2 -dependent regulation of complement receptors (Figure 2), cellular complement protein accumulation (Figure 3), cell-derived C3 colocalized with CD11b, and C3 activation products C3b and C3d in ARPE-19 cells (Figure 4), we hypothesized that the NLRP3 inflammasome and FOXP3 also play an autocrine, complement-dependent role in ARPE-19 cells treated with H 2 O 2 . This regulation would be independent of blood-derived complement components and would involve the release of cytokines and growth factors in stressed ARPE-19 cells (Figure 6). inflammasome and FOXP3 also play an autocrine, complement-dependent role in ARPE-19 cells treated with H2O2. This regulation would be independent of blood-derived complement components and would involve the release of cytokines and growth factors in stressed ARPE-19 cells (Figure 6). Indeed, we detected an increased expression of nlrp3 and foxp3 mRNA after 4 h of H2O2 treatment ( Figure 6A,B). Subsequent enhanced expression of il1β mRNA after 24 h and 48 h was associated with increased nlrp3 levels in stressed ARPE-19 cells ( Figure 6C). However, the mRNA expression of il18 was not changed (Supplementary Materials, Figure S2J). Further, we found higher proinflammatory cytokine levels in the H2O2-treated ARPE-19 cell supernatants compared to the untreated controls ( Figure 6D,E). IL-1β was slightly increased after treatment, while IL-6 was significantly elevated in the supernatant of stressed ARPE-19 cells.
Increased foxp3 expression is an attribute of anti-inflammatory regulatory T-cells, which secrete mainly transforming growth factor (TGF)-β and IL-10. We did not detect a change in tgf-β expression (Supplementary Materials, Figure S2K) or IL-10 secretion in H2O2-treated ARPE-19 cells. Therefore, we assumed a proangiogenic function of foxp3 in the cells, as previously reported [22,23]. In line with this, we observed an increase in IL-8 and VEGF-α concentration in the apical supernatant of stressed ARPE-19 cells (Figure 6F,G). This correlation between complement components, foxp3 expression, and proangiogenic reactions in RPE cells needs to be further investigated.
Olaparib Boosted the Proinflammatory Response of ARPE-19 Cells to Oxidative Stimuli
Oxidative stress-induced cellular reactions have been previously ameliorated by an approved Indeed, we detected an increased expression of nlrp3 and foxp3 mRNA after 4 h of H 2 O 2 treatment ( Figure 6A,B). Subsequent enhanced expression of il1β mRNA after 24 h and 48 h was associated with increased nlrp3 levels in stressed ARPE-19 cells ( Figure 6C). However, the mRNA expression of il18 was not changed (Supplementary Materials, Figure S2J). Further, we found higher proinflammatory cytokine levels in the H 2 O 2 -treated ARPE-19 cell supernatants compared to the untreated controls ( Figure 6D,E). IL-1β was slightly increased after treatment, while IL-6 was significantly elevated in the supernatant of stressed ARPE-19 cells.
Increased foxp3 expression is an attribute of anti-inflammatory regulatory T-cells, which secrete mainly transforming growth factor (TGF)-β and IL-10. We did not detect a change in tgf-β expression (Supplementary Materials, Figure S2K) or IL-10 secretion in H 2 O 2-treated ARPE-19 cells. Therefore, we assumed a proangiogenic function of foxp3 in the cells, as previously reported [22,23]. In line with this, we observed an increase in IL-8 and VEGF-α concentration in the apical supernatant of stressed ARPE-19 cells (Figure 6F,G). This correlation between complement components, foxp3 expression, and proangiogenic reactions in RPE cells needs to be further investigated.
Olaparib Boosted the Proinflammatory Response of ARPE-19 Cells to Oxidative Stimuli
Oxidative stress-induced cellular reactions have been previously ameliorated by an approved anticancer drug, olaparib, which is an inhibitor of poly(ADP-ribose) polymerase (PARP) [45][46][47]. We investigated the effects of olaparib on H 2 O 2 -dependent mRNA expression changes of complement receptors, components, and inflammation-related transcripts (Figure 7, Supplementary Materials, Figure S5). Oxidative stress increased the expression of cd11b, c5ar1, and nlrp3 after 4 h of H 2 O 2 treatment. This was further enhanced by olaparib treatment (Figure 7A-C). An increase in properdin and ctsb transcripts was observed after 24 h following oxidative stress alone (Figures 3A and 5A). A combination of H 2 O 2 and olaparib accelerated this reaction, with a significant increase in properdin and ctsb mRNA expression after only 4 h ( Figure 7D,E). The expression of cfd (Supplementary Materials, Figure S2E) was not altered under oxidative stress; however, H 2 O 2 and olaparib together increased cfd transcript levels ( Figure 7F). Olaparib did not interfere with the transcription of foxp3 ( Figure 7G) and other transcripts (c3, c4a, c5, cfb, cfh, cfi, c3ar, and ctsl) (Supplementary Materials, Figure S5) in ARPE-19 cells treated with H 2 O 2 . Figure S5). Oxidative stress increased the expression of cd11b, c5ar1, and nlrp3 after 4 h of H2O2 treatment. This was further enhanced by olaparib treatment (Figure 7A-C). An increase in properdin and ctsb transcripts was observed after 24 h following oxidative stress alone (Figures 3A and 5A). A combination of H2O2 and olaparib accelerated this reaction, with a significant increase in properdin and ctsb mRNA expression after only 4 h ( Figure 7D,E). The expression of cfd (Supplementary Materials, Figure S2E) was not altered under oxidative stress; however, H2O2 and olaparib together increased cfd transcript levels ( Figure 7F). Olaparib did not interfere with the transcription of foxp3 ( Figure 7G) and other transcripts (c3, c4a, c5, cfb, cfh, cfi, c3ar, and ctsl) (Supplementary Materials, Figure S5) in ARPE-19 cells treated with H2O2.
Discussion
The RPE is exposed to high-energy light, and it conducts the phagocytosis of oxidized photoreceptor outer segments. Both of these processes are accompanied by a rapid release of reactive oxygen species [6,48,49]. Reactive oxygen species, including H2O2, are on the one hand major cellular stressors [6,50] and on the other hand cellular survival factors [3,51]. Antioxidants are decreased in light-exposed retinae, allowing the intraocular accumulation of H2O2 [52]. We used H2O2 treatment
Discussion
The RPE is exposed to high-energy light, and it conducts the phagocytosis of oxidized photoreceptor outer segments. Both of these processes are accompanied by a rapid release of reactive oxygen species [6,48,49]. Reactive oxygen species, including H 2 O 2 , are on the one hand major cellular stressors [6,50] and on the other hand cellular survival factors [3,51]. Antioxidants are decreased in light-exposed retinae, allowing the intraocular accumulation of H 2 O 2 [52]. We used H 2 O 2 treatment to mimic physiological oxidative stress in serum-free cultivated ARPE-19 cells to investigate the endogenous complement response in ARPE-19 cells independent of external complement sources [53,54].
Oxidative stress increased the concentration of the complement regulators CFH and properdin and the central complement protein C3 in ARPE-19 cells in a time-dependent manner, without access to any extracellular complement source. Previous studies have mostly reported a reduced expression of cfh mRNA in RPE cells exposed to oxidative stress [28][29][30][31], but these studies did not include further CFH protein analysis. Our reported CFH protein accumulation after H 2 O 2 treatment in polarized, monolayer ARPE-19 cells (using immunohistochemistry) is in contrast to reduced CFH protein detection results in Western blots of non-in vivo-like cultivated ARPE-19 cells following H 2 O 2 treatment [30].
However, it is known that intracellular CFH can enhance the cleavage of endogenously expressed C3 through a cathepsin L (CTSL)-mediated mechanism [55]. The concentrations of lysosomal protease CTSL and the central complement protein C3 were both enhanced under oxidative stress conditions in ARPE-19 cells. Previous studies of RPE cell-derived complement components only focused on c3 mRNA expression, which was not changed under low H 2 O 2 concentrations [56]. We went a step further and showed that C3 was accumulated in the ARPE-19 cells following oxidative stress. This ARPE-19 cell-dependent local accumulation of C3 was also shown for ARPE-19 cells treated with cigarette smoke [27].
If C3 is activated in the blood, CFH serves as a negative regulator and properdin as a positive regulator. We showed for the first time that oxidative stress increased properdin mRNA expression in ARPE-19 cells. This resulted in a higher properdin protein concentration in these cells, which may promote cellular C3 cleavage. In summary, our data described a local production of complement proteins in ARPE-19 cells and an enhanced cellular storage of complement proteins in the cells after H 2 O 2 treatment. This cellular accumulation suggests an autocrine, cellular function of complement proteins in ARPE-19 cells following oxidative stress that is independent of external complement protein sources.
Our studies revealed a colocalization of accumulated, endogenous C3 with complement receptor 3 (CR3, subunit CD11b) in ARPE-19 cells exposed to oxidative stress and an increase in CD11b after 4 h. CR3 expression has been associated with inflammasome activation as a reaction to complement components and/or oxidative stress in white blood cells and RPE cells [57,58]. In agreement with this association, the addition of H 2 O 2 to ARPE-19 cells increased the time-dependent expression of nlrp3 and il-1β mRNA and subsequently enhanced the secretion of proinflammatory cytokines.
Inflammasome activation can be triggered by reactive oxygen species and has been associated with lipid peroxidation end products and phototoxicity in RPE cells [59,60]. The involvement of the complement components in this oxidative stress response of RPE cells has only been described in relation to extracellularly added anaphylatoxins so far [24], but an endogenous complement of RPE cells has not been suggested as a potential priming factor for the inflammasome. We detected activated C3 cleavage products in ARPE-19 cells, and previous studies have shown that activated C3a can be intracellularly generated in RPE cells independent of the systemic canonical complement system [32][33][34][35]. Further, C3 receptors were expressed (CR3, C3aR) and regulated (CR3) under oxidative stress in ARPE-19 cells, indicating a role for endogenous complement components in stressed ARPE-19 cells. Cellular C3 is cleaved by lysosomal CTSL [18,55], and NLRP3 inflammasome activation depends on this CTSL activity [60]. It has been reported that CTSL inhibition reduces inflammasome activity in ARPE-19 cells exposed to oxidative stress [61]. These findings show the interaction between cell-specific complement component cleavage and inflammasome activity. It is already known that endogenous C3-driven complement activation is required for IL-1β and IL-6 secretion, as well as for inflammasome activation in immune cells [62]. Our data suggest that proinflammatory cytokine secretion may also be an autocrine mechanism in ARPE-19 cells associated with complement components and receptors.
In addition to C3, C5 has been identified as a key player in cell homeostasis [24]. The c5aR1 receptor is expressed in RPE cells [63,64] and was increased after oxidative stress treatment. C5 mRNA expression was not changed, and the biologically highly active C5a fragment, a ligand for C5aR1 with a very low biological half-life (approximately 1 min [65,66]), was not detected in our study. The rapid C5a-C5aR interaction might have interfered with our detection schedule. However, C5aR1 stimulation is associated with IL-8 and VEGF-α secretion in ARPE-19 cells [63,64]. The increased secretion of these proangiogenic factors was also observed following the H 2 O 2 stimuli. The signaling pathway is not exactly known so far, but exclusive C5aR1 activation by non-ARPE-19 cell components can be excluded. In regulatory T-cells, the transcription factor FOXP3 promotes the expression of IL-8 [22], and in bladder cancer cells, a knock-down of foxp3 has resulted in the reduced expression of vegf-α [23]. Foxp3 mRNA was expressed in ARPE-19 cells and increased under oxidative stress conditions. Previous studies have shown that extracellular C5a can activate FOXP3 in ARPE-19 cells, which is associated with increased IL-8 secretion [25]. We showed that this could also be due to the endogenous activation of C5aR1 following oxidative stress in RPE cells.
These changes in expression and cellular complement protein accumulation following oxidative stress were time-dependent (Supplementary Materials, Figure S6). The first changes in complement receptor (CD11b, C5aR1) and component (CFH, C3) levels in the ARPE-19 cells occurred after 4 h and were accompanied by changes in nlrp3 and foxp3 mRNA expression. Downstream alterations in properdin expression, intracellular proteases, and an increase in the epithelial-mesenchymal transition marker as well as a loss of tight junctions were described. This indicates that complement receptor signaling may be involved in the early response of ARPE-19 cells to H 2 O 2 treatment.
Oxidative stress-related cell damage in ARPE-19 cells and retinal degeneration in mouse models of RPE degeneration, as well as hereditary retinal degeneration, were successfully ameliorated using olaparib in previous studies [45][46][47]. Olaparib is a clinically developed poly(ADP-ribose) polymerase inhibitor that was developed for cancer treatment by blocking the DNA repair mechanism. ARPE-19 cells were resistant to H 2 O 2 -induced mitochondrial dysfunction and to energy failure when olaparib was added [45]. We asked the following question: can olaparib also normalize complement-associated proinflammatory expression profiles in H 2 O 2 -treated cells? Surprisingly, olaparib accelerated the effect of oxidative stress in ARPE-19 cells and enhanced the expression of complement receptors, complement components, and nlrp3 mRNA. This shows that the endogenous complement-related, proinflammatory response of ARPE-19 cells could be correlated with defective DNA repair mechanisms.
Finally, it needs to be pointed out that this analysis of endogenous complement components and oxidative stress reaction was primarily set up to generate the first data describing an RPE cell-dependent complement reaction. We took advantage of the most commonly used in vitro RPE model (the ARPE-19 cell line), which expresses well-characterized RPE-specific markers [67,68] and provides an unlimited genetic and environmentally identical availability without any risk of contamination with other or undifferentiated cell types. However, it needs to be considered that this model system bears a higher risk of undergoing an epithelial-mesenchymal transition because of long-term cultivation, showing limitations in measuring transepithelial resistance and less expressed RPE-specific markers compared to primary or stem-cell-derived RPE cells [68]. To follow up with this intriguing line of thinking, future studies are needed to verify these cell-associated complement functions in primary or stem-cell-derived RPE cells with different genetic and environmental backgrounds.
Conclusions
Oxidative stress and activation of the complement system cause retinal degeneration, but the mechanism behind this is still a matter of investigation. We showed for the first time that oxidative stress can increase endogenous ARPE-19 cell complement components and receptors and that the process was associated with the release of proinflammatory and proangiogenic factors.
Our data offer a steppingstone for numerous further investigations regarding the function of a cell-associated complement system in primary human RPE. Many questions were raised during this project: How are the complement components activated? Independent of external complement sources, what is (are) the signaling pathway(s) of the complement receptors? How are inflammasome regulation and FOXP3 activity modulated by endogenous complement components in RPE cells? Can endogenous complement factors be targeted to affect cell-associated signaling pathways? These new perspectives will hopefully help to decipher the function of intracellular complement components in retinal health and disease and offer new strategies for the treatment of retinal degeneration.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/8/11/548/s1, Figure S1: ARPE-19 cells showed a stable, confluent monolayer, and H 2 O 2 treatment increased the expression of epithelial-mesenchymal transition markers; Figure S2: H 2 O 2 treatment did not influence the transcription levels of several genes in ARPE-19 cells; Figure S3: The secretome of ARPE-19 cells was influenced by cell passages and H 2 O 2 addition; Figure S4: The stable expression of complement components and related genes after Olaparib and oxidative stress treatment in ARPE-19 cells; Figure Table S1: Primary and secondary antibodies; Table S2: QuantiTec PrimerAssays; Table S3: In-house-designed RT-qPCR primers. Funding: This research was funded by the Velux Foundation, grant #1103, to V.E. and D.P. | v3-fos-license |
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} | pes2o/s2orc | Chemical Probes for the Functionalization of Polyketide Intermediates**
A library of functionalized chemical probes capable of reacting with ketosynthase-bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl-ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology.
Methyl 2-methyl-3-oxo-6-pent-4-ynamidohexanoate (9)
Compound 5 (1.11 g, 4.64 mmol) was dissolved in THF (150 mL) and potassium carbonate (1.66 g, 12.06 mmol) was added. Methyl iodide (0.29 mL, 4.64 mmol) was added dropwise at 0 °C. After one hour the reaction mixture was allowed to warm at room temperature and was left stirring overnight. The reaction was quenched by addition of water (20 mL). THF and water were removed by evaporation/freeze-drying. The crude residue was purified by preparative HPLC (elution gradient starting from 100% H 2 O and linearly increasing to 100% MeOH over 30 minutes) and 9 was yielded as white solid (238 mg, 20%, R t = 10.9 min).
Methyl 4-(4-azidobutanamido)butanoate (60)
Sodium azide (1.06 g, 16.3 mmol) was added to a solution of methyl 4-(4chlorobutanamido)butanoate (59) (1.81 g, 8.15 mmol) in dimethyl sulfoxide (50 mL). The reaction mixture was stirred at 70 °C for 18 h. The reaction mixture was allowed to cool to room temperature and water (100 mL) was added. The aqueous solution was extracted with ethyl acetate (4 x 50 mL). The combined organic phases were washed with water (2 x 100 mL) and brine (100 mL) and dried over MgSO 4 . The MgSO 4 was filtered off and the ethyl acetate was removed in vacuo to yield the crude product. The crude product was purified by column chromatography
Synthesis of probes 7 and 13 2.4.1 1 4-decanamidobutanoic acid (63)
γ-Aminobutyric acid (49, 1.00 g, 9.7 mmol) was suspended in dry methanol (60 mL) in the presence of triethylamine (5.75 mL, 29 mmol). The mixture was cooled to 0 °C and decanoyl chloride (62, 2.1 mL, 9.9 mmol) was added dropwise. The reaction was stirred at 0 °C for 1 h and at room temperature for 16 h. The solvent was evaporated and the crude N-decanoyl-γ-butyric acid (triethylammonium salt) was suspended in water (100 mL), a solution of aqueous 1 M HCl was added to adjust the pH to 2. The mixture was then extracted with CHCl 3 (3 x 100 mL). The organic phase was dried over MgSO 4 , filtered and evaporated to afford 63 as white powder (2.00 g, 80%).
The organic solvent was removed under reduced pressure and the remaining solution was acidified to pH 1 with hydrochloric acid (1M). The obtained suspension was extracted with ethyl acetate (3 x 20 mL). The organic phase was dried over MgSO 4 , filtered and the solvent was removed under reduced pressure. 4-(10-azidodecanamido)butanoic acid was obtained as white solid and used without further purification for the next step (1.24 g).
The reaction was stirred at room temperature for 48 h.
Synthesis of tert-butyl 5-azidopentylcarbamate (44)
Sodium azide (2.03 g, 31.2 mmol) was dissolved in a solvent mixture of water (9 mL) and dichloromethane (18 mL). [7] Trifluoromethansulphonic anhydride (1.05 mL, 6.24 mmol) was added to the mixture at 0 °C and stirred at room temperature for 2 hours. The aqueous layer was separated and extracted with dichloromethane (2 x 5 mL). The pooled organic layers were washed with brine (1 x 5 mL) and then the trifluoromethansulphonic azide solution was added to a solution of tert-butyl (5-aminopentyl)carbamate (0.65 mL, 3.12 mmol), potassium carbonate (0.98 g, 7.10 mmol) and copper sulphate pentahydrate (5 mg) in methanol (22.5 mL) and water (15 mL). The reaction mixture was stirred overnight. The organic layer was washed with water (2 x 5 mL) and the aqueous layers were extracted with dichloromethane (2 x 10 mL). The pooled organic layers were dried over Na 2 SO 4 , filtered and the solvent was removed in vacuo. 44 was yielded as colorless oil (0.69 g, 91%). Data correspond to literature. [8]
Microbiology methods
All media and glassware were sterilized prior to use by autoclave (Astell). Liquid cultures were grown with shaking in Innova 44 incubator/shaker (New Brunswick scientific).
MYM medium: 1.0 g maltose, 1.0 g yeast extract, 2.5 g malt extract in 250 ml of tap water adjusted to pH 7.1.
Construction of mutant strains of Streptomyces lasaliensis
The cultivation of the wild type lasalocid-producing strain S. lasaliensis NRRL3382 was carried out as previously described. 12 The construction of S. lasaliensis ACP12 (S970A) and ΔlasB ACP12 (S970A) mutant strains was previously reported. 12 The construction of S. lasaliensis ACP5 (S3799A) was similarly accomplished by site-directed mutagenesis utilising primer pairs O5_SDMF, oP5R, O5_SDMR and oP5L (Table 1S) by overlap extension PCR and cloning into pYH7 to give plasmid pA5M. 12 The ligated plasmid was transformed into E. coli strain DH10B and positive colonies were tested by restriction mapping and sequencing before a correct clone was transferred to E. coli ET12567/pUZ8002. Conjugation was carried out with the S. lasaliensis wild type as previously described. [12] Candidate mutants were confirmed by sequencing of the mutant region utilising oligonucleotides oA5SF and oA5SR (Table 1S). The mutant was fermented and LC-MS analysis of the ethyl acetate extracts of each culture showed that lasalocid production was completely abolished. and 1b has been previously reported. [12] 3
LC-HRMS and MS/MS analysis of off-loading of 27 and 38, generated from
S. lasaliensis ACP12 (S970A) and probe 7 extracted ion trace for the off-loaded polyketide 28 is shown (B). Its stereochemistry is yet to be separately determined; further work is in progress to establish this, as well as the origin of two peaks for 28 (possibly arising from the presence of isomers/conformers). These species have also been detected as ammonium adduct (data not shown) and were absent in all the control samples (e.g. in absence of the probe, A, and in extracts obtained from the use of different probes). The high resolution masses and isotopic abundances for 28 are shown (C and D). are shown (B and C, respectively, Analysis Method 3). Their stereochemistry is yet to be separately determined; further work is in progress to establish this, as well as the origin of two peaks for 28 and 30
LC-HRMS and MS/MS
(possibly arising from the presence of isomers/conformers). These species have been also detected as ammonium adducts (data not shown) and were absent in all the control samples (e.g. in absence of the probe, A, and in extracts obtained from the use of different probes). The high resolution masses and isotopic abundances for 30 (D and E) are shown. | v3-fos-license |
2018-04-03T01:28:28.605Z | 2013-10-23T00:00:00.000 | 15322850 | {
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} | pes2o/s2orc | Molecular Docking Study of Conformational Polymorph: Building Block of Crystal Chemistry
Two conformational polymorphs of novel 2-[2-(3-cyano-4,6-dimethyl-2-oxo-2H-pyridin-1-yl)-ethoxy]-4,6-dimethyl nicotinonitrile have been developed. The crystal structure of both polymorphs (1a and 1b) seems to be stabilized by weak interactions. A difference was observed in the packing of both polymorphs. Polymorph 1b has a better binding affinity with the cyclooxygenase (COX-2) receptor than the standard (Nimesulide).
Introduction
Polymorphism "Supramolecular isomerism" is pertinent to supramolecular chemistry, and crystal engineering in the same way as isomerization is pertinent to organic molecules. In the simplest way, polymorphism is the ability of molecules to produce more than one crystal structure [1,2], resulted from interplay of kinetic and thermodynamic parameters [3]. The complexities of the organic solid state and especially the differences of intermolecular forces influence crystal packing [4]. Conformational polymorphism will always be a possibility for molecules that have multiple conformational isomers accessible energetically: every different conformation is a different molecular shape and can, in principle, form its own crystalline polymorph (or polymorphs) [5]. Because of the variation in crystallization environment (e.g., temperature, solvent, using of additives, and concentration), the same molecules can pack differently and form different crystal lattices or polymorphs [6][7][8]. As a result, the physical, chemical, and mechanical properties of the crystals can be dramatically affected. Nicotinonitrile-based crystals are highly influenced by and cooperative effects [9]. Self-assemblies of these derivatives are governed by various weak interactions [10][11][12][13][14][15][16][17][18][19][20]. The presence of various weak interactions leads to the development of polymorphism in compounds [21][22][23][24][25]. Polymorphism in organic and inorganic solids can be of crucial importance in the drug design and pharmaceutical industries due to its regulatory action [26][27][28]. Earlier we had studied weak interactions and its polymorphism in 1,3bis(4,6-dimethyl-1H-nicotinonitrile-1-yl)1,3-dioxy propane, which was symmetrical dimer [29]. This current study is focused on the pharmaceutical property of dissymmetrical molecule, 2-[2-(3-cyano-4,6-dimethyl-2-oxo-2H-pyridin-1yl)-ethoxy]-4,6-dimethyl nicotinonitrile, and its polymorphs (1a and 1b).
Instrumentation
. The X-ray diffraction measurements were carried out using a CrysAlis CCD, Oxford diffractometer. The structure was solved by direct methods with the SHELXS-97 program and refined by the full-matrix least squares method on 2 data using the SHELXL-97 program. Molecular graphics: ORTEP; software used to prepare material for publication: MERCURY-3.1. FT-IR spectra were recorded on a VARIAN 3100 FT-IR spectrometer, which was evacuated to avoid water and CO 2 absorptions, at a 2 cm −1 resolution in KBr. The 1H and 13C NMR spectra were recorded on a JEOL AL300 FTNMR spectrometer operating at 300.40 and 75.46 MHz for proton and carbon 13, respectively. The 1H and 13C chemical shifts were measured CDCl 3 solution relative to TMS. The details of the data collection and final refinement parameters are listed in
Weak aromatic interaction (CH⋅ ⋅ ⋅ N, CH⋅ ⋅ ⋅ , and CH⋅ ⋅ ⋅ O interaction) plays an important role in occupying both the polymorphs conformation. A detailed list of their bond lengths and bond angles are summarized in Table 2.
Intermolecular CH⋅ ⋅ ⋅ N (2.573Å, 131.53 ∘ ) and CH⋅ ⋅ ⋅ O (2.425Å, 174.68 ∘ ) interaction stabilized the network of 1a in a symmetrical manner. However, these interactions are absent in polymorph 1b. The major difference observed in the packing diagram of both the polymorphs (Figure 2) is that intermolecular -interaction present between centroid (C13C14C15N3C11C12) and centroid (C4C3C2C1N1C5) of heteroaromatic ring in 1b is crystallized more closely while The Scientific World Journal in the case of 1a aromatic -interaction is completely absent and packing of this polymorph stabilized by CH⋅ ⋅ ⋅ interaction (Figure 3). Both polymorphs are showing roughness in their morphology due to the formation of zigzag sheets via weak interactions. In other words the crystal packing of molecules seems to achieve maximum crystal density. In the packing of the 1st polymorph 1a, due to CH⋅ ⋅ ⋅ O and CH⋅ ⋅ ⋅ (pibond of CN group) interaction, the molecules linked together and formed a cavity. However, in the case of 1b the ⋅ ⋅ ⋅ and CH⋅ ⋅ ⋅ (pi-bond of CN group) interaction joined the molecules together in packing more tightly and a cavity appears. Presence of different sizes of cavities indicates that both the polymorphs can be used as a host for the different guest molecules. Such kinds of molecular systems will be helpful in many biological systems. Details of intermolecular weak interaction are given in Table 3.
Docking Studies of Synthesized Compound. Firstly, all bound waters, ligands, and cofactors were removed from the proteins. The macromolecule was checked for polar hydrogen; torsion bonds of the inhibitors were selected and defined. Gasteiger charges were computed and the AutoDock atom types were defined using AutoDock 4.2, graphical user interface of AutoDock supplied by MGL Tools [30]. The Lamarckian genetic algorithm (LGA), which is considered one of the best docking methods available in AutoDock [31,32], was employed. This algorithm yields superior docking performance compared to simulated annealing or the simple genetic algorithm and the other search algorithms available The ability of compound 1a-b to interact with the COX-2 was further assessed by in silico studies with AutoDock ( Figure 4). Results indicate that polymorph 1b shows a better binding effect with COX-2 compared with standard (Nimesulide) than 1a (Table 4). It seems that 1b can further be used as an anti-inflammatory drug.
Conclusion
Weak interactions play an important role in stabilizing the structure of both polymorphs due to which they have different crystal packing. The presence of different sizes of cavities, The Scientific World Journal 5 formed via such weak interactions, plays a crucial role in their biological activity. Polymorph 1b has more binding affinity with COX-2 than polymorph 1a. Polymorph 1b can further be explored for anti-inflammatory activity. | v3-fos-license |
2020-11-25T14:06:54.511Z | 2020-11-23T00:00:00.000 | 227159022 | {
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} | pes2o/s2orc | The extracellular lactate-to-pyruvate ratio modulates the sensitivity to oxidative stress-induced apoptosis via the cytosolic NADH/NAD+ redox state
The advantages of the Warburg effect on tumor growth and progression are well recognized. However, the relevance of the Warburg effect for the inherent resistance to apoptosis of cancer cells has received much less attention. Here, we show here that the Warburg effect modulates the extracellular lactate-to-pyruvate ratio, which profoundly regulates the sensitivity towards apoptosis induced by oxidative stress in several cell lines. To induce oxidative stress, we used the rapid apoptosis inducer Raptinal. We observed that medium conditioned by HepG2 cells has a high lactate-to-pyruvate ratio and confers resistance to Raptinal-induced apoptosis. In addition, imposing a high extracellular lactate-to-pyruvate ratio in media reduces the cytosolic NADH/NAD+ redox state and protects against Raptinal-induced apoptosis. Conversely, a low extracellular lactate-to-pyruvate ratio oxidizes the cytosolic NADH/NAD+ redox state and sensitizes HepG2 cells to oxidative stress-induced apoptosis. Mechanistically, a high extracellular lactate-to-pyruvate ratio decreases the activation of JNK and Bax under oxidative stress, thereby inhibiting the intrinsic apoptotic pathway. Our observations demonstrate that the Warburg effect of cancer cells generates an anti-apoptotic extracellular environment by elevating the extracellular lactate-to-pyruvate ratio which desensitizes cancer cells towards apoptotic insults. Consequently, our study suggests that the Warburg effect can be targeted to reverse the lactate-to-pyruvate ratios in the tumor microenvironment and thereby re-sensitize cancer cells to oxidative stress-inducing therapies.
Introduction
The enhancement of aerobic glycolysis (Warburg effect), first described by Otto Warburg in 1924 [1], supports tumorigenesis in numerous ways. First believed to be caused by dysfunctional mitochondria, it is now established to be the consequence of oncogene activation or tumor suppressor inactivation [2,3]. While aerobic glycolysis maintains sufficient glycolytic intermediates for multiple biomolecule synthetic pathways [4], most of the carbon skeleton from glucose is secreted in the form of lactate and does not contribute to biomolecule synthesis [5]. In addition, increased flux through the pentose phosphate pathway provides the bulk of NADPH needed for lipid synthesis and neutralization of reactive oxygen species (ROS) [6]. The latter is important because many cancers display elevated levels of ROS and are in a state of constitutive oxidative stress that would result in apoptosis if not counterbalanced by NADPH-dependent defenses [7,8]. On the other hand, the elevated ROS levels continuously drives DNA mutagenesis for tumor progression and stabilizes HIF-1α for high glycolytic capacity and angiogenesis [9][10][11][12]. Moreover, constitutive ROS-mediated signaling via the MAPK/ERK1/2 or PI3K/Akt/mTOR pathways promotes cell proliferation and survival [13,14].
Resistance to apoptosis is one of the hallmarks of cancer [15]. Despite the usage of cytotoxic agents to specifically induce cancer apoptosis, therapeutic efficacy is counteracted by drug resistance, genomic instability and tumor heterogeneity [16]. Hence, combining cytotoxic agents with therapies targeting other essential biological processes, such as tumor growth, are preferred [17]. The recently characterized compound Raptinal induces apoptosis via oxidative stress in a wide variety of (cancer) cell lines in an unusually rapid fashion and attenuates tumor growth in in vivo tumor xenografts [18]. On the other hand, targeting of tumor metabolism by compounds such as dichloroacetate (DCA) and 3-bromopyruvate are attractive because of the various anti-tumor properties that accompany the inhibition or reversal of the Warburg effect [19,20].
In the present study, we show that the Warburg effect generates an extracellular environment with an elevated lactateto-pyruvate ratio, which protects against oxidative stressinduced apoptosis by reducing the cytosolic NADH/NAD + redox state. Mechanistically, a reduced cytosolic NADH/ NAD + redox state inhibits oxidative stress-induced apoptosis by suppressing mitochondrial outer membrane permeabilization (MOMP) mediated by JNK-Bax signaling. Conversely, oxidizing the cytosolic NADH/NAD + redox state sensitizes cancer cells to oxidative stress-induced apoptosis by enhancing JNK-Bax signaling.
Cell lines and culture conditions
The hepatoma cell line HepG2 and colorectal carcinoma cell line HCT116 were maintained in DMEM (Invitrogen, Landsmeer) with 2 g/L glucose, 1.8 g/L NaHCO 3 , 20 mM HEPES-NaOH (pH 7.4), 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin under 5% CO 2 . The human immortalized cholangiocyte cell line H69 was cultured as described previously [21]. The leukemia cell lines U937 and HL-60 were maintained in IMDM (Invitrogen, Landsmeer) with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin under 5% CO 2 . For the generation of conditioned medium, 6.5 × 10 5 cells were plated in 2 mL culture medium on 6-wells plates. Medium was subsequently harvested at 24, 48 and 72 h after seeding, filtered through 0.22 µm pore filter and stored at -20 °C until further use. All media used for experiments were pre-equilibrated overnight at 37 °C in the 5% CO 2 incubator.
Cell treatment for induction of apoptosis
Cells were cultured in 96-well plates until confluence and refreshed the day before the experiment. On the day of an experiment, cells were refreshed with 100 µL experimental medium or (un)conditioned culture medium. For suspension cells, 5 × 10 5 cells per well were directly seeded in a 96-wells plate in experimental medium. The experimental medium consisted of base DMEM, supplemented with 5.5 mM glucose, 1.5 g/L NaHCO 3 , 20 mM HEPES-NaOH (pH 7.4), 10 µg/mL phenol red and 1% FBS. Cells were pre-incubated for 1 h in media containing different lactate-to-pyruvate ratios in the presence or absence of various pharmacological inhibitors. Following pre-incubation, cells were treated with vehicle (0.1% DMSO), 10 µM Raptinal, 200-750 µM Na-chenodeoxycholate or 40 ng/mL TRAIL in the respective medium for the indicated time points.
Enzymatic determination of glucose, lactate and pyruvate
Spent medium was deproteinized by collecting 50 µL of spent medium in 75 µL of 5% metaphosphoric acid. Samples were incubated for at least 1 h on ice and subsequently spun down at 10,000×g for 10 min. Supernatant was collected and stored at − 20 °C until analysis. Medium glucose, lactate and pyruvate were enzymatically determined as described in [22] using the CLARIOstar microplate reader.
Clamping the cytosolic NADH/NAD + redox state by lactate-to-pyruvate clamping solution
Clamping of the cytosolic NADH/NAD + redox state, or [NADH]/[NAD + ] ratio, was achieved by incubating cells in medium containing a fixed total amount (2.5 mM) of l-lactate plus pyruvate in different lactate-to-pyruvate ratios. Stock solutions of 100 mM Na-l-lactate and 100 mM Napyruvate were prepared in 50 mM NaCl.
Measurement of cytosolic NADH/NAD + redox state with Peredox-mCherry biosensor
HepG2 cells stably transduced with the inducible Peredox-mCherry construct or an empty vector (pCW-MCS-BSD) as described in [23]. Cells were induced with 800 ng/mL doxycycline for 48 h prior to the experiment. Cells were incubated in HBSS modified for ambient air medium containing a fixed total amount (2.5 mM) of l-lactate plus pyruvate but in different lactate-to-pyruvate ratios (1,5,10,20,50 and 100) in the presence of glucose. Fluorescence of Peredox (F 1 ) and mCherry (F 2 ) were monitored as described in [23]. Fluorescence from HepG2 cells transduced with the empty vector was used to correct for background fluorescence (denoted as F 1 ′ and F 2 ′). The background-corrected fluorescence ratio R was defined as (F 1 −F 1 ′)/(F 2 −F 2 ′). R was then linearly transformed by normalizing the minimal fluorescence ratio R min (2.5 mM pyruvate in the absence of glucose) and the maximal fluorescence ratio R max (2.5 mM l-lactate in the absence of glucose) to 100% and 200%, respectively.
Superoxide anion radicals measurement by DHE oxidation
HepG2 cells were pre-incubated in phenol-red free DMEM containing 5.5 mM glucose and a fixed total amount (2.5 mM) of l-lactate plus pyruvate in different lactateto-pyruvate ratios (1, 250) for 45 min at 37 °C. Subsequently, 5 µM DHE was added and after a 15-min incubation, 10 µM Raptinal (or vehicle, 0.1% DMSO) was added. Oxidation of DHE was monitored fluorometrically at λ Ex / λ Em = 520 ± 10/590 ± 20 in the CLARIOstar microplate reader under 5% CO 2 atmosphere. The slope of the changes in fluorescence between 30 and 60 min after Raptinal addition was used to calculate the oxidation rate of DHE.
Cytochrome c release assay
Cytochrome c release assay was performed as previously described in detail [21]. Briefly, following incubation of the cells the plasma membranes were selectively permeabilized by incubation with 75 µg/mL digitonin dissolved in intracellular buffer at 4 °C for 20 min. Solubilized cytosolic proteins were harvested and the remaining fraction, containing mitochondria, were lysed in RIPA buffer. Equal volumes were loaded from each fraction for immunoblotting.
Immunoblotting
Immunoblotting was performed as previously described in detail [21]. Transferred proteins on PVDF membranes were blocked in 5% milk overnight and incubated with primary antibody solutions. Following incubation with primary antibody, membranes were incubated with goat-anti-mouse or goat-anti-rabbit antibodies conjugated to horse-radish peroxidase. Finally, membranes were developed with a homemade enhanced chemiluminescence solution (100 mM Tris-HCl pH 8.5, 1.25 mM luminol, 0.2 mM p-coumarin and freshly added 3 mM H 2 O 2 ) and chemiluminescence was recorded using the LAS4000 machine (GE healthcare, Eindhoven).
Statistical analysis
All data are expressed as mean ± SD. Statistical significance was tested using the one or two-way ANOVA, followed by Sidak or Tukey multiple comparison tests using Graphpad Prism 8.0 software. p-values ≤ 0.05 were considered as significantly different.
HepG2-conditioned medium protects against Raptinal-induced apoptosis
The human hepatoma cell line HepG2 is an established liver cancer cell model for apoptosis and exhibits a strong Warburg effect. The latter property is characteristic of fastgrowing cancers and is associated with increased resistance to cytotoxic agents [19]. Therefore, we used HepG2 to study the influence of the cellular metabolic state on oxidative stress-induced apoptosis.
Raptinal is a rapid inducer of the intrinsic apoptotic pathway in a wide range of cancer cells [18]. Incubating HepG2 Data is normalized to 0 µM Raptinal (vehicle, 0.1% DMSO) condition. Shown are the mean ± SD of a representative experiment (N = 2). One-way ANOVA with multiple comparison (Tukey) with ***p < 0.001, ns; not significant. b HepG2 cells were pre-incubated with the conditioned media for 1 h and thereafter treated with vehicle (0.1% DMSO) or 10 µM Raptinal for 1.5 h. Caspase 3/7 activity was then measured. Data is normalized to vehicle in unconditioned medium and shown are the mean ± SD of a representative experiment (N = 2). Two-way ANOVA with multiple comparison (Tukey) with ****p < 0.001, *p < 0.05, ns; not significant. c The medium glucose, lactate, and pyruvate of cultured HepG2 cells were measured at 24, 48 and 72 h after seeding. Shown are the mean ± SD of a representative experiment (N = 2). d The lactate-to-pyruvate (L/P) ratio in the medium conditioned by HepG2 cells over time. The ratios are derived from (c) cells with Raptinal in fresh medium dose-dependently induced apoptosis as measured by caspase 3/7 activity (Fig. 1a). In contrast, incubation with Raptinal in conditioned medium from a parallel HepG2 culture dramatically suppressed the apoptotic response (Fig. 1b). These data strongly suggest that HepG2 cells can condition their extracellular environment to protect against oxidative stress-induced apoptosis.
We hypothesized that the conditioned medium contains or lacks factors that modulate the sensitivity to Raptinal-induced apoptosis. One prominent difference between conditioned and unconditioned medium is the accumulation of glycolytic endproducts as a result of the Warburg metabolic phenotype of HepG2 cells. To confirm this phenotype in our model system, we measured the concentrations of glucose, pyruvate and lactate in the medium in HepG2 cultures over time (Fig. 1c). During culture, HepG2 cells converted the majority of glucose in the culture medium into lactate but reduced medium pyruvate, thereby elevating the medium lactate-to-pyruvate ratio over time (Fig. 1d). These findings demonstrated a correlation between the extracellular lactate-to-pyruvate ratio and the sensitivity to apoptosis.
The extracellular lactate-to-pyruvate ratio controls the cytosolic NADH/NAD + redox state
The extracellular lactate-to-pyruvate ratio is an established indicator of the cytosolic NADH/NAD + redox state [24]. Via the monocarboxylate transporters (MCTs), extracellular lactate and pyruvate are in near equilibrium with the cytosolic pool of lactate and pyruvate. In turn, the cytosolic pool of lactate and pyruvate is in near equilibrium with the cytosolic free NADH and NAD + via the reversible catalytic action of lactate dehydrogenase (LDH) (Fig. 2a) [24,25]. Due to these metabolic interactions it is expected that the cytosolic free [NADH]/[NAD + ] ratio can be clamped by manipulating the extracellular lactate-to-pyruvate ratio.
To verify that the extracellular lactate-to-pyruvate ratio directly alters the cytosolic free [NADH]/[NAD + ] ratio, we expressed the [NADH]/[NAD + ] redox biosensor Peredox in the cytosol in HepG2 cells. Incubations of HepG2 cells in media with a fixed total concentration (2.5 mM) of lactate and pyruvate, but at varying ratios ranging from low (oxidized cytosolic NADH/NAD + redox state) to high (reduced cytosolic NADH/NAD + redox state), resulted in a stable ratio-dependent change of Peredox fluorescence intensity over time (Figs. 2b; S1). These data show that the extracellular lactate-to-pyruvate ratio indeed controls the cytosolic free [NADH]/[NAD + ] ratio. Assuming a cytosolic pH of 7.0 in HepG2 cells and the equilibrium constant of LDH, the cytosolic [NADH]/[NAD + ] ratio can even be estimated from the extracellular lactate-topyruvate ratio (Fig. 2b).
The extracellular lactate-to-pyruvate ratio modulates the sensitivity to Raptinal-induced apoptosis by regulating the cytosolic NADH/NAD + redox state
Since HepG2-conditioned medium had an elevated medium lactate-to-pyruvate ratio and suppressed Raptinal-induced apoptosis (Fig. 1b, d), we next investigated whether the increased lactate-to-pyruvate ratio by itself confers protection against Raptinal-induced apoptosis. To this end, we incubated HepG2 cells with Raptinal in fresh culture media containing different lactate-to-pyruvate ratios ranging from 1 (oxidized cytosolic NADH/NAD + redox state) to 200 (reduced cytosolic NADH/NAD + redox state) in the presence of glucose. Indeed, elevating the lactate-to-pyruvate ratio in fresh medium stepwise desensitized HepG2 cells to Raptinal-induced apoptosis (Fig. 3a). Since the cytosolic free [NADH]/[NAD + ] ratio is directly affected by the medium lactate-to-pyruvate ratio (Fig. 2b), these results strongly suggest that a high cytosolic free [NADH]/[NAD + ] ratio, i.e. a more reduced cytosolic NADH/NAD + redox state, confers resistance to Raptinal-induced apoptosis.
It has been reported that Raptinal induces apoptosis by imposing oxidative stress [18]. Therefore, we investigated the effect of anti-oxidant N-acetyl-l-cysteine (NAC) on Raptinal-induced apoptosis. Indeed, NAC effectively suppressed Raptinal-induced apoptosis (Fig. 3b), confirming that Raptinal induces apoptosis via oxidative stress. To exclude that the protective effect of the cytosolic NADH/ NAD + redox state is exclusive for Raptinal we used the hydrophobic bile salt sodium chenodeoxycholate (Na-CDC) as another inducer for oxidative stress-induced apoptosis [26]. We therefore treated H69 cholangiocytes, previously shown to be sensitive to bile salt-induced apoptosis [21], and HepG2 cells with Na-CDC in medium containing different lactate-to-pyruvate ratios. Elevation of the extracellular lactate-to-pyruvate ratio proved to be protective against bile salt-induced apoptosis both in H69 cholangiocytes (Fig. 3c) and in HepG2 cells (Fig. 3d). These data strongly imply that the cytosolic NADH/NAD + redox state not only modulates the sensitivity to Raptinal-induced apoptosis, but affects oxidative stress-induced apoptosis in general.
We next examined whether Raptinal-induced apoptosis is affected by manipulations of the cytosolic [NADH]/ [NAD + ] redox other than by clamping the extracellular lactate-to-pyruvate ratio. Cytosolic NADH is primarily generated by glycolysis and oxidized in the cytosol to NAD + by LDH, or shuttled into mitochondria via malate-aspartate or glycerol-3-phosphate shuttles. Since mitochondria are the main consumers of NADH, sustained manipulation of mitochondrial function (in)directly influences the cytosolic 1 3 free [NADH]/[NAD + ] ratio. Indeed, it has been reported that lactate secretion and the cellular [NADH]/[NAD + ] redox ratio are elevated upon inhibition of complex I of the mitochondrial respiratory chain [27]. In contrast, promotion of mitochondrial pyruvate oxidation by dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, has been shown to decrease the cellular [NADH]/[NAD + ] redox ratio [28]. We used these conditions and tested if rotenone can protect against apoptosis by increasing the cytosolic free [NADH]/[NAD + ] ratio. We indeed observed a protection against apoptosis by rotenone (Fig. 3e). Conversely, we tested if decreasing the cytosolic [NADH]/[NAD + ] ratio by co-incubation with DCA re-sensitized cells to apoptosis. Indeed, DCA re-sensitized HepG2 cells to Raptinal-induced apoptosis even in the presence of a high lactate-to-pyruvate ratio (Fig. 3f). Together, these results support the notion that the cytosolic NADH/NAD + redox state regulates oxidative stress-induced apoptosis. The extracellular lactate-to-pyruvate ratio regulates
Raptinal-induced apoptosis by inhibition of JNK activation
We and others have shown that Raptinal induces apoptosis by imposing oxidative stress (Fig. 3b) [18]. We therefore investigated whether the extracellular lactate-to-pyruvate ratio affects Raptinal-induced production of ROS. To this end, we measured the generation of superoxide anion radicals under oxidized and reduced cytosolic NADH/NAD + redox state. We observed no effect of the medium lactateto-pyruvate ratio on the generation of superoxide anion radicals by Raptinal (Fig. 4a). These data indicated that a high extracellular lactate-to pyruvate ratio, and therefore a reduced cytosolic NADH/NAD + redox state, acts downstream of ROS production to protect against Raptinalinduced apoptosis. As Raptinal has been reported to induce apoptosis through the intrinsic mitochondrial pathway, we examined whether the cytosolic NADH/NAD + redox state regulates cytochrome c release, which is required to initiate the caspase activation cascade in intrinsic apoptosis. Indeed, Raptinal induced release of cytochrome c into the cytosol in cells clamped under an oxidized cytosolic NADH/NAD + redox state, but not under a reduced cytosolic NADH/NAD + redox state (Fig. 4b). Next, we investigated the effect of different cytosolic NADH/NAD + redox states on cleavage of caspase-3 and the caspase-3 substrate PARP-1. Consistently, we observed the cleavage of caspase-3 and PARP-1 under an oxidized cytosolic NADH/NAD + redox state by Raptinal, but not under a reduced cytosolic NADH/NAD + redox state (Fig. 4c). Interestingly, clamping cells under a reduced cytosolic NADH/NAD + redox state did not protect cells from apoptosis by TNF-related apoptosis-inducing ligand (TRAIL), an activator of the extrinsic pathway of apoptosis (Fig. 4d) [29]. These results indicate that the cytosolic NADH/NAD + redox state only regulates the intrinsic mitochondrial apoptotic pathway.
Whilst we have verified that Raptinal induces apoptosis via the intrinsic mitochondrial pathway by generating oxidative stress (Figs. 3b, 4a) [18], the mechanism has not been characterized in detail. JNK is known to promote different forms of oxidative stress-induced apoptosis by coordinating the activation of Bax, an effector protein for MOMP, and several BH3-only Bcl-2 proteins, such as Bim, and Bmf [30,31]. Therefore, we examined whether Raptinal induces activation of JNK and used the phosphorylation of Bax at Thr167 as a readout for JNK-dependent activation of MOMP proteins [32]. Indeed, we observed a clear increase of JNK and Bax phosphorylation by Raptinal under an oxidized cytosolic NADH/NAD + redox state, which was strongly decreased under a reduced cytosolic NADH/NAD + redox state (Fig. 5a). To test if JNK activation is an obligatory step for Raptinal-induced apoptosis, we treated HepG2 cells with the JNK inhibitor SP600125 and found that JNK inhibition strongly decreased Raptinal-induced apoptosis under an oxidized cytosolic NADH/NAD + redox state (Fig. 5b). Furthermore, JNK inhibition not only effectively prevented phosphorylation of Bax at Thr167, but also prevented activation of JNK itself in HepG2 cells (Fig. 5c). As observed in HepG2 cells, similar protective effects of a reduced cytosolic NADH/NAD + redox state and of the JNK inhibitor SP600125 were noted on Raptinal-induced apoptosis in U937 and HL-60 cells (Fig. S2A, B). In contrast, the sensitivity of HCT116 cells to Raptinal-induced apoptosis was hardly affected by reducing the cytosolic NADH/NAD + redox state or inhibition of JNK (Fig. S2C). Taken together, our results demonstrate that JNK mediates Raptinal-induced apoptosis and that the cytosolic NADH/NAD + redox state regulates oxidative stress-induced apoptosis downstream of ROS production, but upstream of the activation of JNK.
Discussion
Accumulation of lactic acid in the tumor microenvironment is typical of tumors that exhibit a strong Warburg effect. Many aerobic glycolytic cancers have increased expression of LDHA, which preferentially converts pyruvate into lactate and thereby increases lactate secretion but decreases pyruvate secretion [33]. We demonstrate here that this high lactate but low pyruvate extracellular environment elevates the cytosolic free [NADH]/[NAD + ] ratio (Fig. 2b), which is in line with the observation that cancerous tissues have an elevated cytosolic free [NADH]/[NAD + ] ratio in comparison to healthy (adjacent) tissue [34,35]. We further show that this elevated cytosolic NADH/NAD + redox state confers resistance to oxidative stress induced apoptosis by Raptinal via decreased JNK activation (Fig. 5a). Extracellular lactate levels in the internal tumor core can increase by approximately 20-40 times in comparison to healthy tissues [36,37]. Increased lactate content in cancer tissue correlates with resistance to radiotherapy in solid tumors [38]. Moreover, administration of lactate has been shown to be protective in different cellular models of apoptosis [39,40] but the metabolic background of this phenomenon has not been studied. It has been reported that cholangiocarcinoma and colorectal cancer cells maintain low pyruvate by levels by oncogenic c-myc-enhanced
mM lactate
Dichloroacetate (mM) Relative caspase 3/7 activity ns ** *** ** expression of LDHA and PKM2 and by limiting pyruvate entry through epigenetic silencing of SLC5A8. This decrease in intracellular pyruvate activates HDAC1/3mediated transcriptional upregulation of anti-apoptotic proteins Bcl2 and survivin, and downregulation of proapoptotic proteins p53 and Bax [41][42][43]. However, it is unlikely that transcriptional regulation is responsible for the protective effect observed in our study because of the rapid action of Raptinal (within 2 h). In vivo, it is possible that prolonged increased lactate and decreased pyruvate exposure (high lactate-to-pyruvate ratio) and subsequent changes on the cytosolic NADH/NAD + redox state and transcriptional regulation synergistically protect cancers from apoptosis. Moreover, via "paracrine" signaling, lactate may also affect the cytosolic NADH/NAD + redox state and confer resistance to oxidative stress-induced apoptosis in cells that are not (epi)genetically programmed to exhibit the Warburg effect (Fig. 6).
Our data indicate signaling via the oxidative stress sensor JNK as an important effector for modulating the sensitivity of Raptinal-induced apoptosis exerted by the cytosolic NADH/NAD + redox state (Fig. 5a). Under oxidative stress, JNK-mediated phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and downstream Bcl2-family proteins including Bax itself, leads to liberation and mitochondrial translocation of Bax to initiate MOMP [30,32,44]. In HCT116 cells, Raptinal is capable of inducing cytochrome c release even in the absence of the essential components (Bak/Bax/Box) required for canonical pore formation [45], suggesting a yet unidentified mechanism for Raptinal induced cytochrome c release in these cells. Consistently, our data indicate that this unidentified mechanism is rather insensitive to changes in the cytosolic [NADH]/[NAD + ] ratio or JNK inhibition (Fig. S2C). Changes of the cytosolic [NADH]/[NAD + ] ratio also had no effect on TRAIL-induced cleavage of Bid (data not shown), which is then capable of bypassing the JNKdependent Bax regulation and initiate MOMP [29]. The lack of a protective effect of a reduced cytosolic NADH/ NAD + redox state (and JNK inhibition) in HCT116 cells by Raptinal or in HepG2 under TRAIL-induced apoptosis imply that canonical JNK-Bax signaling of the intrinsic mitochondrial pathway is pivotal for its protective effect. Interestingly, we also observed decreased Raptinal-induced JNK phosphorylation in HepG2 cells treated with the JNK inhibitor SP600125, which is consistent with the reported positive feedback loop of JNK signaling under oxidative stress [46,47].
Apart from the decrease in JNK activation by Raptinal under a Warburg effect environment, we did not directly identify the target(s) responsible for the protective effect. However, because of its rapid action, the protective effect likely involve a post-translational regulation that is sensitive to manipulations of the cytosolic NADH/NAD + redox state. Recently, Sarikhani et al. demonstrated that deacetylation of JNK by cytosolic NAD + -dependent SIRT2 activates the kinase activity of JNK and thereby promotes oxidative stress-induced apoptosis [48]. This is consistent with the protective effect of SIRT2 inhibition described in other models of oxidative stress-induced apoptosis [49][50][51] Fig. 3 The cytosolic NADH/NAD + redox state alters sensitivity to oxidative stress-induced apoptosis. a HepG2 cells were pre-incubated for 1 h in fresh media with different L/P ratios. Following preincubation, cells were treated with vehicle (0.1% DMSO) or 10 µM Raptinal for 1.5 h. Caspase 3/7 activity was determined and normalized to no Raptinal condition. Data represent mean ± SD of a representative experiment (N = 3). b HepG2 cells were pre-incubated with 5 mM NAC under an oxidizing cytosolic NADH/NAD + redox state (L/P = 1) for 1 h. Then, vehicle (0.1% DMSO) or 10 µM Raptinal was added for 1.5 h and caspase 3/7 activity was measured. Data are normalized to vehicle condition and shown are the mean ± SD of a representative experiment (N = 3). c Caspase 3/7 activity assay of H69 cells pre-incubated for 1 h in media with different L/P ratios and then treated with vehicle (0.1% DMSO) or 750 µM Na-CDC for 1 h. Data are normalized to L/P = 1 vehicle condition and are representative of N = 3 experiments. d Similar treatment as in (c), only in HepG2 cells and with 200 µM Na-CDC. Data are normalized to L/P = 1 vehicle condition and are representative of N = 3 experiments. e HepG2 cells were pre-incubated for 1 h in the presence or absence of 10 nM rotenone, an inhibitor of complex I of the electron transport chain, under an oxidizing redox clamp (L/P = 1). Subsequently, cells were treated with vehicle (0.1% DMSO) or 10 µM Raptinal for 1.5 h and caspase 3/7 activity was measured at the end of incubation. Data are normalized to vehicle in the absence of rotenone. Shown are the mean ± SD from a representative experiment (N = 2). f HepG2 cells were preincubated with 0, 1, and 5 mM DCA for 1 h under reducing redox clamp (2.5 mM lactate). Subsequently, cells were incubated with vehicle (0.1% DMSO) or 10 µM Raptinal for 1.5 h and caspase 3/7 activity was measured at the end of incubation. Data are normalized to vehicle in the absence of DCA. Difference in salt concentration was compensated by NaCl. Total added salt concentration was fixed at 5 mM across incubations. Shown are mean ± SD from a representative experiment (N = 3). Statistical analysis: a one-way ANOVA with multiple comparison (Tukey) (b-f) Two-way ANOVA with multiple comparison (Tukey) with ****p < 0.0001, ***p < 0.001, **p < 0.01, ns not significant ◂ redox ratio than NAD + . The buildup of NADH in cancers is proposed to competitively inhibit NADPH-dependent thioredoxin-reductase, which is important to sustain the phosphatase action of tumor suppressor Phosphatase and tensin homolog (PTEN). Inactivation of PTEN leads to Akt activation and resistance to arsenic trioxide-induced apoptosis [19,52]. Indeed, lactate-induced Akt activation protected cancer cells from glucose starvation-induced apoptosis [39]. However, supplementation of lactic acid still protected colon cancer cells from pan-Akt inhibitor uprosertib-induced apoptosis, suggesting that lactate-mediated apoptosis protection may also occur independent of Akt signaling [53]. Future studies are required to further characterize the effects of the cytosolic NADH/NAD + redox state, potentially exerted via SIRT2 or PTEN, on JNK and its modulatory effect on apoptosis.
Finally, pharmacological inhibitors of mitochondria such as rotenone and DCA, which (indirectly) affect cytosolic NADH/NAD + redox state in opposite directions, reversed the sensitizing and desensitizing effects of oxidized and . The oxidation rate of DHE was monitored fluorometrically. Rates of DHE oxidation were normalized to vehicle-treated cells under oxidized redox clamp (L/P = 1) set to 100%. Data are represented as mean ± SD and are representative of two independent experiments. b HepG2 cells were preincubated for 1 h in media containing different L/P ratios. Following pre-incubation, vehicle (0.1% DMSO) or 10 µM Raptinal was added for 2 h. Cytosolic fractions were separated from non-cytosolic fractions (containing mitochondria). To assess cytochrome c release from mitochondria, equal volumes of cytosolic and non-cytosolic fractions were immunoblotted for cytochrome c. Shown is a representative experiment of N = 3. c Treatment as in (b) but whole cell lysates were prepared and immunoblotted for cleaved caspase-3 and its substrate, PARP-1. Shown is a representative experiment of 3 independent experiments (N = 3). d HepG2 cells were pre-incubated for 1 h in media with different L/P ratios after which 40 ng/mL TRAIL was added for 4 h. Caspase 3/7 activity was then measured. Data are normalized to vehicle condition under L/P = 1 and presented as mean ± SD of a representative experiment (N = 3). Statistical analysis (a, d): two-way ANOVA with multiple comparison (Tukey) with ****p < 0.0001, ns not significant reduced cytosolic NADH/NAD + redox state, respectively (Fig. 3e, f). Consistently, high-throughput compound screening with the NADH/NAD + biosensor SoNar showed that compounds lowering cytosolic free [NADH]/[NAD + ] ratio are associated with cancer cell cytotoxicity [35]. In addition, restoration of the pyruvate transporter SLC5A8 in colorectal cancer cells sensitizes them to pyruvate-induced apoptosis whereas pyruvate administration restored the cytotoxic effects of doxorubicin, 5-fluorouracil, and oxaliplatin in otherwise chemo-resistant cancer cells.
In summary, we show that the crosstalk between metabolism and apoptosis can be exploited to overcome resistance to anti-cancer therapy, where the balance of extracellular metabolites (pyruvate and lactate) acts as a determinant of apoptosis tolerance. This apoptosis tolerance, which is dependent on the cytosolic NADH/NAD + redox state and the activation of key enzyme JNK under oxidative stress, may also be relevant for other stimuli that induce oxidative stress, including chemotherapeutic agents. Consequently, oxidizing the cytosolic NADH/NAD + redox state by pyruvate supplementation or by pharmacological inhibitors such as DCA may aid as adjuvant therapy in sensitizing cancer cells with an intact apoptotic signaling machinery to oxidative stress-inducing therapies. Data availability Raw data is available on request.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
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} | pes2o/s2orc | Eosinophil Peroxidase Nitrates Protein Tyrosyl Residues
Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase have primarily focused on its unusual substrate preference for bromide (Br−) and the pseudohalide thiocyanate (SCN−) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO2 −), a major end-product of nitric oxide (⋅NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically relevant concentrations of NO2 −. Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO2 − with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and NO2 −. EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN− concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o′-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory conditions characterized by recruitment and activation of eosinophils.
Eosinophil peroxidase (EPO) has been implicated in promoting oxidative tissue injury in conditions ranging from asthma and other allergic inflammatory disorders to cancer and parasitic/helminthic infections. Studies thus far on this unique peroxidase have primarily focused on its unusual substrate preference for bromide (Br ؊ ) and the pseudohalide thiocyanate (SCN ؊ ) forming potent hypohalous acids as cytotoxic oxidants. However, the ability of EPO to generate reactive nitrogen species has not yet been reported. We now demonstrate that EPO readily uses nitrite (NO 2 ؊ ), a major end-product of nitric oxide ( ⅐ NO) metabolism, as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. EPO-catalyzed nitration of tyrosine occurred more readily than bromination at neutral pH, plasma levels of halides, and pathophysiologically relevant concentrations of NO 2 ؊ . Furthermore, EPO was significantly more effective than MPO at promoting tyrosine nitration in the presence of plasma levels of halides. Whereas recent studies suggest that MPO can also promote protein nitration through indirect oxidation of NO 2 ؊ with HOCl, we found no evidence that EPO can indirectly mediate protein nitration by a similar reaction between HOBr and NO 2 ؊ . EPO-dependent nitration of tyrosine was modulated over a physiologically relevant range of SCN ؊ concentrations and was accompanied by formation of tyrosyl radical addition products (e.g. o,o-dityrosine, pulcherosine, trityrosine). The potential role of specific antioxidants and nucleophilic scavengers on yields of tyrosine nitration and bromination by EPO are examined. Thus, EPO may contribute to nitrotyrosine formation in inflammatory conditions characterized by recruitment and activation of eosinophils.
Eosinophils play a central role in host defenses against a variety of cancers and both parasitic and helminthic infections (1)(2)(3). Increased levels of circulating and tissue eosinophils are also implicated in promoting cellular injury during asthma and other allergic inflammatory disorders (1)(2)(3)(4)(5)(6). Eosinophils are thought to mediate many of their cytotoxic and tissue-destroying effects through their exceptional ability to generate oxidizing species (1)(2)(3)(4)(7)(8)(9). Indeed, the respiratory burst of eosino-phils generates several times as much superoxide (O 2 . ) and hydrogen peroxide (H 2 O 2 ) as a corresponding number of neutrophils (9,10). Despite their known proclivity for producing reactive oxygen species and a wealth of clinical evidence linking eosinophils to host defenses and inflammatory tissue injury, structural identification of specific oxidation products formed by these leukocytes is lacking. Eosinophil peroxidase (EPO), 1 an abundant heme protein secreted from activated eosinophils, plays a central role in oxidant production by eosinophils (1-4, 8, 11, 12). EPO amplifies the oxidizing potential of H 2 O 2 produced during the respiratory burst by using it as a co-substrate to generate cytotoxic oxidants. Studies thus far have focused on the unusual substrate preference of EPO for physiological levels of bromide (Br Ϫ ) (8,(13)(14)(15)(16)(17) and the pseudohalide thiocyanate (SCN Ϫ ) (18 -20), even in the presence of a vast molar excess of chloride (Cl Ϫ ), as is seen in vivo. Studies with proteins incubated in the presence of radioactive Br Ϫ or SCN Ϫ salts and either activated eosinophils or the EPO-H 2 O 2 system have demonstrated covalent incorporation of radiolabel into target proteins (13,14). However, structural identification of the oxidation products formed on proteins following exposure to EPO-generated oxidants is essentially unexplored. We recently identified 3-bromotyrosine and 3,5-dibromotyrosine as major products of protein oxidation by the EPO-H 2 O 2 -Br Ϫ system (21). The role of brominating oxidants in promoting tissue injury in eosinophilic inflammatory disorders has not yet been established.
Another potential pathway for oxidative tissue damage by eosinophils may involve formation of nitrating intermediates by EPO. Immunohistochemical studies using antibodies raised against nitrotyrosine, a global marker for protein damage by reactive nitrogen species, intensely stain eosinophils present in the inflamed lung tissues of asthmatics (22). Klebanoff (23) demonstrated that the antimicrobicidal properties of eosinophil peroxidase-H 2 O 2 systems are enhanced in the presence of nitrite (NO 2 Ϫ ), the autoxidation product of ⅐ NO. Moreover, recent studies have shown that myeloperoxidase (MPO), a neutrophiland monocyte-specific peroxidase, can use NO 2 Ϫ and H 2 O 2 as substrates to generate a reactive intermediate capable of nitrating phenolic compounds and protein tyrosyl residues (25-28). However, MPO and EPO are distinct gene products with divergent physical properties. Although both peroxidases contain heme prosthetic groups, structural studies have established that MPO contains a six-coordinate, high spin chlorin, while EPO possesses a high spin, six-coordinate protoporphyrin (24). Moreover, although human MPO is tetrameric and comprised of two heavy and two light chains, EPO is dimeric, having only one heavy and one light chain (7,24). Whether these structural differences underlie the distinct halide and substrate specificities observed between MPO and EPO remains unknown. Furthermore, although increased levels of NO 2 Ϫ and eosinophils have been reported in a variety of conditions, the ability of EPO to generate nitrating intermediates has not yet been explored.
The biological roles and potential targets of mammalian peroxidases are defined by their unique and often nonoverlapping substrate preferences (29,30). We therefore sought to determine whether the EPO-H 2 O 2 system of eosinophils could promote protein oxidative damage through formation of reactive nitrogen species. We now show that isolated EPO readily uses NO 2 Ϫ as substrate to generate a reactive intermediate that nitrates protein tyrosyl residues in high yield. We demonstrate that EPO is more efficient than MPO at nitrating tyrosine and that EPO-dependent protein nitration is a preferred activity of the enzyme. Finally, we provide evidence that EPO also generates tyrosyl radical and that tyrosine nitration by EPO may involve a tyrosyl radical intermediate. Isolation and Characterization of EPO and MPO-Porcine EPO was isolated according to the method of Jorg (31) employing guaiacol oxidation as the assay (32). Purity of EPO preparations was assured before use by demonstrating a RZ of Ͼ0.9 (A 415 /A 280 ), SDS-polyacrylamide gel electrophoresis analysis with Coomassie Blue staining, and in-gel tetramethylbenzidine peroxidase staining to confirm no contaminating MPO activity (33). MPO was initially purified from detergent extracts of human leukocytes by sequential lectin affinity and gel filtration chromatography as described (34). Trace levels of contaminating EPO were then removed by passage over a CM-52 ion exchange column (20). Purity of isolated MPO was established by demonstrating a RZ of 0.87 (A 430 /A 280 ), SDS-polyacrylamide gel electrophoresis analysis with Coomassie Blue staining, and in-gel tetramethylbenzidine peroxidase staining to confirm no contaminating EPO activity (33). Enzyme concentrations were determined spectrophotometrically utilizing extinction coefficients of 89,000 and 112,000 M Ϫ1 cm Ϫ1 /heme of MPO (35) and EPO (36,37), respectively. The concentration of the MPO dimer was calculated as half of the indicated concentration of hemelike chromophore.
Materials-Organic
General Procedures-SDS-polyacrylamide gel electrophoresis analysis was performed as described by Laemmli (38). Protein content was measured by the Markwell-modified Lowry assay with bovine serum albumin (BSA) as the standard (39). HOBr free of Br Ϫ and bromate was prepared from liquid bromine the day of use as described (40). HOBr was quantified spectrophotometrically (⑀ 331 ϭ 315 M Ϫ1 cm Ϫ1 ) (41) as its conjugate base, hypobromite (OBr Ϫ ), immediately prior to use. The concentration of reagent H 2 O 2 (⑀ 240 ϭ 39.4 M Ϫ1 cm Ϫ1 ) (42) was determined spectrophotometrically. Production of H 2 O 2 by the glucose/glucose oxidase system was determined by oxidation of Fe(II) and formation of a Fe(III)-thiocyanate complex (25). Western blot analysis of nitrated proteins was determined as described using immunopurified rabbit polyclonal antibody against nitrotyrosine (Upstate Biotechnology, Inc., Lake Placid, NY) (43). The specificity of the primary antibody was confirmed by blocking immunoreactivity in coincubations with either 10 mM nitrotyrosine or 1 mM of the tripeptide Gly-nitro-Tyr-Ala as described (43). o,oЈ-Dityrosine, pulcherosine, trityrosine, and isodityrosine were prepared and quantified as described previously (44). 3-Bromotyrosine and 3,5-dibromotyrosine standards were synthesized from L-tyrosine and isolated by preparative HPLC as recently described (21). [ 13 C 6 ]Ring-labeled analogs of 3-bromotyrosine and 3,5-dibromotyrosine were similarly prepared using L-[ 13 C 6 ]tyrosine as starting material. UV-visible studies on authentic and EPO-generated 3-nitrotyrosine were performed on a Bio Spectrophotometer (Perkin-Elmer).
5,5Ј-Dithiobis(2-nitrobenzoic acid) (DTNB) Formation by EPO-5-
Thio-2-nitrobenzoic acid (TNB) was prepared fresh by reduction of 1 mM DTNB in 100 ml of sodium phosphate buffer (50 mM, pH 7.0) with 4 l of 2-mercaptoethanol (46). EPO (14.2 nM) was then incubated at 37°C in sodium phosphate buffer (50 mM, pH 7.0) with TNB (53 M) in the absence or presence of the indicated concentrations of Br Ϫ , SCN Ϫ , NO 2 Ϫ , or Cl Ϫ . Oxidation of TNB to DTNB was initiated by the addition of H 2 O 2 (30 M) and followed spectrophotometrically at 412 nm (⑀ 412 ϭ 27,200 M Ϫ1 cm Ϫ1 ) (46). Oxidation of Free L-Tyrosine and Protein-bound Tyrosyl Residues-Unless otherwise specified, reactions were initiated by the addition of oxidant (H 2 O 2, HOBr or glucose to the glucose/glucose oxidase system) and performed in sodium phosphate buffer (20 mM, pH 7.0) at 37°C for 60 min under the conditions described in the figure and table legends. Reactions were stopped by the addition of phenol (1% final) and catalase (30 nM; for glucose/glucose oxidase studies) to the reaction mixture. The pH dependence of 3-nitrotyrosine formation was assayed in phosphate buffer (50 mM final) composed of mixtures of phosphoric acid and monobasic and dibasic sodium phosphate. The pH of each reaction mixture was determined at the end of the incubation and did not change by more than 0.2 pH units over the course of the reaction. Experiments utilizing the glucose/glucose oxidase system for H 2 O 2 generation were performed in the presence of 100 g/ml glucose and 20 ng/ml glucose oxidase (grade II; Roche Molecular Biochemicals) at 37°C for the indicated times. Preliminary studies demonstrated that under these conditions, a constant flux of H 2 O 2 (0.18 M/min) was generated.
Protein Hydrolysis-Preliminary studies confirmed that removal of NO 2 Ϫ prior to acidification of proteins was essential to avoid artifactual nitration of proteins. Proteins oxidized in vitro were therefore prepared for analysis by first precipitating and desalting them in a single-phase extraction mixture composed of H 2 O/methanol/H 2 O-saturated diethyl ether (1:3:7, v/v/v) as described (47). Proteins were typically hydrolyzed by incubating the desalted protein pellet with 6 N HCl (0.5 ml) supplemented with 1% phenol for 24 h (47). Prior to initiating hydrolysis, acid mixtures were degassed under vacuum and then sealed under a blanket of argon. When chlorotyrosine and/or bromotyrosine determinations were performed, formation of trace levels of halogenated tyrosine analogs during acid hydrolysis was avoided by hydrolysis in 4 N methane sulfonic acid (0.5 ml) supplemented with 1% phenol for 24 h at 100°C under a blanket of argon (21). Control experiments utilizing proteins supplemented with L-[ 2 H 4 ]tyrosine as internal standard added prior to acid hydrolysis and then analyzed by GC/MS (48) Reverse Phase HPLC Quantification of Tyrosine Oxidation Products-Quantification of oxidation products from free L-tyrosine (3-nitrotyrosine, 3-bromotyrosine, 3,5-dibromotyrosine, 3-chlorotyrosine, and o,oЈ-dityrosine) was performed on a Beckman Gold HPLC system equipped with diode array and fluorescence detectors. Separations were performed on a C18 column (Beckman Ultrasphere, 5 m, 4.6 ϫ 250 mm) equilibrated with solvent A (0.1% trifluoroacetic acid, pH 2.5). L-Tyrosine and its oxidation products were eluted at a flow rate of 1 ml/min with a linear gradient generated with solvent B (0.1% trifluoroacetic acid in methanol, pH 2.5) as follows: 0% solvent B for 5 min, 0 -100% solvent B over 30 min, 100% solvent B for 10 min. Products were quantified employing standard curves constructed with authentic synthetic standards. N ␣ -acetyl-L-tyrosine-derived oxidation products were isolated, and their structures were confirmed by on-line electrospray ionization mass spectrometric analysis as described below. Routine quantification of N ␣ -acetyl-L-tyrosine oxidation products was determined by reverse phase HPLC with UV detection (A 280 ) as above, utilizing external calibration curves constructed with known amounts of their nonacetylated counterparts (and assuming identical extinction coefficients). The retention times of N ␣ -acetyl-L-tyrosine analogs of tyrosine oxidation products were determined using authentic standards isolated at the time of on-line liquid chromatography/mass spectrometric analysis (LC/MS).
3-Nitrotyrosine, 3-bromotyrosine, 3,5-dibromotyrosine, o,oЈ-dityrosine, and 3-chlorotyrosine in protein hydrolysates were routinely quantified by reverse phase HPLC with electrochemical (coulometric) detection on an ESA (Cambridge, MA) CoulArray HPLC instrument equipped with UV detector and electrochemical cells (six channels) arranged in series and set to increasing specified potentials: channel 1 (300 mV); channel 2 (350 mV); channel 3 (480 mV); channel 4 (650 mV); channel 5 (800 mV); channel 6 (880 mV). Chromatographic separation of amino acids was performed on a a Projel TSK ODS-80 TM column (5 m, 4.6 ϫ 250 mm) equilibrated with mobile phase A (15 mM lithium phosphate, 3 mg/liter lithium dodecyl sulfate, pH 3.2). Products were eluted at a flow rate of 1 ml/min with a nonlinear gradient generated with mobile phase B (50% methanol, 15 mM lithium phosphate, 3 mg/liter lithium dodecyl sulfate, pH 3.2) as follows: isocratic elution at 0% mobile phase B for 10 min, 0 -15% mobile phase B over 10 min, isocratic elution at 15% mobile phase B for 10 min, 15-20% mobile phase B over 10 min, isocratic elution at 20% mobile phase B for 10 min, 20 -100% mobile phase B over 20 min, isocratic elution at 100% mobile phase B for 10 min. Peak identity was established by demonstrating the appropriate retention time, redox potential, and ratio of integrated currents in adjacent channels and by the method of standard additions for each analyte. L-Tyrosine, 3-nitrotyrosine, 3-bromotyrosine, 3,5-dibromotyrosine, o,oЈ-dityrosine, and 3-chlorotyrosine standards (0.5-50 pmol each on column) were also dissolved together and used to generate external calibration curves. The experimental error in data obtained by this assay is less than Ϯ5%. Control experiments comparing quantitation of the tyrosine analogues by stable isotope dilution GC/MS (45) versus reverse phase with electrochemical detection demonstrated comparable results.
Mass Spectrometry-LC/MS was performed using electrospray ionization (ESI) and detection with a Quatro II triple quadrupole mass spectrometer (Micromass, Inc.) interfaced with an HP 1100 high performance liquid chromatograph (Hewlett Packard, Wilmington, DE) equipped with diode array detector. L-Tyrosine oxidation products were resolved on an Ultrasphere C18 column (Beckman; 5 m, 4.6 ϫ 250 mm) at a flow rate of 1 ml/min and a linear gradient between H 2 O (with 0.3% formic acid) and methanol (with 0.3% formic acid) over 30 min. Column eluent was split (970 l/min to diode array, 30 l/min to mass detector) and analyzed in the positive ion mode with a cone potential of 30 eV. GC/MS analyses of HPLC-isolated L-tyrosine oxidation products were performed following derivatization to their n-propyl perheptafluorylbutyryl or n-propyl perpentafluorylproprionyl derivatives (49). 3-Ni-trotyrosine was also analyzed by GC/MS analysis as its n-propyl perheptafluorylbutyryl derivative following reduction to 3-aminotyrosine (50). Negative ion chemical ionization GC/MS studies were performed utilizing a Perkin Elmer (Norwalk, CT) TurboMass spectrometer equipped with chemical ionization probe.
3-Nitrotyrosine and Tyrosyl Radical Addition Products Are Formed during L-Tyrosine Oxidation by the Eosinophil Peroxidase-H 2 O 2 -NO 2
Ϫ System-The addition of purified EPO and H 2 O 2 to reaction buffer (20 mM sodium phosphate, 100 M diethylenetriaminepentaacetic acid, pH 7.0) supplemented with L-tyrosine generated several new species with distinct retention times on reverse phase HPLC analysis with diode array detection (Fig. 1, upper panel, peaks a-c). The ultraviolet absorption maximum of each peak was nearly indistinguishable from L-tyrosine ( max 275-285 nm). LC/MS analyses were consistent with formation of the tyrosyl radical addition products o,oЈ-dityrosine and isodityrosine (peak a; m/z 361 (M ϩ H) ϩ ), pulcherosine (peak b; m/z 540 (M ϩ H) ϩ ), and trityrosine (peak c; m/z 540 (M ϩ H) ϩ ) (44). Likewise, HPLC analysis with UV and fluorescence detection demonstrated co-migration with authentic synthetic standards prepared as described under ؊ system. Top, the major L-tyrosine oxidation product generated by the EPO-H 2 O 2 -NO 2 Ϫ system (peak I, Fig. 1, bottom) was isolated by reverse phase HPLC as described under "Experimental Procedures." The isolated product was dried, resuspended at the indicated pH, and then analyzed by UV-visible spectroscopy (200 -600 nm). Bottom, the major L-tyrosine oxidation product generated by the EPO-H 2 O 2 -NO 2 Ϫ system (peak I in Fig. 1, bottom) was analyzed by LC/MS as described under "Experimental Procedures." The positive ion ESI mass spectrum is shown. The structure and predicted m/z of protonated 3-nitrotyrosine is also shown (inset). "Experimental Procedures." Following the addition of NO 2 Ϫ , the reaction mixture developed an intense yellow color. Reverse phase HPLC analysis demonstrated the formation of an additional major (peak I) and minor (peak d) product (Fig. 1, bottom panel) as well as enhanced production of the tyrosyl radical addition products (peaks a-c). The UV-visible spectra of the new products (peaks I and d) both illustrated similar absorbance maxima in the visible wavelength range ( max 350 -355 nm), consistent with nitration of the aromatic amino acid (51). Use of heat-inactivated EPO or omission of either L-tyrosine or H 2 O 2 from the reaction mixture both resulted in no detectable production of the products. The reaction products formed (peak I and peaks a-d) were stable to treatment with acid (4 N HCl at 100°C for 24 h), incubation at 37°C with H 2 O 2 , and the addition of a molar excess of either reducing agents or nucleophilic scavengers (e.g. NaCNBH 3 , methionine, 2-mercaptoethanol, taurine, ammonium acetate).
To characterize the structures of the L-tyrosine oxidation products generated by the EPO-H 2 O 2 -NO 2 Ϫ system, a variety of analytical techniques were employed. The pH dependence of the UV-visible absorbance spectrum of HPLC-isolated peak I is illustrated in Fig. 2 (upper panel) and is identical to that observed with authentic 3-nitrotyrosine. To confirm the structure of the major L-tyrosine oxidation product (peak I) as 3-nitrotyrosine, LC/MS analysis was performed. The retention time and positive ion mass spectrum of peak I was identical to that of authentic 3-nitrotyrosine and demonstrated a m/z of 227 (M ϩ H) ϩ (Fig. 2, lower panel). LC/MS analysis of the minor products generated during oxidation of L-tyrosine by the complete EPO-H 2 O 2 -NO 2 Ϫ system was consistent with the prior structural assignments of peaks a-c and suggested that peak d was composed of a nitrated analog of o,oЈ-dityrosine (e.g. 3-nitro-o,oЈ-dityrosine, m/z 406 (M ϩ H) ϩ ). These results, combined with the chemical stability and fluorescence spectra (data not shown) of the compounds formed, suggest that the major compounds generated during oxidation of free L-tyrosine by EPO and H 2 O 2 are tyrosyl radical addition products. In contrast, in the presence of EPO, H 2 O 2 , and NO 2 Ϫ , 3-nitrotyrosine is the major product formed.
Characterization of Reaction Requirements for EPO-dependent Formation of 3-Nitrotyrosine-The reaction requirements for EPO-catalyzed nitration of L-tyrosine yielding 3-nitrotyrosine are illustrated in Fig. 3. At pH 7.0 and 100 M L-tyrosine and NO 2 Ϫ , 3-nitrotyrosine synthesis by EPO (following the bolus addition of 100 M H 2 O 2 ) was linear over the first 15 min and reached a plateau within 30 min (Fig. 3A). At higher L-tyrosine concentrations, the yield of 3-nitrotyrosine decreased, consistent with the competing bimolecular mechanism of o,oЈ-dityrosine formation. EPO-catalyzed nitration was optimal between pH 6 and 6.5, where approximately 0.35 mol of product was formed for each mole of oxidant consumed (Fig. 3B). A dose-dependent increase in the synthesis of 3-nitrotyrosine was observed over a (patho)physiologically relevant range of NO 2 Ϫ concentrations (Fig. 3C). The H 2 O 2 concentration dependence for 3-nitrotyrosine production by EPO yielded a similarly shaped plot and overall yield (Fig. 3D). At high concentrations of H 2 O 2 , however, the total amount of 3-nitrotyrosine formed decreased, consistent with either substrate inhibition or an interaction between H 2 O 2 and an intermediate in 3-nitrotyrosine formation (data not shown).
3-Nitrotyrosine Is a Preferred Product of Eosinophil Peroxidase at Physiologically Relevant Concentrations of L-Tyrosine, Halides, and NO 2
Ϫ -Thiocyanate (SCN Ϫ ) and bromide (Br Ϫ ) are reported to be preferred substrates for EPO (13)(14)(15)(16)18). To estimate the relative substrate preferences of isolated EPO for NO 2 Ϫ versus other reported substrates for the enzyme, we ex- amined the initial rate of DTNB formation in reaction mixtures containing EPO, TNB, H 2 O 2 , and various concentrations of anionic substrates (SCN Ϫ , Br Ϫ , NO 2 Ϫ , Cl Ϫ ) at neutral pH. The relative rates of TNB oxidation observed by EPO were as follows: SCN Ϫ Ͼ Br Ϫ Ͼ NO 2 Ϫ Ͼ Ͼ Cl Ϫ (Fig. 4). Although EPO does not conform to Michaelis-Menten kinetics (e.g. high concentrations of H 2 O 2 inhibit tyrosine nitration), a measure of the relative ability of EPO to discriminate in favor of a particular substrate in the presence of a mixture of competing substrates can be calculated as an apparent specificity constant (k x Ϫ) for each substrate (33). Because high levels of H 2 O 2 can inhibit oxidant production, we estimated the kinetic parameters under conditions that minimize inhibition and mimic physiological conditions (e.g. neutral pH and low concentrations of H 2 O 2 ) ( Table I). SCN Ϫ was the preferred substrate for EPO, demonstrating an approximately 2.7-fold higher apparent specificity constant than Br Ϫ , the next best substrate examined. EPO preferred Br Ϫ approximately 4-fold more than NO 2 Ϫ (Table I). Implicit in this type of kinetic analysis is the assumption that the reactive nitrogen species formed by the EPO-H 2 O 2 -NO 2 Ϫ system oxidize TNB at a rate and stoichiometry comparable with that of other hypohalous acids formed by the enzyme.
As an alternative means of assessing the potential significance of EPO-dependent tyrosine nitration, we performed product analyses of the major L-tyrosine oxidation products formed by the enzyme system (Table I). When plasma levels of Ltyrosine and halides were incubated with isolated EPO, H 2 O 2 , and concentrations of NO 2 Ϫ as might be observed in inflammatory tissues or fluids (tyrosine ϭ Br Ϫ ϭ NO 2 Ϫ ϭ 100 M, Cl Ϫ ϭ 100 mM; Refs. 25 and 52-56), 3-nitrotyrosine was formed in high yield (Table II). In fact, 3-nitrotyrosine was the major product formed and was present at 2-3-fold greater levels than either 3-bromotyrosine or o,oЈ-dityrosine (Table II). Despite that vast molar excess of Cl Ϫ , no detectable formation of 3-chlorotyrosine was observed under all of the conditions examined, consistent with the low apparent specificity constant observed (Tables I and II). The highest yields of 3-nitrotyrosine production were observed during incubations in the absence of Br Ϫ , consistent with the unusual substrate preference of EPO for this halide.
3-Nitrotyrosine Formation by Eosinophil Peroxidase Occurs in the Presence of Nucleophilic Scavengers and Is Inhibited by
Ascorbate and Urate-Reactive oxidants and diffusible radical species generated by peroxidases in vivo will encounter a variety of nucleophilic scavengers and antioxidant compounds. To characterize the nature of the reactive halogenating and nitrating intermediates generated by EPO, we incubated the enzyme with H 2 O 2 , Br Ϫ , and NO 2 Ϫ under a variety of conditions and then quantified 3-bromotyrosine and 3-nitrotyrosine production. Again, when L-tyrosine was incubated with EPO, H 2 O 2 (100 M), and equivalent amounts of Br Ϫ and NO 2 Ϫ (100 M each), 3-nitrotyrosine was the major oxidation product formed and was produced in 2-3-fold higher yield than 3-bromotyrosine (Fig. 5). The addition of methionine, a potent scavenger of reactive halogenating species, completely ablated 3-bromotyrosine production. In contrast, the addition of the thiol ethercontaining amino acid to the reaction mixture had no significant effect on 3-nitrotyrosine production (Fig. 5). Likewise, the addition of reduced thiol compounds such as glutathione totally inhibited tyrosine bromination but only partially blocked aromatic nitration by the peroxidase. The addition of either ascorbic acid or uric acid potently inhibited both 3-bromotyrosine and 3-nitrotyrosine production. The addition of primary aminecontaining species (e.g. N ␣ -acetyl lysine) to the reaction mixture did not attenuate either 3-bromotyrosine or 3-nitrotyrosine production. These results are consistent with our recent report that N-monobromamines serve as brominating intermediates for tyrosine (21). They also suggest that the nitrating intermediate formed by the EPO-H 2 O 2 -NO 2 Ϫ system is not scavenged by amine groups. Finally, these results suggest that aromatic nitration reactions may be mediated by EPO in inflammatory fluids and tissues where NO 2 Ϫ levels are increased and antioxidants such as ascorbate and urate may be depleted.
Eosinophil Peroxidase Nitrates Protein Tyrosyl Residues in High Yield-The ability of EPO to nitrate protein tyrosyl residues was first examined by incubating a target protein (e.g. BSA) with the complete EPO-H 2 O 2 -NO 2 Ϫ system and then performing SDS-polyacrylamide gel electrophoresis analysis with Western blot analysis using affinity-purified polyclonal antibodies specific for 3-nitrotyrosine (43). A NO 2 Ϫ -dependent increase in the intensity of staining was observed following exposure of the protein to the enzymatic system (Fig. 6). To confirm that 3-nitrotyrosine was produced in proteins exposed to the EPO-H 2 O 2 -NO 2 Ϫ system, protein hydrolysates were directly analyzed for 3-nitrotyrosine production by GC/MS in the negative-ion chemical ionization mode employing synthetic 3-nitro-[ 13 C 6 ]tyrosine as an internal standard, as described under "Experimental Procedures." Ions with the appropriate mass-to-charge ratio (m/z 464 (M) . and 444 (M Ϫ HF) . ) and identical retention time to that observed with their corresponding stable isotope-labeled fragment ions were readily observed for the n-propyl-perheptafluorylbutyryl derivative of 3-nitrotyrosine. Moreover, reduction of the products prior to analysis by GC/MS demonstrated ions with identical retention time and appropriate m/z for that of the n-propyl perheptafluorylbutyryl derivative of 3-aminotyrosine (m/z 806 (M Ϫ HF) . , m/z 762 (M Ϫ HF Ϫ CO 2 ) . and m/z 628 (M Ϫ heptafluorylbutyryl) . ). Collectively, these results unambiguously confirm that EPO catalyzes nitration of protein tyrosyl residues in the presence of the co-substrates, H 2 O 2 and NO 2 Ϫ . To characterize the significance of EPO-dependent protein nitration, the content of protein-bound 3-nitrotyrosine produced by the enzyme was determined by reverse phase HPLC with electrochemical detection as described under "Experimental Procedures." BSA was incubated with the complete EPO-H 2 O 2 -NO 2 Ϫ system, while the concentrations of either NO 2 Ϫ (Fig. 7, upper panel) or H 2 O 2 (Fig. 7, lower panel) were varied. Nitration of protein tyrosyl residues occurred readily in a concentration-dependent manner for both co-substrates. The overall yield of EPO-catalyzed nitration of protein tyrosyl residues as a function of each substrate is shown in Fig. 8. The highest yields (10 -20%) of protein nitration were noted when NO 2 Ϫ concentrations were high and H 2 O 2 concentrations were low. These results suggest that protein nitration would be highest under conditions where H 2 O 2 production occurs at a low rate, similar to the conditions that are likely to be present in vivo.
To more fully examine the potential physiological significance of protein nitration by EPO, we quantified the levels of formation of multiple tyrosyl residue oxidation products (3nitrotyrosine, 3-bromotyrosine, 3-chlorotyrosine, and o,oЈ-dityrosine) in proteins incubated with isolated EPO, a hydrogen in sodium phosphate buffer (20 mM, pH 7.0) at 37°C for 60 min. After incubation, 1.2 g of protein was loaded on 10% SDS-polyacrylamide gels for electrophoresis, and proteins were transferred and immunostained using a rabbit polyclonal antibody against 3-nitrotyrosine (top) or stained with Coomassie Blue (bottom). peroxide-generating system (glucose/glucose oxidase), and concentrations of halides and NO 2 Ϫ as might be observed in inflammatory fluids (i.e. 100 mM Cl Ϫ , 100 M Br Ϫ , and 100 M NO 2 Ϫ ). Again, EPO nitrated protein tyrosyl residues to a greater extent than that of all other tyrosine oxidation products examined (Table III). Under the conditions employed, only minimal amounts of o,oЈ-dityrosine and no detectable levels of 3-chlorotyrosine were formed (Table III). EPO-catalyzed bromination of protein tyrosyl residues occurred, albeit at a level approximately one-third that of tyrosine nitration. Generation of the tyrosine oxidation products demonstrated an absolute requirement for EPO and the H 2 O 2 -generating system. Collectively, these results demonstrate that EPO uses NO 2 Ϫ as a preferred substrate to form a reactive nitrogen intermediate(s) even in the presence of physiological levels of halides.
The pseudohalide SCN Ϫ is a preferred substrate of EPO (18 -20). We were therefore interested in examining the poten-tial influence of SCN Ϫ levels on EPO-catalyzed nitration. Extracellular fluid and tissue levels of this unusual anion have not been reported; however, plasma levels of SCN Ϫ vary considerably (normal values are Ͻ69 M) depending upon diet and smoking habits in normal individuals (57). In a separate set of experiments, either free L-tyrosine (Fig. 9, left) or BSA (Fig. 9, right) was incubated with NO 2 Ϫ (100 M) and plasma levels of halides (100 mM Cl Ϫ and 100 M Br Ϫ ) in the absence and presence of varying amounts of SCN Ϫ , and the content of 3-nitrotyrosine was determined. EPO-mediated nitration was attenuated by the addition of SCN Ϫ over the reported range of plasma levels of SCN Ϫ (Fig. 9). In the presence of equivalent levels of NO 2 Ϫ and SCN Ϫ (100 M each), 3-nitrotyrosine formation was significantly inhibited; however, nitration of both free and protein-bound tyrosine residues was still detectable.
Eosinophil Peroxidase Is More Effective Than Myeloperoxidase at Generating Nitrotyrosine in the Presence of NO 2 Ϫ , H 2 O 2 , and Plasma Levels of Halides-Since MPO-dependent formation of nitrotyrosine may also contribute to protein nitration at sites of inflammation (25-28, 58, 59), we sought to compare the ability of isolated MPO and EPO to nitrate target proteins. BSA was incubated with equal concentrations of each peroxidase in the presence of plasma levels of halides (100 mM Cl Ϫ and 100 M Br Ϫ ) and an H 2 O 2 -generating system (glucose/ glucose oxidase) to more closely mimic the low steady flux of peroxide that might be formed in vivo. Kinetic analyses comparing the ability of each peroxidase to use NO 2 Ϫ as substrate for protein nitration demonstrated that EPO formed at least 4-fold more 3-nitrotyrosine on target proteins than MPO at all times examined (Fig. 10, left). Moreover, regardless of the concentration of NO 2 Ϫ examined (0 -100 M), EPO was significantly more effective than MPO at nitrating protein tyrosyl residues (Fig. 10, right). Thus, at sites of eosinophilic inflammatory disorders, EPO may contribute to 3-nitrotyrosine formation.
The Mechanism of Eosinophil Peroxidase-dependent Production of 3-Nitrotyrosine Involves Formation of a Tyrosyl Radical
Intermediate-To examine the mechanism of protein tyrosyl residue nitration by the EPO-H 2 O 2 -NO 2 Ϫ system, we initially performed a variety of experiments utilizing N ␣ -acetyl-L-tyrosine, a low molecular weight surrogate for a protein-bound tyrosyl residue. In the absence of NO 2 Ϫ , incubation of N ␣ -acetyl-L-tyrosine with EPO and H 2 O 2 resulted in formation of the N ␣ -acetyl dityrosine analog (Table IV). The addition of Br Ϫ to the reaction mixtures resulted in production of the N ␣ -acetyl-L-tyrosine analogs of 3-bromotyrosine and 3,5-dibromotyrosine but no detectable production of the N ␣ -acetyl-L-tyrosine analog of the tyrosyl radical-addition product o,oЈ-dityrosine. In contrast, the addition of NO 2 Ϫ to N ␣ -acetyl-L-tyrosine and the EPO-H 2 O 2 system formed predominantly the N ␣ -acetyl-L-tyrosine analog of 3-nitrotyrosine and modest levels of the N ␣ -acetyl-Ltyrosine analog of o,oЈ-dityrosine (Table IV). The addition of both NO 2 Ϫ and Br Ϫ to N ␣ -acetyl-L-tyrosine and the EPO-H 2 O 2 system formed all of the expected tyrosine oxidation products (N ␣ -acetyl-L-tyrosine analogs of 3-nitrotyrosine, o,oЈ-dityrosine, 3-bromotyrosine, and 3,5-dibromotyrosine). Thus, in every instance where tyrosine nitration occurred, concurrent formation of tyrosyl radical addition products was observed. In contrast, during tyrosine bromination (in the absence of NO 2 Ϫ ), no formation of tyrosyl radical addition products (i.e. N ␣ -acetyl-Ltyrosine analog of o,oЈ-dityrosine) could be detected. These results are consistent with 3-nitrotyrosine production through an addition reaction between an intermediate tyrosyl radical and reactive nitrogen species such as ⅐ NO 2 , the one electron oxidation product of NO 2 Ϫ .
Co-incubation of HOCl and NO 2
Ϫ has been reported to form a nitrating and chlorinating intermediate, presumably NO 2 Cl (58). Moreover, we observed modest increases in the levels of free and protein-bound nitrotyrosine formed during incubation with the EPO-H 2 O 2 system in the presence of NO 2 Ϫ and Cl Ϫ compared with NO 2 Ϫ alone (Tables II and III). However, the mechanism for the increase does not appear to involve reaction of HOCl with NO 2 Ϫ , since comparable increases in tyrosine nitration were observed in the presence of Cl Ϫ during incubations that contained excess methionine, a potent scavenger of HOCl and other halogenating intermediates (data not shown).
To determine if EPO might indirectly promote protein nitration by forming a nitrating intermediate by oxidation of NO 2 Ϫ with HOBr, we performed the following experiment. N ␣ -Acetyl-Ltyrosine was incubated with HOBr in the presence and absence of NO 2 Ϫ , and the extent of tyrosine nitration was determined. No detectable formation of the 3-nitrotyrosine analog was observed, even in the presence of 1 mM concentrations of both HOBr and NO 2 Ϫ (Table IV). Similar results were observed in reactions performed under acidic (pH 4) conditions (data not shown). Collectively, these results suggest that the nitrating intermediate formed by EPO arises from direct oxidation of NO 2 Ϫ and not by secondary oxidation of NO 2 Ϫ by HOCl, HOBr, or some other halogenating agent.
In a final series of experiments, we sought to further explore Ϫ BSA (0.4 mg/ml) was incubated with eosinophil peroxidase (57 nM), glucose (100 g/ml), glucose oxidase (20 ng/ml), and the indicated anionic substrates (Br Ϫ ϭ NO 2 Ϫ ϭ 100 M, Cl Ϫ ϭ 100 mM) in sodium phosphate buffer (20 mM, pH 7.0) at 37°C for 4 h. Following reaction, protein was desalted and subjected to acid hydrolysis, and the content of 3-nitrotyrosine, 3-bromotyrosine, o,oЈ-dityrosine, and 3-chlorotyrosine was determined by reverse phase HPLC with electrochemical detection as described under "Experimental Procedures." Results are the mean Ϯ S.D. of three independent experiments. The limit of detection for these analyses is Ͻ0.05 mmol/mol tyrosine for each analyte examined. the potential role of a tyrosyl radical intermediate in 3-nitrotyrosine formation by monitoring the extent of aromatic nitration using tyrosine analogs that do not form tyrosyl radicals. Because of its acidic character, the phenoxyl hydrogen of Ltyrosine is the preferred site of hydrogen atom abstraction from tyrosine; consequently, O-methyl-L-tyrosine is resistant to formation of tyrosyl radical (60). If 3-nitrotyrosine production by the EPO-H 2 O 2 -NO 2 Ϫ system instead occurred by an electrophilic addition reaction (e.g. through a NO 2 ϩ intermediate), use of the O-methyl-L-tyrosine analog should not significantly effect the yield of 3-nitrotyrosine formation. Incubation of the complete EPO-H 2 O 2 -NO 2 Ϫ system with L-tyrosine readily formed 3-nitrotyrosine; in contrast, no nitrated products were formed from O-methyl-L-tyrosine at all concentrations of NO 2 Ϫ examined, as determined by reverse phase HPLC with on-line UV and ESI/MS (Fig. 11). Collectively, these results strongly support the hypothesis that the EPO-H 2 O 2 -NO 2 Ϫ system nitrates tyrosyl residues through a tyrosyl radical intermediate. DISCUSSION Eosinophils play an essential role in tissue surveillance and host defense mechanisms (1-3). These cells have evolved enzymatic mechanisms to inflict oxidative damage upon invading parasites, pathogens, and cancer cells. The reactive species they form, however, also have the potential to harm host tissues and contribute to inflammatory tissue injury. Microbicidal and cytotoxic oxidants generated by the EPO-H 2 O 2 system are thought to participate in promoting many of these functions (2)(3)(4)(5)(6)(7)(8). However, despite the numerous links between EPO, oxidant production, and tissue injury in eosinophilic inflammatory disorders, structural identification of oxidation products formed by the action of EPO on target proteins is lacking. The present studies identify aromatic nitration reactions as a new category of oxidation reactions mediated by EPO. EPO readily used NO 2 Ϫ , a major end product of ⅐ NO metabolism, as substrate to generate a reactive intermediate that nitrates free and protein-bound tyrosyl residues in high yield. Multiple independent analytical methods confirmed the structure of the major tyrosine oxidation product formed by the EPO-H 2 O 2 -NO 2 Ϫ system as 3-nitrotyrosine: (i) studies employing LC/MS with electrospray ionization detection demonstrated that the retention time and mass spectrum of the oxidation product were identical to that of authentic 3-nitrotyrosine; (ii) multiple distinct derivatives of the major tyrosine oxidation product formed by the EPO-H 2 O 2 -NO 2 Ϫ system demonstrated identical GC retention times and negative ion electron impact mass spectra to that of similarly derivatized authentic 3-nitrotyrosine and co-migrated with synthetically prepared 3-nitro-[ 13 C 6 ]tyrosine (both in native form and following reduction of both to 3-aminotyrosine); (iii) the UV-visible absorbance spectra of the tyrosine oxidation product was identical to that of authentic 3-nitrotyrosine at acidic, neutral, and basic pH; (iv) the retention time and electrochemical properties (i.e. hydrodynamic voltamogram) of the EPO-generated product were identical to those of authentic 3-nitrotyrosine when analyzed by reverse phase HPLC with electrochemical (coulometric array) detection; and finally (v) antibodies raised against 3-nitrotyrosine specifically recognize protein oxidized by the EPO-H 2 O 2 -NO 2 Ϫ system. Collectively, these results unambiguously identify 3-nitrotyrosine as a product formed by action of the EPO-H 2 O 2 -NO 2 Ϫ system on free and protein-bound tyrosine residues.
The present results also provide the first evidence for tyrosyl radical production by EPO. EPO-dependent formation of this diffusible radical species is strongly supported by the structural identification of multiple EPO-generated phenolic addition products of tyrosyl radical (e.g. o,oЈ-dityrosine, isodityrosine, pulcherosine, and trityrosine). Moreover, the mechanism of aromatic nitration reactions mediated by the EPO-H 2 O 2 -NO 2 Ϫ system is consistent with formation of a tyrosyl radical intermediate. Thus, tyrosyl radical-dependent oxidative damage of biological targets represents another potential pathway that EPO may contribute to inflammatory tissue injury.
One of the remarkable features of protein nitration by the EPO-H 2 O 2 -NO 2 Ϫ system is the overall high yield of 3-nitrotyrosine formation. In the presence of plasma levels of halides and levels of NO 2 Ϫ that approximate those found in inflammatory tissues and fluids, 3-nitrotyrosine was a preferred product formed with both free and protein-bound tyrosine residues as targets (e.g. Tables II and III). Moreover, tyrosine nitration was optimal in a physiological pH range (6.0 -7.0) and accounted for between 10 and 20% of the H 2 O 2 consumed during oxidation of proteins by the EPO-H 2 O 2 -NO 2 Ϫ system at neutral pH. Finally, common nucleophilic scavengers such as primary amines, thiols, and thiol ethers only modestly attenuated nitrotyrosine formation; however, physiological levels of glutathione, ascorbate, and SCN Ϫ are anticipated to have a major impact on the extent of nitrotyrosine formation by EPO. Taken together, these results suggest that EPO-dependent nitration of protein tyrosyl residues is likely to occur in vivo, particularly at sites where eosinophilic infiltration and ⅐ NO (and hence NO 2 Ϫ ) production are enhanced and ascorbate, glutathione, and SCN Ϫ levels are limiting. Indeed, recent immunohistochemical studies using antibodies raised against nitrotyrosine demonstrated intense staining over eosinophils and throughout eosinophilrich tissues of individuals with severe asthma (22), a condition where eosinophils are implicated in promoting inflammatory tissue injury and enhanced oxidant stress and ⅐ NO production occur (2)(3)(4)(5)(6)(61)(62)(63)(64). Although the authors interpreted the 3-nitrotyrosine immunostaining as evidence of peroxynitrous acid production (22), the current results suggest that an alternative source may be the EPO-H 2 O 2 -NO 2 Ϫ system of eosinophils. It is interesting to note that the extent of 3-nitrotyrosine formation by EPO is modulated by physiologically relevant levels of SCN Ϫ . Thiocyanate ion is produced following the enzymatic hydrolysis of certain plant glycosides. Thus, eating certain vegetables (e.g. cabbage) results in a transient increase in the concentration of thiocyanate in blood and urine (57). Normal plasma levels of SCN Ϫ are under 70 M (57), but higher levels may be observed following excessive tobacco smoking or prolonged intravenous administration of sodium nitroprusside, a potent vasodilator used in critically ill patients (57,65). Eosinophils perform their biological functions in a large variety of tissues and fluids whose concentration of halides, NO 2 Ϫ , and SCN Ϫ may differ considerably from plasma. Indeed, our kinetic studies (Table I) suggest that at plasma concentrations of substrates (SCN Ϫ , Br Ϫ , NO 2 Ϫ , and Cl Ϫ ), EPO is far from saturated. Thus, the relative contribution of EPO to 3-nitrotyrosine formation in vivo may be tissue-specific, even at sites of inflammation characterized by eosinophil recruitment and activation.
The formation of reactive nitrogen species by direct peroxidasedependent oxidation of NO 2 Ϫ , or through oxidation of NO 2 Ϫ by oxidants such as HOCl, has recently been reported (25-28, 58, 59). Indeed, the ability of MPO secreted from activated neutrophils to catalyze aromatic nitration reactions has suggested that nitrotyrosine formation at sites of inflammation may arise from pathways independent of peroxynitrous acid generation (26). However, whether EPO could catalyze similar reactions given the unusual substrate preferences, distinct prosthetic heme group, and unique physical properties of this highly cationic protein was unclear. The results of the present studies demonstrate that aromatic nitration reactions are indeed a preferred activity of the enzyme. Moreover, the nitrating capacity of EPO was shown to exceed that of the related leukocyte-derived peroxidase, MPO, by at least 4-fold at physiological concentrations of halides and every concentration of NO 2 Ϫ examined. Finally, it should be noted that O 2 . and H 2 O 2 production by activated eosinophils is severalfold greater than that of an equal number of comparably stimulated neutrophils (9,10) and that the total content of EPO in eosinophils is reported to be 2-4-fold higher than that of MPO in neutrophils (e.g. 30 -33 pmol of MPO per 1 ϫ 10 6 neutrophils versus 70 -135 pmol of EPO per 1 ϫ 10 6 eosinophils) (16,66,67). Thus, EPOdependent formation of reactive nitrogen intermediates is likely to occur in vivo, particularly during inflammatory disorders characterized by the presence of eosinophils.
Another interesting distinction between EPO-and MPO-dependent nitration reactions arises from their differing halide specificity. Although MPO generates chlorinating oxidants at physiological concentrations of halides (46, 68 -70), and HOCl is reported to react with NO 2 Ϫ to form a nitrating intermediate (58), we found no evidence for a parallel reaction between NO 2 Ϫ and HOBr, the primary halogenating intermediate formed by EPO at plasma levels of halides. Moreover, although EPO-dependent nitration of tyrosine was optimal in the presence of both NO 2 Ϫ and plasma levels of Cl Ϫ (e.g. see Tables II and III), the addition of methionine, a potent scavenger of halogenating intermediates (46,71,72), failed to attenuate nitrotyrosine formation by EPO. The mechanism of the modest but reproducible increase in both free and protein-bound 3-nitrotyrosine formation observed in the presence versus absence of Cl Ϫ is unclear. However, the inability of methionine to attenuate tyrosine nitration by EPO in the presence of either Br Ϫ or Cl Ϫ suggests that indirect oxidation of NO 2 Ϫ by an EPO-generated hypohalous acid does not contribute significantly to tyrosine nitration.
One interesting question is the chemical nature of the reactive nitrogen species formed by EPO-catalyzed oxidation of NO 2 Ϫ . Peroxidases catalyze both one-and two-electron oxidation reactions, and both the one-electron (i.e. nitrogen dioxide) and two-electron (e.g. peroxynitrous acid) oxidation products of NO 2 Ϫ have been suggested as potential intermediates formed by MPO (25, 28). Both intermediates can form nitrotyrosine, as well as abstract a phenoxyl hydrogen atom to form tyrosyl radical intermediates. Future studies aimed at elaborating the oxidant(s) formed and the mechanisms of free and proteinbound tyrosine nitration mediated by peroxidase-dependent oxidation of NO 2 Ϫ are warranted. Eosinophils are typically rare leukocytes with a limited circulating life span (t1 ⁄2 ϳ 2 h) before they take residence in tissues (1). However, they do play a prominent role in certain forms of inflammation (e.g. allergic), where their numbers in tissues can greatly exceed those of other leukocytes (1)(2)(3)(4)(5)(6). Indeed, some of the most devastating inflammatory disorders in humans are associated with intense eosinophilic infiltration (1-6, 73, 74). Identifying the mechanisms and products of EPO-dependent oxidative damage is a critical step toward development of targeted interventions designed to interrupt oxidative tissue injury in eosinophilic inflammatory disorders. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2000-10-31T00:00:00.000 | 96664352 | {
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} | pes2o/s2orc | A New Pathway to 3-Hetaryl-2-oxo2H-chromenes: On the Proposed Mechanisms for the Reaction of 3-Carbamoyl-2iminochromenes with Dinucleophiles. Molecules 2000
The present account summarizes the author's studies to elucidate the mechanisms of the recently reported rearrangements resulting from inter- and/or intramolecular reactions of 2-imino-2H-chromene-3-carboxamides with different dinucleophiles.
Introduction
The coumarin (2H-chromen-2-one) moiety is often found in natural products [1].In view of the ubiquity of this fragment in a variety of biologically active compounds, the synthesis of various 2Hchromen-2-one analogs is important in gauging their potential as a source of chemotherapeutics [2].As part of our investigations on the reactivity of 3-carbamoyl-2-imino-2H-chromenes [3], we recently introduced a new method for synthesis of 3-hetaryl-2-oxo-2H-chromenes [4].This method was based on the rearrangements of 2-imino-2H-chromene-3-carboxamides into 3-hetaryl-2-oxo-2H-chromenes under the action of dinucleophiles.In this account, results of our studies on clarification of the mechanism of the above-mentioned rearrangements are summarized and exemplified by utilizing anthranilic acid, its derivatives, and arylhydrazides as N-nucleophiles.In order to elucidate the mechanisms of the applied rearrangements, a model system approach based on isolation of stable reaction intermediates or their structural analogs was used.
A method for synthesis of 2-(arylimino)chromenes 19 was recently introduced in our laboratory and it was shown that a variety of 2-(aryl-or alkylimino)-substituted 2H-chromen-2-ones of type 19 could be prepared [3].This method is based on aminolysis of cyclic imido esters and is similar to the reaction of simple imidates with amines [16].This type of reactions should also be similar to the acid hydrolysis of 2-imino-2H-chromenes to 2H-chromen-2-ones which proceeds through the formation of the corresponding benzopyrylium salts [17,18].
Conclusion
The results obtained in the study of the rearrangements of 2-imino-2H-chromene-3-carboxamides with different N-nucleophiles clearly indicate that the reactions studied follow the mechanisms described in Schemes 1 and 5. Finally, this work opened a new avenue for the synthesis of a variety of new 3-hetaryl substituted 2-oxo-2H-chromene derivatives.
General
Melting points (°C) were measured on a Büchi melting point apparatus and are uncorrected.Thin layer chromatography (TLC) was performed on aluminum sheets precoated with silica gel (Merck, Kieselgel 60 F-254). 1 H-NMR spectra were recorded on Bruker WP-100 SY, Bruker DPX-250, Bruker AMX-400 or Varian WXR-400 spectrometers in DMSO-d 6 or DMSO-d 6 -CDCl 3 using TMS as an internal standard (chemical shifts in δ ppm).Mass spectra (MS) were obtained with Finnigan MAT-4615B spectrometer at an ionization potential of 70 eV.Combustion analyses of all compounds synthesized gave satisfactory microanalytical data.Infrared spectra (IR) were recorded in KBr pellets on Nicolet Protege 460 FT-IR or an IBM 486 computer-controlled Specord M-80 spectrometers.
Method C: A solution of 30 (309 mg, 1.0 mmol) in glacial (99.8%) acetic acid (5 mL) was refluxed for 30 min.The mixture was cooled, the yellow precipitate was filtered off, washed with water and recrystallized from DMF/BuOH to afford 201 mg (70%) of 7a.According to 1 H-NMR and IR spectral data as well as the melting points, the products obtained by Methods A, B and C are identical.
Amidines 16a,b were prepared from carbamoyliminochromenes 1a,b and 1-aminopyridone 15 [27] using the reaction conditions described in Method A for the synthesis of 12.
General procedures
Method A: A mixture of 1a or 1b (1.5 mmol) and anthranilic acid 2 (275 mg, 2 mmol) in aqueous (80%) acetic acid (10 mL) was refluxed for 2 h.After the reaction finished, the mixture was cooled and the precipitate was filtered off, washed with water and cold propan-2-ol (2 x 5 mL).The products obtained were recrystallized from an appropriate solvent.
Method B: To a well stirred solution of ethyl 2'-carboxymalonanilate (37) [23] (4 mmol) in ethanol (10 mL) was added an equivalent amount of salicylaldehydes 9a or 9b and a few drops of piperidine as a catalyst.The reaction mixture was stirred at room temperature for ca. 1 day and then poured into water.The products precipitated were filtered off and recrystallized from an appropriate solvent. | v3-fos-license |
2018-04-03T04:59:15.669Z | 2016-03-24T00:00:00.000 | 205356841 | {
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} | pes2o/s2orc | Importance of a Potential Protein Kinase A Phosphorylation Site of Na+,K+-ATPase and Its Interaction Network for Na+ Binding*
The molecular mechanism underlying PKA-mediated regulation of Na+,K+-ATPase was explored in mutagenesis studies of the potential PKA site at Ser-938 and surrounding charged residues. The phosphomimetic mutations S938D/E interfered with Na+ binding from the intracellular side of the membrane, whereas Na+ binding from the extracellular side was unaffected. The reduction of Na+ affinity is within the range expected for physiological regulation of the intracellular Na+ concentration, thus supporting the hypothesis that PKA-mediated phosphorylation of Ser-938 regulates Na+,K+-ATPase activity in vivo. Ser-938 is located in the intracellular loop between transmembrane segments M8 and M9. An extended bonding network connects this loop with M10, the C terminus, and the Na+ binding region. Charged residues Asp-997, Glu-998, Arg-1000, and Lys-1001 in M10, participating in this bonding network, are crucial to Na+ interaction. Replacement of Arg-1005, also located in the vicinity of Ser-938, with alanine, lysine, methionine, or serine resulted in wild type-like Na+ and K+ affinities and catalytic turnover rate. However, when combined with the phosphomimetic mutation S938E only lysine substitution of Arg-1005 was compatible with Na+,K+-ATPase function, and the Na+ affinity of this double mutant was reduced even more than in single mutant S938E. This result indicates that the positive side chain of Arg-1005 or the lysine substituent plays a mechanistic role as interaction partner of phosphorylated Ser-938, transducing the phosphorylation signal into a reduced affinity of Na+ site III. Electrostatic interaction of Glu-998 is of minor importance for the reduction of Na+ affinity by phosphomimetic S938E as revealed by combining S938E with E998A.
M8 and M9 (L8-9) at the interphase between the membrane and the cytoplasm (Fig. 1C) in the vicinity of the C terminus. Molecular dynamics computational studies based on the K ϩ -bound E 2 structure have suggested that the introduction of the bulky and negatively charged phosphate group at Ser-938 will affect the structure of the C terminus and promote hydration of a cavity in the transmembrane region that connects the cytoplasm and the ion-binding sites (17), which might lead to defective interaction with Na ϩ . This idea was examined by electrophysiological measurements of the apparent affinity for Na ϩ in a mutant with glutamate replacement of Ser-938, which due to the negative charge and considerable size of the glutamate commonly is considered to mimic phosphorylation. However, no significant effect of the phosphomimetic glutamate substitution was revealed in the electrophysiological study (17). Therefore the mechanism underlying the putative effect of Ser-938 phosphorylation on Na ϩ ,K ϩ -ATPase function has remained elusive. A limitation of the electrophysiological study is that only the affinity for Na ϩ binding from the extracellular side was examined, thus leaving the effect of the phosphomimetic mutation on the affinity for activating Na ϩ binding from the intracellular side uncharacterized. In fact, it is the interac-tion with intracellular Na ϩ and extracellular K ϩ , and not extracellular Na ϩ , that fine-tunes the Na ϩ ,K ϩ -ATPase activity in vivo.
The present study was undertaken to provide a deeper understanding of the function and mechanism of the potential Ser-938 PKA site on the basis of the recently solved structure of Na ϩ ,K ϩ -ATPase in the E 1 form with bound Na ϩ (8), which is the most relevant structure in relation to possible effects on Na ϩ affinity. Fig. 1C shows the location in this structure of Ser-938 and surrounding charged residues that might be affected by the introduction of the negatively charged phosphate group. Closest to the side chain hydroxyl group of Ser-938 are the Glu-998 carboxylate group and a guanidino group nitrogen of Arg-1005 of transmembrane segment M10. A bit further away are Asp-997, Arg-1000, and Lys-1001. We have studied the functional consequences (including Na ϩ affinities on both sides of the membrane) of alanine scanning mutagenesis of these residues as well as other mutations to Arg-1005 and Ser-938, including the phosphomimetic S938D and S938E mutations. Furthermore, we have combined S938E with mutations to Glu-998 or Arg-1005 to investigate the importance of electrostatic interaction between the phosphate group on the FIGURE 1. Reaction cycle and structure of Na ؉ ,K ؉ -ATPase. A, simplified scheme of the Na ϩ ,K ϩ -ATPase reaction cycle (reactions 1-6). E 1 and E 2 denote the two major conformational states of the enzyme. P represents phosphorylation of the conserved aspartate in the catalytic site of the P-domain. Occluded ions are shown in parentheses, and boxed ATP indicates low affinity binding of ATP. B, overall structure of the Na ϩ -bound E 1 form of Na ϩ ,K ϩ -ATPase with the L8-9 loop containing the potential PKA site highlighted in yellow. Details of this region (framed by a broken line) are shown in C. The cytoplasmic A-, P-, and N-domains of the ␣-subunit as well as the and ␥-subunits are indicated. C, close-up showing the relations of the potential PKA site at Ser-938 in L8-9 (yellow), the C terminus (light blue), and the ion-binding transmembrane segments M4, M5, M6, and M8. The view is from the cytoplasmic side of the membrane. The residues studied by mutagenesis are depicted as sticks (Ser-938, Asp-997, Glu-998, Arg-1000, Lys-1001, and Arg-1005). In addition, Thr-1016 and the C-terminal tyrosine (Tyr-1018) are shown as sticks as are the interaction partners of the C terminus, Arg-935 in L8-9 and Lys-768 in M5. The three bound Na ϩ ions are depicted as purple spheres labeled I, II, and III according to site nomenclature. B and C were prepared from the Protein Data Bank structure with code 3WGV (8) using PyMOL.
phosphorylated Ser-938 (Ser(P)-938) and the glutamate and arginine, respectively. Our results demonstrate that the phosphomimetic mutation reduces the affinity for cytoplasmic Na ϩ significantly as do mutations to some of the charged residues in the vicinity. The positive charge of Arg-1005 is shown to be essential for accommodation of the negative charge of S938E, indicating a critical role of the arginine-phosphate electrostatic interaction in Ser(P)-938. This arginine is fully conserved among all Na ϩ ,K ϩ -ATPases and H ϩ ,K ϩ -ATPases. Hence, the electrostatic interaction may well be part of a general regulatory mechanism that also pertains to the closely related H ϩ ,K ϩ -ATPases.
Experimental Procedures
Mutagenesis and Expression-The QuikChange site-directed mutagenesis procedure was used to introduce the desired base substitutions into the cDNA encoding the ouabain-resistant rat ␣1 isoform of Na ϩ ,K ϩ -ATPase. The resultant cDNA constructs were transfected into COS-1 cells by the calcium phosphate precipitation method (18). Because the endogenous Na ϩ ,K ϩ -ATPase of the COS-1 cells is ouabain-sensitive, stable cell lines expressing the recombinant ouabain-resistant wild type or mutants could be isolated by ouabain selection (19 -21). To confirm the stable integration of the cDNA carrying the intended single or double mutations, the genomic DNA was isolated, and the integrated cDNA was amplified by PCR using primers that span exon-exon boundaries as described previously (22). In a few cases where the expressed exogenous mutant Na ϩ ,K ϩ -ATPase did not support cell growth in the presence of ouabain, transient co-expression with siRNA to knock down the endogenous COS-1 Na ϩ ,K ϩ -ATPase was carried out as described previously (23,24).
Plasma Membrane Vesicles-Plasma membrane vesicles, harboring the recombinant wild type or mutant Na ϩ ,K ϩ -ATPase, were isolated by differential centrifugation (20). Prior to functional analysis, the vesicles were permeabilized by treatment with sodium deoxycholate or alamethicin to make both sides of the membrane accessible to substrates and inhibitors.
ATPase Activity Assays-ATPase activity measurements were carried out at 37°C for 15 min in 30 mM histidine (pH 7.4), 3 mM MgCl 2 , 1 mM EGTA, and various concentrations of NaCl, KCl, ATP, and ouabain according to a modification (20 -22) of the method originally described by Baginski and co-workers (25). For determination of the maximal catalytic turnover rate, the respective NaCl, KCl, and ATP concentrations were 130, 20, and 3 mM, and the specific activity was related to the active site concentration obtained by stoichiometric phosphorylation from [␥-32 P]ATP at 0°C in the presence of 150 mM NaCl and oligomycin (see below) (26). In studies of K ϩ dependence of Na ϩ ,K ϩ -ATPase activity, 40 mM NaCl, 3 mM ATP, and 0 -30 mM KCl were included in the reaction buffer, whereas 130 mM NaCl, 20 mM KCl, and 0.01-3 mM ATP were used in studies of ATP dependence. In measurements of so-called "Na ϩ -ATPase" activity, KCl was omitted from the reaction mixture, which included 3 mM ATP and 0 -1000 mM NaCl. All measurements of ATPase activity were carried out in the presence of 10 M ouabain, which specifically inhibits the endogenous ouabainsensitive COS-1 Na ϩ ,K ϩ -ATPase. Background ATPase mea-surements were performed in the presence of 10 mM ouabain, which inhibits all Na ϩ ,K ϩ -ATPase activity, and subtracted from that measured at 10 M ouabain.
Phosphorylation and Dephosphorylation Assays-The autophosphorylation of the P-type ATPase signature aspartate in the P-domain and dephosphorylation of wild type and mutants were studied at 0°C using [␥-32 P]ATP. To examine the Na ϩ dependence of phosphorylation, the enzyme was incubated for 15 s in 20 mM Tris (pH 7.5), 3 mM MgCl 2 , 1 mM EGTA, 2 M [␥-32 P]ATP, 20 g/ml oligomycin (to block dephosphorylation), and various concentrations of NaCl as described previously (22,24,26). In this assay, the ionic strength was kept constant at 150 mM by addition of various concentrations of N-methyl-D-glucamine.
The distribution of the phosphoenzyme between the ADPsensitive E 1 P and ADP-insensitive/K ϩ -sensitive E 2 P was examined following phosphorylation for 15 s in 20 mM Tris (pH 7.5), 150 mM NaCl, 3 mM MgCl 2 , 1 mM EGTA, and 2 M [␥-32 P]ATP. The time course of dephosphorylation was followed by adding 2.5 mM ADP and 1 mM unlabeled ATP (9,22). Under these conditions, phosphoenzyme present as the E 1 P form will react rapidly with ADP in the backward direction (Fig. 1A, reaction 2), whereas that present as the E 2 P form will dephosphorylate slowly in the forward direction by hydrolysis (Fig. 1A, reaction 5), thus giving rise to biphasic curves for which the amplitudes of the fast and slow components represent E 1 P and E 2 P, respectively.
In all phosphorylation and dephosphorylation assays, 10 M ouabain was included to inhibit the endogenous COS-1 Na ϩ ,K ϩ -ATPase. The background level of phosphorylation was determined by phosphorylation in the presence of 50 mM KCl and absence of NaCl.
Phosphorylation of Na ϩ ,K ϩ -ATPase by PKA-Plasma membrane vesicles containing stably expressed wild type or mutant were preincubated for 30 min at 30°C in the presence of 20 mM Tris-HCl (pH 7.5), 1 mM EGTA, and 10 mM MgCl 2 followed by addition of 100 ng of PKA (bovine PKA catalytic subunit from Sigma-Aldrich), 2% (v/v) Triton X-100, and 1 mM dithiothreitol (DTT). The phosphorylation reaction was started by addition of 50 M [␥-32 P]ATP and allowed to proceed for 15 min at 30°C in a total volume of 15 l. The reaction was stopped by addition of 5 l of 4ϫ sample buffer (200 mM Tris-HCl (pH 6.8), 8% SDS, 32% glycerol, 400 mM DTT, and bromphenol blue), and the reaction products were separated by gel electrophoresis using 4 -15% polyacrylamide gels (Bio-Rad). Purified pig kidney Na ϩ ,K ϩ -ATPase (27) was used as positive control to indicate the migration position of PKA-phosphorylated Na ϩ ,K ϩ -ATPase. In this case, the Triton X-100 concentration present during the phosphorylation reaction was 0.2%.
Data Analysis-Data were processed and analyzed using the SigmaPlot program (SPSS, Inc.) for non-linear regression using the complete set of normalized data as described in detail previously (21). Ligand concentration dependences were fitted by use of the appropriate form of the Hill equation. For Na ϩ dependence of phosphorylation, the following equation was used.
Importance of Ser-938 and Its Network for Na ؉ Binding
EP is the phosphorylation level, EP max is the maximum level of phosphorylation, K 0.5 is the Na ϩ concentration giving halfmaximum activation ("apparent affinity"), and n is the Hill coefficient (ranging between 1.0 and 2.0 in the present study). For ATP dependence of ATPase activity, the following equation was used.
V is the ATPase activity, V max is the extrapolated activity corresponding to infinite ATP concentration, K 0.5 is the ATP concentration giving half-maximum activation (apparent affinity), and n is the Hill coefficient (ranging between 0.9 and 1.2 in the present study). For K ϩ dependence of ATPase activity, a two-component Hill equation was used.
where the second term representing inhibition was omitted for wild type and mutants not showing inhibition. V is the ATPase activity, V 0 is the activity in the absence of K ϩ ("Na ϩ -ATPase activity"), V max is the extrapolated activity corresponding to infinite K ϩ concentration in the absence of inhibition, K 0.5 is the K ϩ concentration giving half-maximum activation (apparent affinity corresponding to the rising phase), K 2 0.5 is the K ϩ concentration giving half-maximum inhibition (apparent affinity for inhibition), n and n2 are the corresponding Hill coefficients. Time courses of dephosphorylation were fitted using a double exponential decay function.
EP is the total amount of phosphoenzyme, E 1 P and E 2 P are the ADP-sensitive and -insensitive fractions, and k1 and k2 are the respective decay constants. The results of the non-linear Functional analysis of mutants -Fold changes relative to WT are shown in red parentheses. The number of independent experiments n/total number of data points and mean Ϯ S.E. are indicated. ϩϩ, pronounced inhibition: ϩ, partial inhibition; and Ϫ, no inhibition of ATPase activity by high K ϩ or Na ϩ concentrations. regression analysis (K 0.5 values and E 2 P fraction) ϮS.E. from the regression are reported in Table 1.
Results
Ser-938 Is Important for Binding of Intracellular Na ϩ but Not Extracellular Na ϩ -Ser-938 of the potential PKA site was replaced by either aspartate or glutamate to mimic phosphorylation by PKA. To study the importance of side chain size and charge, the serine was furthermore replaced by alanine and arginine. All four mutants S938A/D/E/R could be stably expressed in COS-1 cells at levels similar to that of the wild type enzyme under ouabain selection pressure, showing that the ␣and -subunits are correctly assembled and transported to the plasma membrane where they are capable of transporting Na ϩ and K ϩ across the membrane at rates compatible with cell viability. The maximal catalytic turnover rate, calculated as the ratio between the specific Na ϩ ,K ϩ -ATPase activity measured under optimal conditions and the active site concentration obtained by phosphorylation (26), was wild type-like for mutant S938A and slightly increased (1.2-and 1.3-fold) for S938D, S938E, and S938R (Table 1).
To obtain information about the Na ϩ binding properties of the sites on the E 1 form that in the intact cell face the intracellular side (Fig. 1A, reaction 1), advantage was taken of the dependence of activation of phosphorylation from ATP (Fig. 1A, reaction 2) on the binding of all three Na ϩ ions at these sites. The measurements of the Na ϩ dependence of phosphorylation were carried out in the presence of oligomycin to prevent dephosphorylation and in the absence of K ϩ to exclude competition from K ϩ . Whereas mutant S938A displayed a slight (1.7-fold) decrease of the apparent affinity for Na ϩ relative to wild type, the effects of the substitutions S938D, S938E, and S938R were more pronounced, resulting in 2.3-, 3.1-, and 3.8-fold reductions, respectively, of the apparent affinity for Na ϩ ( Fig. 2A and Table 1). In principle, such a reduction of apparent Na ϩ affinity can be due to either a reduction of the intrinsic affinity of the E 1 form for intracellular Na ϩ or a shift of the E 1 -E 2 conformational equilibrium away from the Na ϩ -binding E 1 form (Fig. 1A, reaction 6). To distinguish between these two possibilities, we determined the ATP concentration dependence of Na ϩ ,K ϩ -ATPase activity, which can be used as a measure of the distribution of the enzyme between the E 1 and E 2 conformations because E 1 binds ATP with high affinity, whereas E 2 only binds ATP with low affinity and without being phosphorylated (1, 28). As seen in Fig. 2B, the mutations S938D, S938E, and S938R caused a left shift of the ATP titration curves, which is indicative of an increased apparent ATP affinity relative to wild type and hence a shift of the E 1 -E 2 equilibrium in favor of E 1 (Fig. 2B and Table 1). Mutant S938A, in contrast, displayed a wild typelike apparent affinity for ATP ( Fig. 2B and Table 1). These latter findings allow us to conclude that the reduced apparent Na ϩ affinities observed for mutants S938D, S938E, and S938R are caused by mutation-induced disturbance of the binding of Na ϩ to the E 1 form rather than being caused indirectly by a shift of the conformational equilibrium in favor of E 2 .
Experiments with sided membrane vesicles have shown that besides binding to the intracellularly facing Na ϩ sites, activating phosphorylation from ATP, Na ϩ also binds to extracellu-larly facing activating and inhibitory sites (29 -31). Having observed that the phosphomimetic mutations S938E and S938D as well as mutation S938R disturb the interaction with Na ϩ at the intracellularly facing sites of the E 1 form, we next investigated whether these mutations also interfere with the binding of Na ϩ to the E 2 P form of the enzyme at the sites that in the intact cell face the extracellular side. To this end, we determined the Na ϩ concentration dependence of the ATPase activity in the absence of K ϩ . When K ϩ is omitted from the reaction medium, Na ϩ is able to bind to E 2 P, activating dephosphorylation, although much less efficiently than K ϩ (Fig. 1A, reactions 4 and 5) (1). Consequently, the catalytic turnover rate measured in the mere presence of Na ϩ (Na ϩ -ATPase activity) is only a small fraction of that measured with K ϩ present (less than 5% for the wild type). For the wild type enzyme, Na ϩ concentrations in the 50 -400 mM range activate dephosphorylation, whereas high Na ϩ concentrations are inhibitory (Fig. 2C) due to the binding of Na ϩ to the E 2 P form driving the E 1 P 3 E 2 P transition backward (Fig. 1A, reaction 3) (30 -32). Similar results with both an activating and an inhibitory phase were obtained for the S938A/D/E/R mutants ( Fig. 2C and Table 1). For the S938A/D/E mutants, the apparent affinity for Na ϩ inhibition was similar to that of the wild type or slightly higher. Thus, the extracellularly facing Na ϩ sites on E 2 P appear to be intact in all S938 mutants, indicating that this serine is not essential to binding of extracellular Na ϩ and that the phosphomimetic mutations S938D and S938E only interfere with Na ϩ binding from the intracellular side of the membrane and not with Na ϩ binding from the extracellular side. Mutant S938R displayed a slight right shift of the inhibitory part of the curve relative to wild type, indicating a minor reduction of the affinity for binding of extracellular Na ϩ .
Ser-938 Is Less Important for Binding of K ϩ than for Binding of Na ϩ -By binding at sites on E 2 P that in the intact cell face the extracellular side, K ϩ triggers dephosphorylation, thereby stimulating the ATPase activity (Fig. 1A, reactions 4 and 5). Compared with the significant effects on the binding of Na ϩ from the intracellular side, only relatively small changes in the apparent K ϩ affinities for activation were detected ( Fig. 2D and Table 1): the S938D, S938E, and S938R mutants exhibited 1.6 -1.8-fold reductions of the K ϩ affinity relative to wild type, whereas S938A displayed wild type-like K ϩ affinity. Thus, the substitutions S938D, S938E, and S938R seem primarily to affect the interaction with Na ϩ binding from the intracellular side.
There are in principle two possible explanations of the above described slight reductions of the apparent K ϩ affinity observed for the Ser-938 mutants: 1) reduced intrinsic affinity of E 2 P for K ϩ or 2) a conformational shift away from the K ϩ -binding E 2 or E 2 P form in favor of E 1 and E 1 P, respectively. As described above, ATP titrations of Na ϩ ,K ϩ -ATPase activity (Fig. 2B) disclosed a shift of the E 2 -E 1 equilibrium in favor of E 1 . This shift can account for the slightly reduced K ϩ affinities of mutants S938D, S938E, and S938R; hence, there is no need to assume any reduction of the intrinsic K ϩ affinity of E 2 P. We furthermore investigated the distribution of the phosphoenzyme between the intermediates E 1 P and E 2 P by taking advantage of the ability of E 1 P to dephosphorylate upon addition of ADP (1,9,33). None of the Ser-938 mutants differed significantly from the wild type with respect to the fraction of the phosphoenzyme that was ADP-insensitive (compare 52-59 with 62% for the wild type; Fig. 2E and Table 1), meaning that the slightly reduced K ϩ affinities of S938D, S938E, and S938R cannot be attributed to a displacement of the E 1 P-E 2 P equilibrium toward E 1 P.
Charged Residues Asp-997, Glu-998, Arg-1000, and Lys-1001 in the Vicinity of Ser-938 Are Crucial for Na ϩ Binding-As shown in Fig. 1C, transmembrane segment M10 contains two negatively charged residues, Asp-997 and Glu-998, and three positively charged residues, Arg-1000, Lys-1001, and Arg-1005, relatively close to Ser-938. These residues might be electrostatically repulsed and attracted, respectively, when a negatively charged phosphate group is attached at Ser-938. To investigate the function of Asp-997, Glu-998, Arg-1000, Lys-1001, and Arg-1005, alanine scanning mutagenesis of these residues was performed. In addition, Asp-997 and Glu-998 were simultaneously replaced by alanine. The results obtained with R1005A encouraged us to study also the mutants R1005K/M/S. All the above mentioned single mutations were compatible with cell viability in COS-1 cells in the presence of ouabain, whereas the double mutant D997A/E998A did not sustain cell growth. Functional analysis of the maximal turnover rates revealed wild type-like catalytic rates for D997A, E998A, R1000A, K1001A, and R1005A/K/ M/S (0.8 -1.3-fold changes relative to wild type) ( Table 1).
Investigation of the Na ϩ dependences of activation of phosphorylation from ATP disclosed marked effects of mutations D997A, E998A, R1000A, and K1001A on the binding of Na ϩ , amounting to 4.4-, 8.8-, 15-, and 7.6-fold reductions of the Na ϩ affinity, respectively, compared with wild type (Fig. 3A and Table 1. B, ATP dependence of Na ϩ ,K ϩ -ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K 0.5 values are listed in Table 1. C, Na ϩ dependence of Na ϩ -ATPase activity. Inhibition of Na ϩ -ATPase activity by high Na ϩ concentrations is indicated semiquantitatively in Table 1. D, K ϩ dependence of Na ϩ ,K ϩ -ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K 0.5 values are listed in Table 1. E, distribution of the phosphoenzyme between E 1 P and E 2 P at 150 mM NaCl. Each line shows the best fit of a biexponential decay function. The initial amounts of E 2 P, which correspond to the amplitude of the slow phase, are listed in Table 1. A-E, experimental conditions and equations used for data fitting are described under "Experimental Procedures." Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean Ϯ S.E. Dotted lines reproduce the wild type for direct comparison in the same panel. Table 1). These effects are not caused by a conformational shift away from the Na ϩ -binding E 1 form as the apparent affinity for ATP was in fact increased, indicating that the E 1 -E 2 equilibrium is shifted in favor of E 1 (Fig. 3B and Table 1). The effect is rather specific for Na ϩ binding, as D997A, E998A, R1000A, and K1001A all displayed a close to wild type-like apparent affinity for K ϩ activation of Na ϩ ,K ϩ -ATPase activity (1.1-1.5-fold reductions of the K ϩ affinity relative to wild type; Fig. 3C and Table 1). As regards Arg-1005, none of the investigated mutations replacing Arg-1005 with alanine, lysine, methionine, or serine affected the Na ϩ or K ϩ binding properties substantially (Figs. 3 and 4A and Table 1).
Unlike the wild type, the mutants D997A, E998A, and R1000A were inhibited at high K ϩ concentrations; this was most distinct for D997A and R1000A (Fig. 3C). This inhibition may be explained by the low intrinsic Na ϩ affinity of these mutants (see Table 1), allowing K ϩ to bind in competition with Na ϩ at a site(s) on the E 1 form, thus leading to displacement of the E 1 -E 2 equilibrium toward E 2 (34). However, the inhibition is not strictly correlated with the reduction of Na ϩ affinity (e.g. the D997A mutant shows more K ϩ inhibition than E998A, whereas the order is reversed for Na ϩ affinity). Therefore, the Na ϩ selectivity of site III might be affected in these mutants (see Discussion).
Mutations D997A, E998A, and R1000A also had an impact on binding of extracellular Na ϩ as seen from the Na ϩ -ATPase measurements in Fig. 4A. In contrast with the wild type enzyme, which is completely inhibited at a Na ϩ concentration of 1 M, these mutants were not inhibited at all (R1000A) or only partly inhibited (D997A and E998A) at high Na ϩ concentrations ( Fig. 4A and Table 1), indicating that the extracellularly facing Na ϩ sites on E 2 P are less efficient in binding Na ϩ and Table 1. B, ATP dependence of Na ϩ ,K ϩ -ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K 0.5 values are listed in Table 1. C, K ϩ dependence of Na ϩ ,K ϩ -ATPase activity. Each line shows the best fit of the Hill equation or, for the mutants showing inhibition at high K ϩ concentrations, a two-component Hill equation with the inhibition represented by a negative term. K 0.5 values for the rising parts of the curves are listed in Table 1. A-C, experimental conditions and equations used for data fitting are described under "Experimental Procedures." Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean Ϯ S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.
reversing the E 1 P 3 E 2 P transition and thereby impeding ATPase activity (Fig. 1A, reaction 3). Conversely, K1001A and the Arg-1005 mutants displayed wild type-like behavior with respect to binding of extracellular Na ϩ . Based on these findings, it may be concluded that Asp-997, Glu-998, and Arg-1000, the latter in particular, are important for the binding of Na ϩ from both sides of the membrane, whereas Lys-1001 only is important for Na ϩ binding from the intracellular side, and Arg-1005 is not, per se, crucial for cation interaction.
These mutations had limited influence on the distribution of the phosphoenzyme intermediates between E 1 P and E 2 P; the largest deviation was seen for R1000A (83% E 2 P versus 62% in wild type). K1001A and the various Arg-1005 mutants showed slightly lower accumulation of E 2 P than the wild type (33-52%), thus indicating slowing of the E 1 P to E 2 P conversion ( Fig. 4B and Table 1). The increased steady-state level of E 2 P seen for R1000A is likely the consequence of the inability of this mutant to react with Na ϩ at the extracellularly facing sites, which would have driven the E 1 P-E 2 P equilibrium in the direction of E 1 P.
The Phosphomimetic Mutation S938E Mediates Its Effect
Through Interaction with Arg-1005-To examine whether the phosphorylated side chain of Ser-938 senses the potential interaction partners Arg-1005 and Glu-998, we made several double mutants where the phosphomimetic mutation S938E was combined with either of the single mutations R1005A/K/M/S and E998A (see Table 1 for an overview). Of the four mutants combining S938E with substitutions of Arg-1005, only S938E/ R1005K was capable of sustaining cell viability in the presence of ouabain (Table 1). Hence, mutants S938E/R1005A, S938E/ R1005M, and S938E/R1005S are unable to transport Na ϩ and K ϩ , or the transport rate is below a critical level (5-10% of wild type activity seems to be required to sustain cell viability based on previous experience of our laboratory). Expression of these mutants was then carried out by transient co-transfection with siRNA knocking down the endogenous Na ϩ ,K ϩ -ATPase in the COS-1 cells, a technique allowing analysis of the partial reaction steps in the enzyme cycle despite severe reduction of transport activity that we previously used for other transportdeficient mutants (23,24). Hence, the ability to form a phos- . Several charged residues in M10 near Ser-938, but not Arg-1005, are important for binding of Na ؉ from the extracellular side of the membrane. A, Na ϩ dependence of Na ϩ -ATPase activity. Inhibition of Na ϩ -ATPase activity by high Na ϩ concentrations is indicated semiquantitatively in Table 1. B, distribution of the phosphoenzyme between E 1 P and E 2 P at 150 mM NaCl. Each line shows the best fit of a biexponential decay function. The initial amounts of E 2 P, which correspond to the amplitude of the slow phase, are listed in Table 1. A and B, experimental conditions and equations used for data fitting are described under "Experimental Procedures." Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean Ϯ S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.
phorylated intermediate from ATP at the conserved aspartate in the catalytic site was examined (Fig. 1A, reaction 2). As seen in Fig. 5A, none of the transiently expressed mutants S938E/ R1005A, S938E/R1005M, and S938E/R1005S could be phosphorylated in the presence of oligomycin and a Na ϩ concentration of 100 or 300 mM with the latter being more than 20-fold higher than required for saturation of the wild type enzyme. This finding is remarkable in the context of the more or less wild type-like behavior of the mutants R1005A, R1005M, and R1005S with single substitutions.
The viable double mutant S938E/R1005K exhibited a maximal catalytic turnover rate only slightly decreased compared with that of the wild type (7313 versus 8474 min Ϫ1 ; Table 1), whereas a substantial disturbance of Na ϩ binding at the sites on E 1 was found, amounting to as much as a 10-fold reduction of the apparent affinity for Na ϩ activation of phosphorylation ( Fig. 5B and Table 1). This effect is particularly noteworthy because the single mutation S938E reduced the Na ϩ affinity only 3-fold, and R1005K was without effect, suggesting that the presence of the lysine reinforced the effect of the phosphomimetic S938E mutation. Again the effects on apparent Na ϩ affinity are not caused by a conformational shift away from the Na ϩbinding E 1 form as the apparent affinity for ATP was increased ( Fig. 5C and Table 1). These findings support the hypothesis FIGURE 5. The phosphomimetic mutation S938E mediates its effect through interaction with Arg-1005. A, representative phosphorimaging autoradiograph of transiently expressed double mutants. The double mutants S938E/R1005A, S938E/R1005M, and S938E/R1005S, which could not sustain cell viability in the presence of ouabain, were transiently expressed in the presence of siRNA-targeted knockdown of the endogenous COS-1 Na ϩ ,K ϩ -ATPase. The autoradiograph shows 32 P incorporation from [␥-32 P]ATP at the catalytic site aspartate in the P-domain following separation by SDS-PAGE of enzyme phosphorylated in the presence of the Na ϩ concentrations indicated in mM. It can be seen that all the double mutants tested were phosphorylation-inactive. B, Na ϩ dependence of phosphorylation from [␥-32 P]ATP. Each line shows the best fit of the Hill equation, and the extracted K 0.5 values are listed in Table 1. C, ATP dependence of Na ϩ ,K ϩ -ATPase activity. Each line shows the best fit of the Hill equation, and the extracted K 0.5 values are listed in Table 1. D, K ϩ dependence of Na ϩ ,K ϩ -ATPase activity. Each line shows the best fit of a two-component Hill equation with the inhibition represented by a negative term. K 0.5 values for the rising parts of the curves are listed in Table 1. E, Na ϩ dependence of Na ϩ -ATPase activity in the absence of K ϩ . Inhibition of Na ϩ -ATPase activity by high Na ϩ concentrations is indicated semiquantitatively in Table 1. B-E, experimental conditions and equations used for data fitting are described under "Experimental Procedures." Statistical information is given in Table 1. Symbol and error bars (seen only when larger than the size of the symbols) represent mean Ϯ S.E. Dotted lines reproduce the wild type for direct comparison in the same panel.
that electrostatic interaction between the introduced phosphate on Ser-938 and the positive side chain of Arg-1005 is central to the PKA regulatory effect on the binding of intracellular Na ϩ . The K ϩ dependence of the Na ϩ ,K ϩ -ATPase activity of S938E/R1005K showed only a minor reduction of K ϩ affinity for activation on par with the K ϩ affinity observed for the S938E single mutation (Fig. 5D and Table 1). Furthermore, the extracellularly facing low affinity sites on E 2 P were still capable of binding Na ϩ , leading to inhibition of Na ϩ -ATPase activity; however, the apparent affinity for the inhibitory Na ϩ appears slightly reduced relative to wild type, and the maximal Na ϩ -ATPase activity was clearly reduced in S938E/R1005K (Fig. 5E), indicating a reduced efficacy of Na ϩ activation of dephosphorylation. Thus, the combination of S938E with R1005K also leads to an altered Na ϩ interaction at the extracellularly facing sites, although neither of the single mutations had such an effect.
Information about the influence of the electrostatic repulsion between S938E and Glu-998 was obtained by studying the double mutation S938E/E998A, which turned out to further compromise Na ϩ affinity relative to the single mutations S938E and E998A (14-fold in the double mutant versus 3-and 9-fold in the respective mutants with single substitutions) (Fig. 5B and Table 1). It is, however, important to note that the E998A mutation does not diminish the effect of the S938E substitution on the Na ϩ affinity. This result indicates that electrostatic interaction between S938E and Glu-998 is of minor importance for the effect of the S938E mutation on Na ϩ affinity. The S938E/E998A mutant displayed a wild type-like turnover rate and apparent affinity for K ϩ activation (Fig. 5D). A distinct inhibition phase was observed at high K ϩ concentrations, which is in accordance with the marked reduction of Na ϩ affinity because, as explained above, inhibition by K ϩ results from K ϩ binding in competition with Na ϩ (Fig. 5D and Table 1).
In addition, we constructed the swap mutant S938R/ R1005S, which also displayed a larger reduction of Na ϩ affin-ity (6-fold; Fig. 5B and Table 1) than the corresponding mutants with single substitutions S938R and R1005S (4-and 2-fold, respectively). Hence, the swap of the serine and the arginine did not recover the Na ϩ binding properties of the Na ϩ ,K ϩ -ATPase, indicating that interaction of the unphosphorylated serine with the arginine is not a major determinant of the normal Na ϩ affinity in the absence of phosphorylation.
Direct Measurement of PKA-mediated Phosphorylation of Mutants-To examine whether Arg-1005 is required for PKAmediated phosphorylation of Ser-938, PKA phosphorylation experiments were performed with selected mutants (Fig. 6). Conditions were chosen under which almost no phosphorylation was observed in the absence of PKA (Fig. 6, A, lanes 1 and 4, and B, lanes 1, 3, and 5). As a positive control, highly purified renal Na ϩ ,K ϩ -ATPase was subjected to PKA-mediated phosphorylation, giving rise to a single phosphorylated band (Fig. 6B, lanes 8 and 9). PKA-mediated phosphorylation of the microsomes from COS-1 cells expressing the wild type Na ϩ ,K ϩ -ATPase showed a distinct band migrating at the same position as the band corresponding to the purified Na ϩ ,K ϩ -ATPase in addition to a number of other bands reflecting the fact that the expressed Na ϩ ,K ϩ -ATPase constitutes only a few percent of the total microsomal protein (Fig. 6A, lane 2). The microsomes from cells expressing the phosphorylationdeficient S938A mutant showed a weaker phosphorylated Na ϩ ,K ϩ -ATPase band corresponding to the background of endogenous COS-1 cell Na ϩ ,K ϩ -ATPase (Fig. 6, A, lane 3, and B, lane 2). For all the Arg-1005 mutants examined (R1005A, R1005K, and R1005M; Fig. 6B, lanes 4, 6, and 7, respectively), a distinct phosphorylated Na ϩ ,K ϩ -ATPase band stronger than the background corresponding to S938A could be seen. Hence, the presence of Arg-1005 is not obligatory for the PKA-mediated phosphorylation even though the arginine appears to be mechanistically involved in the signal transduction.
Discussion
Although it is well established that PKA regulates Na ϩ ,K ϩ -ATPase activity in vivo, the underlying molecular mechanism has remained obscure. The current study provides new information about this mechanism. Our data show that the phosphomimetic mutations S938D and S938E reduce the apparent affinity of the E 1 form for Na ϩ 2-3-fold relative to wild type ( Fig. 2A and Table 1). In the intact cell, the Na ϩ sites on E 1 face the intracellular side. The sites on E 2 P that bind extracellular Na ϩ and K ϩ were unaffected by these mutations (Fig. 2, C and D, and Table 1). In agreement with our findings, previous electrophysiological investigations of S938E did not disclose any defect in the binding of extracellular Na ϩ , which was attributed to the smaller size and charge of the glutamate substituent compared with a phosphate group having a charge of Ϫ2 at physiological pH (17). However, the latter study did not investigate the binding of Na ϩ from the intracellular side to the E 1 form probably due to the difficulty in controlling the intracellular Na ϩ concentration in whole cells. The presently observed reduction of Na ϩ affinity of the intracellularly facing E 1 sites caused by the phosphomimetic mutations S938D and S938E is within the range expected for physiological regulation of the Na ϩ ,K ϩ -ATPase (15). Because the Na ϩ ,K ϩ -ATPase works under suboptimal conditions in vivo where the intracellularly facing Na ϩ sites are not saturated with respect to Na ϩ , the moderate 2-3-fold reduction of Na ϩ affinity observed here is expected to have a significant effect on the Na ϩ ,K ϩ -ATPase turnover rate in intact cells. Thus, altogether, the present results suggest that PKA phosphorylation of Na ϩ ,K ϩ -ATPase, mimicked by the glutamate substituent, exerts its role in cells through modification of the intracellular Na ϩ affinity. According to a recent study from our group, this modification will have consequences for the intracellular concentration of Na ϩ , which we have shown correlates with the apparent Na ϩ affinity (24). Our findings are in agreement with a previous study showing that the Na ϩ clearance rate decreases upon modulation of the Na ϩ ,K ϩ -ATPase ␣3 isoform by PKA (35).
In the vicinity of Ser-938 in L8-9 are five charged residues, Asp-997, Glu-998, Arg-1000, Lys-1001, and Arg-1005, of M10 (Fig. 1C). Our results demonstrate that alanine substitution of Asp-997, Glu-998, and Arg-1000 affects the binding of Na ϩ from both sides of the membrane, whereas alanine substitution of Lys-1001 only affects the binding of Na ϩ from the intracellular side. In contrast, Arg-1005 is not required for proper Na ϩ or K ϩ binding (Figs. 3 and 4 and Table 1). The double sidedness of the effects seen for Asp-997, Glu-998, and Arg-1000 indicates that these residues are involved in stabilizing the Na ϩoccluded state, which is reached from both sides of the membrane. The most defective Na ϩ binding is seen for the R1000A mutant, which displays a 15-fold reduced apparent Na ϩ affinity of the E 1 form (Fig. 3A) as well as a complete disruption of Na ϩ binding from the extracellular side (Fig. 4A). These observations suggest a key role for the cytoplasmic half of M10 in the regulation of Na ϩ binding. M10 is physically connected to the C terminus whose interaction with M5 through Lys-768 (see Fig. 1C) is known to stabilize the so-called Na ϩ site III, which is specific for Na ϩ and is believed to be the first Na ϩ -binding site occupied during the sequential binding of three Na ϩ ions to Na ϩ ,K ϩ -ATPase (8). Because replacement of each of the residues Asp-997, Glu-998, Arg-1000, and Lys-1001 only affects the interaction with Na ϩ and not the interaction with K ϩ , the observed mutational effects may be ascribed to modification of the Na ϩ -specific site III (Fig. 3). In the Na ϩ ,K ϩ -ATPase structure with bound Na ϩ , the side chain of Asp-997 seems to form salt bridges with the side chains of both Arg-1000 and Lys-1001 (Figs. 1C and 7). Arg-1000 is hydrogen-bonded to Thr-1016 near the C terminus (Fig. 1C), and Lys-1001 is bonded to backbone carbonyl oxygens of M8 (Thr-934 and Ile-931; Fig. 7). Furthermore, Glu-998 is hydrogen-bonded to the backbone amide of Val-939 in L8-9, and the C terminus is tied up to Arg-935 in L8-9 (6,9). Thus, Asp-997, Glu-998, Arg-1000, and Lys-1001 of M10 are part of an elaborate bonding network that involves M10, L8-9, the C terminus, and the M5 and M8 helices providing liganding residues (Tyr-773, Thr-776, and Ser-777 in M5, Gln-925, and Asp-928 in M8) at Na ϩ site III (8,34,36). This extended network likely allows changes in the configuration of the M10 residues to be transmitted to Na ϩ site III.
The inhibition at high K ϩ concentrations, which is most distinct for the alanine substitutions of Arg-1000 and its interaction partner Asp-997 (Fig. 3C), may also arise from disturbance of the above mentioned bonding network. Several mutations affecting the C terminus have been found to exhibit this pattern of K ϩ inhibition (6,9,24,34,36), and the marked K ϩ inhibition of D997A and R1000A may be seen as a consequence of interference by the mutations with the bond between Arg-1000 and FIGURE 7. Structural relations of L8-9 containing the potential PKA site Ser-938. Shown is the relevant part of the structure of the Na ϩ -bound E 1 form of Na ϩ ,K ϩ -ATPase (Protein Data Bank code 3WGV) (8) viewed from the side along the membrane surface. Depicted is the extensive bonding network that connects L8-9 (yellow) harboring the potential PKA site at Ser-938 with M10 (gray), the C terminus (light blue), and the Na ϩ binding segment M8 (gray). The residues studied by mutagenesis are depicted as sticks (Ser-938, Asp-997, Glu-998, Arg-1000, Lys-1001, and Arg-1005). Stick representation of backbone atoms (blue nitrogen and red oxygen atoms) is shown for the entire L8-9. In addition, Gln-925 and Asp-928 known to contribute to the binding of the Na ϩ ion at site III are shown as sticks as are Ile-931 and Thr-934 that interact with Lys-1001 in M10. The Na ϩ ion bound at site III is depicted as a large purple sphere. The figure was prepared using PyMOL.
Thr-1016 next to the C-terminal tyrosines (Fig. 1C). Hence, analysis of the crystal structure has indicated that K ϩ binding at Na ϩ site III and resulting inhibition, i.e. loss of the Na ϩ selectivity of site III, will occur if the cytoplasmic half of M5 is inclined toward M10 by 10°as a consequence of disturbance of the stabilizing interaction of the C terminus with M5 (8).
In this context, it is worth mentioning that a mutation replacing the arginine corresponding to Arg-1000 with glutamine in the Na ϩ ,K ϩ -ATPase ␣2 isoform has been found in patients with familial hemiplegic migraine (37). Asp-997 has also been implicated in neurological disease as substitutions of this residue have been identified both in ␣2 in association with familial hemiplegic migraine and in the ␣3 isoform of patients with alternating hemiplegia of childhood (38,39).
The side chain hydroxyl of Ser-938 seems to form hydrogen bonds to the two backbone amides of Phe-940 and Gln-941 that may serve to stabilize a rigid loop structure of L8-9 (Figs. 1C and 7). The loss of these hydrogen bonds may be the reason for the 2-fold reduction of Na ϩ affinity caused by the S938A mutation. The introduction of the phosphomimetic S938E mutation led to a more pronounced, 3-fold reduction of Na ϩ affinity. In the Na ϩ -bound structure, the side chains of Glu-998 and Arg-1005 are 4.12 and 4.91 Å away, respectively, from the Ser-938 hydroxyl group. The glutamate substituent or in vivo phosphorylation of the serine might disrupt the interactions with the L8-9 backbone, which would provide opportunities for electrostatic attraction to the arginine side chain or repulsion by the glutamate Glu-998. To test these ideas, we generated double mutants of S938E combined with substitutions of either Arg-1005 or Glu-998 to probe the interactions. None of the three double mutants S938E/R1005A, S938E/R1005M, and S938E/ R1005S could support cell growth (Table 1) despite the fact that the mutants R1005A, R1005M, and R1005S with single substitutions behaved more or less wild type-like (Figs. 3 and 4 and Table 1). Even when expressed transiently, S938E/R1005A, S938E/R1005M, and S938E/R1005S failed to phosphorylate in a Na ϩ -dependent reaction at the conserved P-domain aspartate, explaining their inability to support cell growth (Fig. 5A). In contrast, mutant S938E/R1005K was able to transport Na ϩ and K ϩ across the membrane at rates sufficiently high to support cell growth, and the maximal turnover rate of this mutant was almost wild type-like (Table 1), thus underscoring the importance of the positive charge of the side chain at position 1005, which apparently interacts with the phosphomimetic negatively charged glutamate. Arginine side chains are well known and common interaction partners of phosphate groups as analysis of phosphate interactions in several proteins has shown that phosphate groups preferentially interact with one or more arginine residues (40 -42). Due to the planar structure of the guanidino group of the side chain, which has the ability to form several hydrogen bonds, the phosphate-arginine interaction is extraordinarily strong. Besides arginines, phosphate groups can also interact with lysines although generally less strongly. It is therefore of note that even though R1005K exhibited wild typelike Na ϩ affinity the Na ϩ affinity was even more reduced in S938E/R1005K (10-fold) than in single mutant S938E (3-fold) where arginine is present at position 1005 (Figs. 2, 3, and 5 and Table 1). Hence, replacement of Arg-1005 with lysine rein-forced the effect of the phosphomimetic S938E mutation on Na ϩ affinity. The lysine side chain is more flexible than that of arginine, which may increase the probability of "catching" its electrostatic interaction partner. Furthermore, due to the shorter length of the bridging lysine side chain compared with that of arginine, the interaction of the phosphomimetic glutamate of S938E with the lysine at position 1005, near the edge of M10, may cause larger structural perturbation of the elaborate bonding network that connects L8-9 to M10, the C terminus, and the Na ϩ binding region than the interaction with the longer arginine side chain.
The importance of Arg-1005 in the signal transduction resulting from the PKA-mediated phosphorylation of Ser-938 raises the question whether Arg-1005 is obligatory as interaction partner with the phosphate group for the PKA-mediated phosphorylation to occur? Our results showed that the PKAmediated phosphorylation is possible in the Arg-1005 mutants (Fig. 6), which is reasonable because the latter residue is located in transmembrane helix M10 outside the PKA consensus sequence in the loop between transmembrane helices M8 and M9. In the wild type Na ϩ ,K ϩ -ATPase, the phosphorylation of Ser-938 conceivably occurs without requirement for the arginine in the binding of the phosphate, but upon phosphorylation of the serine the phosphate group attracts the arginine, thereby propagating structural perturbation via the surrounding binding network to Na ϩ site III.
Although the structural closeness of Glu-998 to Ser-938 might suggest a role for electrostatic repulsion between a negative charge at position 938 and Glu-998 in the mechanism generating reduced Na ϩ affinity, such an effect was not confirmed experimentally. Hence, the substitution of Glu-998 with alanine did not attenuate the effect of S938E on Na ϩ affinity (Figs. 2, 3, and 5 and Table 1). The marked effect of the E998A single substitution seems to result from disruption of a hydrogen bond between one of the side chain oxygen atoms of Glu-998 and the backbone amide of Val-939 (Fig. 7), thus again pointing to the importance of bonds that stabilize a rigid L8-9 loop structure.
The swap mutant S938R/R1005S was constructed to test whether S938R and R1005S interact in the absence of phosphorylation of the serine. Because the swap mutant displayed a larger reduction of Na ϩ affinity than each of the single mutants S938R and R1005S, interaction of the unphosphorylated serine with the arginine is most likely not a major determinant of the normal Na ϩ affinity in the absence of phosphorylation.
In conclusion, the biochemical data presented in this study, particularly the effects of combining the phosphomimetic S938E mutation with Arg-1005 mutations, support a scenario where a phosphate group introduced at Ser-938 by PKA-mediated phosphorylation would reduce the affinity of Na ϩ site III in the internally facing configuration by way of a mechanism involving electrostatic interaction with the Arg-1005 side chain and destabilization of the rigid loop structure of L8-9, thereby conveying changes to M8 with its Na ϩ -liganding residues. The role of the positive charge at position 1005 is emphasized by the strengthening of the effect of the phosphomimetic mutation on Na ϩ affinity seen upon substitution of Arg-1005 with lysine. The reduced Na ϩ affinity at the internally facing sites resulting from phosphorylation of Ser-938 is expected to increase the intracellular Na ϩ concentration. | v3-fos-license |
2016-10-26T03:31:20.546Z | 2016-09-30T00:00:00.000 | 5387751 | {
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} | pes2o/s2orc | Controlling Properties and Cytotoxicity of Chitosan Nanocapsules by Chemical Grafting
The tunability of the properties of chitosan-based carriers opens new ways for the application of drugs with low water-stability or high adverse effects. In this work, the combination of a nanoemulsion with a chitosan hydrogel coating and the following poly (ethylene glycol) (PEG) grafting is proven to be a promising strategy to obtain a flexible and versatile nanocarrier with an improved stability. Thanks to chitosan amino groups, a new easy and reproducible method to obtain nanocapsule grafting with PEG has been developed in this work, allowing a very good control and tunability of the properties of nanocapsule surface. Two different PEG densities of coverage are studied and the nanocapsule systems obtained are characterized at all steps of the optimization in terms of diameter, Z potential and surface charge (amino group analysis). Results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their amino terminal moiety. An improvement in nanocapsule stability in physiological medium is observed with the highest PEG coverage density obtained. Cytotoxicity tests also demonstrate that grafting with PEG is an effective strategy to modulate the cytotoxicity of developed nanocapsules. Such results indicate the suitability of chitosan as protective coating for future studies oriented toward drug delivery.
Introduction
In the last few decades, many kinds of nanocarriers have been developed for delivery and targeting of therapeutic or diagnostic agents, thanks to some important advantages that they offer depending on their physico-chemical properties [1,2].
According to Vrignaud and co-workers, nanocapsules are vesicular systems, composed of an oily or an aqueous core that can be considered as a reservoir in which the drug is confined to a cavity, surrounded by a polymeric shell [12]. Nanocapsules can be obtained combining nanoemulsion and a polymeric coating. Nanoemulsion particles are stable colloidal suspensions obtained by mixing an organic phase containing oil and a lipophilic surfactant with an aqueous one containing a hydrophilic surfactant, resulting in a particle size ranging from 20 to 600 nm. The characteristics of the obtained particles depend on the spontaneity of the emulsification process that is affected by the nature of the single components of the reaction mixture and also by the rate of the mixing process [13,14]. Depending on the desired application, a coating is necessary to further stabilize the nanoscaled particles resulting from this spontaneous process and improve surface properties. The most commonly used coatings are natural polymers, which are deposited on the nanoemulsion template surface to produce a rigid and dense shell [15]. The shell can be easily tailor-made to achieve desired characteristics and its surface chemistry can be tuned to obtain a proper functionalization for biological targeting [16][17][18].
Natural polymers are among the most used for these kind of coatings since they usually provide a high colloidal stability in water suspensions. Active research is now focused on the use of hydrophilic biopolymers as carrier coatings because of their biocompatibility and biodegradability [19,20]. Chitosan (CS) has been used for the development of sustained release carriers, mucoadhesive formulations, and peptide drug absorption systems [21][22][23][24]. It is currently employed to prepare nanomaterials with mucoadhesive properties since its positive charges allow the interaction of particles with the negative charge of mucin, resulting in a better interaction with mucosal tissues and with epithelial cells. Moreover, it is known that the positive charge of the polymer can promote the paracellular transport by tight-junction regulation [25,26].
In this work, core-shell nanocapsules made of a nanoemulsion core and a chitosan shell were synthesized and characterized with the aim of obtaining a multipocket nano-reservoir carrier to be used in future applications for sustained release of different drugs. The secondary effects-toxicity, poor solubility, and bioavailability-of new drugs lead to the need of their encapsulation to protect them from degradation and to enhance their stability and solubility [27,28].
Thanks to the presence of chitosan amino groups, the surface of the obtained nanocarrier can also be grafted with specific moieties in order to tune the net charge to introduce specific functional groups and/or improve the carrier stability in biological media and physiologic solutions for intravenous administration [29][30][31][32][33]. The development of a smart nanocarrier is strictly related with controlling its surface properties since they are responsible for specific recognition of targeted sites but also for non-specific adsorption of serum proteins. A decrease in protein adsorption leads to a reduced uptake by the mononuclear phagocytic system, leading to a prolonged circulation time in the blood stream and to a higher residence time of the encapsulated drug. Poly (ethylene glycol) (PEG) coatings are known to prevent aggregation and serum protein adsorption by steric and hydration repulsions leading to more stable colloidal suspensions of nanocapsules in physiological media [33][34][35]. In this work, the surface of the developed chitosan-coated nanocapsules was grafted covalently with PEG molecules through a novel, simple, and reproducible strategy based on the use of a homobifunctional crosslinker, bis (sulfosuccinimidyl) suberate (BS 3 ), that links aminated PEG molecules to amino groups on nanocapsule surface. Nanocapsule behavior in different media was evaluated in terms of aggregation degree before and after grafting and the effect of PEG on their cytotoxicity was also assessed.
Chitosan-Coated Nanocapsules
A nanoemulsion method was developed to obtain small nanoparticles (smaller than 200 nm) to be used as a template for the following polymer coating and reinforcement. The aim was to obtain capsules with a lipophilic core and a hydrophilic cationic shell of chitosan hydrogel.
The synthesis of the nanocapsules was carried out in two steps. The formation of the nanoemulsion template particles was carried out simply by adding a water-miscible organic solution of Span ® 85/oleic acid (Croda International PLC, Cowick Hall Snaith, Goole, East Yorkshire, UK) to a Tween ® 20 (Croda International PLC, Cowick Hall Snaith, Goole, East Yorkshire, UK) aqueous solution under stirring. Optimal ratios between components have been found adapting a method reported by Bouchemal et al. [14]. The particles are immediately and spontaneously formed. Chitosan has been chosen for the coating of the nanoemulsion template since it is one of the richest in amino groups' natural polymers.
The presence of such functional groups is responsible for the ability of the polymer to gelify in presence of multi-anions leading to the formation of hydrogel. Moreover, the amino groups exposed on hydrogel shell surface allow the easy functionalization of the nanocapsule with the desired moiety.
Chitosan is directly added to the nanoemulsion that is subsequently mixed with a sodium sulfate solution to obtain the coating with a chitosan shell. This treatment has been used in several works to obtain chitosan particles [36][37][38]. In our case, the method allowed to obtain a hydrogel polymer shell as a result of the interaction of chitosan polyelectrolyte structure with sodium sulfate. Sodium sulfate acts as a bridge promoting interactions between polymeric chains.
The result of the synthesis is a water-stable suspension of chitosan-coated nanocapsules (CS-NCs). A schematic representation of the hypothesized nanocapsule structure is reported in Figure 1A. Moreover, to investigate the morphology of the obtained material, an electron microscopy characterization was carried out on chitosan-coated nanocapsules using both Bright Field Transmission Electron Microscopy (BF-TEM) and Environmental Scanning Electron Microscopy (ESEM) ( Figure 1B,C respectively). Due to the sensitive nature of the sample common in soft materials, previous fixation, dehydration, dyeing and resin embedding were necessary. solution under stirring. Optimal ratios between components have been found adapting a method reported by Bouchemal et al. [14]. The particles are immediately and spontaneously formed. Chitosan has been chosen for the coating of the nanoemulsion template since it is one of the richest in amino groups' natural polymers.
The presence of such functional groups is responsible for the ability of the polymer to gelify in presence of multi-anions leading to the formation of hydrogel. Moreover, the amino groups exposed on hydrogel shell surface allow the easy functionalization of the nanocapsule with the desired moiety.
Chitosan is directly added to the nanoemulsion that is subsequently mixed with a sodium sulfate solution to obtain the coating with a chitosan shell. This treatment has been used in several works to obtain chitosan particles [36][37][38]. In our case, the method allowed to obtain a hydrogel polymer shell as a result of the interaction of chitosan polyelectrolyte structure with sodium sulfate. Sodium sulfate acts as a bridge promoting interactions between polymeric chains.
The result of the synthesis is a water-stable suspension of chitosan-coated nanocapsules (CS-NCs). A schematic representation of the hypothesized nanocapsule structure is reported in Figure 1A. Moreover, to investigate the morphology of the obtained material, an electron microscopy characterization was carried out on chitosan-coated nanocapsules using both Bright Field Transmission Electron Microscopy (BF-TEM) and Environmental Scanning Electron Microscopy (ESEM) ( Figure 1B,C respectively). Due to the sensitive nature of the sample common in soft materials, previous fixation, dehydration, dyeing and resin embedding were necessary. The sample turned out to be composed of spherical capsules of a quite homogeneous size. The polymeric shell can be appreciated in BF-TEM and ESEM images ( Figure 1B,C). ESEM estimation of the diameter distribution is reported in Figure 1D. The number of capsules of different sizes, as percentages over the total number of measured capsules, is reported in the graph as a function of the diameter. The calculated mean diameter of NCs is 104 nm.
As optimization of the process, the importance of the sonication treatment during hydrogel shell formation was evaluated by substituting it with a gentle stirring while adding nanocapsules to Na2SO4 solution. The elimination of the sonication step in the synthesis process could represent an The sample turned out to be composed of spherical capsules of a quite homogeneous size. The polymeric shell can be appreciated in BF-TEM and ESEM images ( Figure 1B,C). ESEM estimation of the diameter distribution is reported in Figure 1D. The number of capsules of different sizes, as percentages over the total number of measured capsules, is reported in the graph as a function of the diameter. The calculated mean diameter of NCs is 104 nm.
As optimization of the process, the importance of the sonication treatment during hydrogel shell formation was evaluated by substituting it with a gentle stirring while adding nanocapsules to Na 2 SO 4 solution. The elimination of the sonication step in the synthesis process could represent an advantage in terms of applicability of the process for future applications to industrial production with high levels of scale-up. Nanocapsules obtained with the modified process have been characterized in terms of hydrodynamic diameter and surface charge and they have been compared with the sonicated ones. In Figure 2 Hydrodynamic diameter of chitosan-coated nanocapsules always increases with respect to the nanoemulsion template ( Figure 2A). Nevertheless, in the case of sonicated capsules the final hydrodynamic diameter is much higher than the non-sonicated sample ( Figure 2B). The value is also higher than the diameter distribution obtained from SEM images referring to sonicated nanocapsules. Moreover, the presence of a significant percentage of big aggregates is observed in the sCS-NC. The increase in the diameter observed can be attributed to aggregation phenomena, indicating a lower stability of this sample in water suspension. In the case of non-sonicated samples, a certain amount of aggregates are detected too, even though such aggregates have a smaller diameter than the ones obtained by sonication. In any case, the presence of a low percentage (i.e., 5%-10%) of aggregates could be considered acceptable for future purposes. The hydrodynamic diameter of the nanocapsules stored in water suspension has been found to be reproducible over at least two months, indicating the suitability of chitosan to successfully stabilize the nanoemulsion.
It is supposed that the hydrogel formation treatment affects the properties of the polymer-coated surface of the nanocapsules depending on the degree of the interaction that could be established between -NH2 and -OH on polymer chains and Na2SO4 molecules. Moreover, it should be taken into account that the presence of salts can also promote hydrophobic interactions between polymer chains themselves. As a consequence of the establishment of such hydrophobic interactions, the exposure amino groups on the surface of the nanocapsule would be favored. To evaluate how the sonication during hydrogel formation can improve the interactions and so affect the nanocapsule surface properties, sonicated and non-sonicated nanocapsules have been compared in terms of surface potential. In particular, analysis of Z-potential and amino group spectrophotometric determination analysis are reported. Both capsules showed a positive potential when measured in a 10 mM KCl solution, being slightly more positive the surface of sCS-NC (+21.1 mV) than the surface of nsCS-NC (+13.8 mV). The observed variation in the surface potential depending on the sonication treatment could be explained in this case by the presence of a higher number of amino groups exposed on the outer surface of chitosan shell in the case of sCS-NC. This hypothesis was confirmed by the quantification of amino groups of the chitosan shell of nanocapsules by the spectrophotometric method of Orange II dye, previously reported and already optimized for inorganic nanoparticles [39]. Briefly, the method is based on a pH-dependent interaction between positively charged amino groups and -SO 3− group of Orange II dye. This spectrophotometric method is simple, inexpensive, and easy, and its most important advantage over other methods for amino quantification consists in Hydrodynamic diameter of chitosan-coated nanocapsules always increases with respect to the nanoemulsion template ( Figure 2A). Nevertheless, in the case of sonicated capsules the final hydrodynamic diameter is much higher than the non-sonicated sample ( Figure 2B). The value is also higher than the diameter distribution obtained from SEM images referring to sonicated nanocapsules. Moreover, the presence of a significant percentage of big aggregates is observed in the sCS-NC. The increase in the diameter observed can be attributed to aggregation phenomena, indicating a lower stability of this sample in water suspension. In the case of non-sonicated samples, a certain amount of aggregates are detected too, even though such aggregates have a smaller diameter than the ones obtained by sonication. In any case, the presence of a low percentage (i.e., 5%-10%) of aggregates could be considered acceptable for future purposes. The hydrodynamic diameter of the nanocapsules stored in water suspension has been found to be reproducible over at least two months, indicating the suitability of chitosan to successfully stabilize the nanoemulsion.
It is supposed that the hydrogel formation treatment affects the properties of the polymer-coated surface of the nanocapsules depending on the degree of the interaction that could be established between -NH 2 and -OH on polymer chains and Na 2 SO 4 molecules. Moreover, it should be taken into account that the presence of salts can also promote hydrophobic interactions between polymer chains themselves. As a consequence of the establishment of such hydrophobic interactions, the exposure amino groups on the surface of the nanocapsule would be favored. To evaluate how the sonication during hydrogel formation can improve the interactions and so affect the nanocapsule surface properties, sonicated and non-sonicated nanocapsules have been compared in terms of surface potential. In particular, analysis of Z-potential and amino group spectrophotometric determination analysis are reported. Both capsules showed a positive potential when measured in a 10 mM KCl solution, being slightly more positive the surface of sCS-NC (+21.1 mV) than the surface of nsCS-NC (+13.8 mV). The observed variation in the surface potential depending on the sonication treatment could be explained in this case by the presence of a higher number of amino groups exposed on the outer surface of chitosan shell in the case of sCS-NC. This hypothesis was confirmed by the quantification of amino groups of the chitosan shell of nanocapsules by the spectrophotometric method of Orange II dye, previously reported and already optimized for inorganic nanoparticles [39]. Briefly, the method is based on a pH-dependent interaction between positively charged amino groups and -SO 3− group of Orange II dye. This spectrophotometric method is simple, inexpensive, and easy, and its most important advantage over other methods for amino quantification consists in the use of a molecule with low steric hindrance-an especially important aspect for porous materials materials. Moreover, the relationship between amino groups and reactant is in this case of 1:1, allowing a direct and reliable quantification [40]. In the present work, the method has been slightly modified to use syringe filters as support for the separation of nanocapsules from the solution during all the washing steps. Results from the spectrophotometric assay for the measurement of amino groups through the interaction with Orange II dye also proved the presence of a high number of positively charged amino groups in the case of sCS-NC (0.35 µmol·mg −1 ) while a value of amino groups of only 0.2 µmol·mg −1 was obtained in the case of nsCS-NC.
It should be taken into account that results from Orange II interaction only represented the amount of amino groups available for the interaction with dye molecules and that the amount of these groups could be lower than the total amount of -NH 2 of the nanocapsules. Data from the Orange II assay were in good agreement with Z potential analysis. sCS-NC presented a number of moles mg −1 of -NH 2 groups almost double with respect to the moles mg −1 of -NH 2 of nsCS-NC.
Thermo Gravimetric Analysis (TGA) of chitosan-based nanocapsules sonicated or not during the synthesis process is reported in Figure S3 of the Supplementary Materials. The weight loss corresponding to chitosan shell can be appreciated at~200 • C and it corresponded to 50% weight of the sCS-NC. Surprisingly in the case of nsCS-NC this percentage is even higher, where chitosan represents 65% of the total weight of the sample. This evidence demonstrated that the observed decrease of amino groups on the surface of non-sonicated nanocapsules was not due to a decrease in the chitosan total amount. Consequently, it is reasonable to suppose that the organization of chitosan layer and interactions between polymer chains themselves and with the surface of the nanoemulsion template are slightly different depending on the condition applied during the synthesis process.
Grafting of the Surface of Chitosan-Coated Nanocapsule
PEG coating has been chosen in this work for the further optimization of the carrier. As previously stated in the introduction and well documented in the literature, the prevention of unspecific adsorption of serum proteins on carrier surface represents a very important goal to allow a prolonged circulation time in the blood stream. From this point of view, pegylation is reported to be one of the most successful strategies [33][34][35].
Nanocapsules obtained without sonication during the synthesis have been selected for the process of grafting of the surface due to their better stability in water suspension. The surface was grafted with α-methoxy-ω-amino poly (ethylene glycol) (5000 Da) (aminated PEG) through a previous reaction with the homobifunctional linker bis (sulfosuccinimidyl) suberate (BS 3 ) (Scheme 1). As the colloidal stability of nanocapsules strongly depends on their surface properties, which in turn greatly affect nanocapsule cytotoxicity in animal cells, the possibility of obtaining nanocapsule surface with a low-density coverage of PEG molecules (5 nmol/mg initially added) (ldCS-NC) and a high-density coverage (100 nmol/mg initially added) (hdCS-NC) was explored. Consecutively, the final properties of the obtained materials were studied. A schematical representation of the strategy used for grafting is reported in Scheme 1.
The determination of the accessible amino groups was fundamental for the optimization of the grafting protocol. In fact, the method utilized here implied the use of a homobifunctional linker in which the main drawback lays in the possibility of crosslinking of amino groups from different nanocapsules and production of aggregates if the amount of reactants is not strictly controlled. For this reason, the value of accessible amino groups has been used as the starting point to adjust the amount of reactants in the following steps of the process. In particular, the amount of BS 3 has been always maintained below the total moles of functional groups of nanocapsules to reduce the possibility of process of grafting of the surface due to their better stability in water suspension. The surface was grafted with α-methoxy-ω-amino poly (ethylene glycol) (5000 Da) (aminated PEG) through a previous reaction with the homobifunctional linker bis (sulfosuccinimidyl) suberate (BS 3 ) (Scheme 1). As the colloidal stability of nanocapsules strongly depends on their surface properties, which in turn greatly affect nanocapsule cytotoxicity in animal cells, the possibility of obtaining nanocapsule surface with a low-density coverage of PEG molecules (5 nmol/mg initially added) (ldCS-NC) and a high-density coverage (100 nmol/mg initially added) (hdCS-NC) was explored. Consecutively, the final properties of the obtained materials were studied. A schematical representation of the strategy used for grafting is reported in Scheme 1. In the FTIR spectrum of nsCS-NC ( Figure S4A), it is possible to recognize the presence of the four starting components, meaning that the final composition is compatible with the hypothesized structure of the nanocapsule ( Figure 1A). Moreover, reported in Figure S6 of the Supplementary Materials a comparison of sCS-NCs and nsCS-NC demonstrates that the sonication process is not affecting the chemical interaction between nanoemulsion template and chitosan shell.
The grafting process consists of three steps. The first one is the incubation with the linker to provide the surface with the sulfosuccinimidyl ester group sensitive to the linking of PEG. During the second step the ligand is added and finally the grafted nanocapsules are incubated with Tris-HCl buffer to quench the free sulfosuccinimidyl ester groups eventually not linked to any PEG molecule. This last step is fundamental to avoid the crosslinking between nanocapsules due to the reaction between free sulfosuccinimidyl ester groups and amino group on a different capsule.
To determine if the grafting with PEG successfully occurred, the FTIR spectrum of the intermediate step of nanocapsules after the incubation with BS 3 is also reported ( Figure S4B). It should be noted that at this intermediate step the sample is incubated with Tris-HCl buffer after the linking of BS 3 to avoid the crosslinking with amino groups on other nanocapsule surface through the reactive groups still available for ligand reaction. In the case of samples incubated with ligand in the second step, Tris-HCl was added at the end of the process (after incubation with the ligand) but in this case it can be supposed that BS 3 , which should have reacted previously with PEG, is not available to react with Tris. This hypothesis was confirmed by comparing FTIR spectra of nanocapsules after the incubation with BS 3 ( Figure S4B) and spectra in Figure S4C,D referring to nanocapsules grafted with a low density and high density of PEG, respectively. Peaks at 3180 and 3100 cm −1 , as well as peaks at 1630 and 1550 cm −1 referring to Tris are present in the intermediate step and (in ldCS-NC with lower intensities) but they completely disappeared in hdCS-NC spectrum.
Differences in the spectra could also be appreciated, comparing peaks and intensities ratios between 1150 and 1030 cm −1 . In this region the C-O-C peak (1105 cm −1 ) presents an increased intensity, compared to other peaks in the same region, especially in the hdCS-NC spectrum. Moreover, in this sample peak at 1055 cm −1 is lost. This peak is supposed to refer to R-SO 3− group that is released from BS 3 in the linking reaction. In the BS 3 intermediate step the peak is still appreciable and it can be observed as a low intensity peak in ldCS-NCs too. It is possible that in these samples not all BS 3 reactive groups are quenched after Tris-HCl incubation and that some of them are still present on the surface. In any case this state did not represent a problem for the quality of the sample since any appreciable crosslinking and consequent aggregation are observed.
The successful grafting on nanocapsule surface was proved also by measuring the Z potential of the material surface before and after the modification with PEG. Using both a low amount and high amount of PEG, a decrease in the potential was observed, indicating a decrease in the free amino groups on the surface and an increase in the total electronegativity of the surface.
A confirmation of this evidence is the small difference in the decrease of amino groups (measured by Orange II spectrophotometric assay) registered between low-density and high-density PEG-covered nanocapsules. The measured amounts of amino groups are 0.1 and 0.08 µmol/mg respectively, corresponding to 50% and 40% of the amounts of amino groups on ungrafted nanocapsules. These values are considered as the apparent disappearing of amino groups probably due to a screening effect for the presence of PEG molecules on the surface. Orange molecule interaction with -NH 3 + groups would be hampered by the presence of the polymer chains since, in the case of low-density PEG coverage, it can be supposed that polymer molecules stand in a conformation that lays around nanocapsule surface, probably thanks to a possible hydrogen bond interaction of the PEG chain with positively charged amino groups on the nanocapsule surface (see Scheme 1). The tendency to form such interactions could also be responsible for a lower than expected efficiency of linking in the case of high-density coverage sample.
The effect of the grafting on the behavior of nanocapsules in physiological medium was further evaluated by measuring the degree of aggregation of grafted and not-grafted nanocapsules in water and phosphate saline buffer (PBS) by means of hydrodynamic diameter measurement. Ungrafted nanocapsules are sensitive to the presence of salts in the medium and they tend to aggregate during incubation in physiological media like PBS. The grafting with PEG chains is a common strategy to improve the stability of nanoparticles in physiological and in vitro culture media [33]. Both phosphate ions and proteins can adsorb onto nanocapsule surface due to the presence of amino groups, producing the crosslinking between different capsules. Moreover, the presence of salts can produce aggregation due to a salting-out-like effect. The presence of PEG on nanocapsule surface would screen the amino groups on the surface from interaction with salts in the medium. All samples were measured in water and PBS and their diameters were compared to assess the effect of PEG on prevention of aggregation ( Figure 3).
Mar. Drugs 2016, 14, 175 7 of 14
A confirmation of this evidence is the small difference in the decrease of amino groups (measured by Orange II spectrophotometric assay) registered between low-density and high-density PEG-covered nanocapsules. The measured amounts of amino groups are 0.1 and 0.08 μmol/mg respectively, corresponding to 50% and 40% of the amounts of amino groups on ungrafted nanocapsules. These values are considered as the apparent disappearing of amino groups probably due to a screening effect for the presence of PEG molecules on the surface. Orange molecule interaction with -NH3 + groups would be hampered by the presence of the polymer chains since, in the case of low-density PEG coverage, it can be supposed that polymer molecules stand in a conformation that lays around nanocapsule surface, probably thanks to a possible hydrogen bond interaction of the PEG chain with positively charged amino groups on the nanocapsule surface (see Scheme 1). The tendency to form such interactions could also be responsible for a lower than expected efficiency of linking in the case of high-density coverage sample.
The effect of the grafting on the behavior of nanocapsules in physiological medium was further evaluated by measuring the degree of aggregation of grafted and not-grafted nanocapsules in water and phosphate saline buffer (PBS) by means of hydrodynamic diameter measurement. Ungrafted nanocapsules are sensitive to the presence of salts in the medium and they tend to aggregate during incubation in physiological media like PBS. The grafting with PEG chains is a common strategy to improve the stability of nanoparticles in physiological and in vitro culture media [33]. Both phosphate ions and proteins can adsorb onto nanocapsule surface due to the presence of amino groups, producing the crosslinking between different capsules. Moreover, the presence of salts can produce aggregation due to a salting-out-like effect. The presence of PEG on nanocapsule surface would screen the amino groups on the surface from interaction with salts in the medium. All samples were measured in water and PBS and their diameters were compared to assess the effect of PEG on prevention of aggregation ( Figure 3). In Figure 3A, a strong aggregation effect in PBS is reported for ungrafted chitosan-based nanocapsules. The mean hydrodynamic diameter changed from 103 nm in water to 471 nm in PBS. On the contrary, the diameter and the PDI of grafted nanocapsules are maintained in PBS, even in the case of ldCS-NCs, confirming that the grafting successfully occurred in both cases and that it was effective for nanocapsule stabilization.
To further demonstrate the stabilizing effect of PEG grafting on nanocapsule surface, an aggregation test has been carried out by measuring the hydrodynamic diameter of grafted and ungrafted nanocapsules in the presence of increasing concentrations of Bovine Serum Albumin (BSA). As in the case of the above reported stability assay in presence of PBS, the high density of amino groups on nanocapsule surface can be considered responsible for the adsorption of proteins and aggregation observed.
The comparison of the behaviors of ungrafted nanocapsules, ldPEG-CS NC and hdPEG-CS NC, In Figure 3A, a strong aggregation effect in PBS is reported for ungrafted chitosan-based nanocapsules. The mean hydrodynamic diameter changed from 103 nm in water to 471 nm in PBS. On the contrary, the diameter and the PDI of grafted nanocapsules are maintained in PBS, even in the case of ldCS-NCs, confirming that the grafting successfully occurred in both cases and that it was effective for nanocapsule stabilization.
To further demonstrate the stabilizing effect of PEG grafting on nanocapsule surface, an aggregation test has been carried out by measuring the hydrodynamic diameter of grafted and ungrafted nanocapsules in the presence of increasing concentrations of Bovine Serum Albumin (BSA). As in the case of the above reported stability assay in presence of PBS, the high density of amino groups on nanocapsule surface can be considered responsible for the adsorption of proteins and aggregation observed.
The comparison of the behaviors of ungrafted nanocapsules, ldPEG-CS NC and hdPEG-CS NC, has been reported in Figure 4. Data reported in the graph demonstrated without any doubt the strong stabilizing effect of PEG on nanocapsules in the presence of proteins. The grafting with PEG, and so the strong decrease of the free amino groups on the surface lead to a very good stability of the nanocapsules in a protein-rich medium. Both ldPEG-CS NC and hdPEG-CS NC showed a significant increase in the hydrodynamic diameter only at very high concentrations of proteins (higher than 0.1 mg/mL). Comparatively, the concentration of BSA one order of magnitude lower is enough to produce the same increase in the diameter in the case of ungrafted NCs.
Cell Viabily and Internalization Assays
The cytotoxicity of chitosan-based nanocapsules before and after the grafting was tested on Vero cells through MTT spectrophotometric assay. Cells were incubated for 24 h with different concentrations of nsCS-NC, selected as ungrafted nanocapsules over the sonicated ones due to their better stability in water, and with the same concentrations of ldCS-NC and hdCS-NC. In Figure 5, the comparison between the grafted nanocapsules and the ungrafted ones is reported in terms of percentage of viability of cell culture after 24 h incubation. Data reported in the graph demonstrated without any doubt the strong stabilizing effect of PEG on nanocapsules in the presence of proteins. The grafting with PEG, and so the strong decrease of the free amino groups on the surface lead to a very good stability of the nanocapsules in a protein-rich medium. Both ldPEG-CS NC and hdPEG-CS NC showed a significant increase in the hydrodynamic diameter only at very high concentrations of proteins (higher than 0.1 mg/mL). Comparatively, the concentration of BSA one order of magnitude lower is enough to produce the same increase in the diameter in the case of ungrafted NCs.
Cell Viabily and Internalization Assays
The cytotoxicity of chitosan-based nanocapsules before and after the grafting was tested on Vero cells through MTT spectrophotometric assay. Cells were incubated for 24 h with different concentrations of nsCS-NC, selected as ungrafted nanocapsules over the sonicated ones due to their better stability in water, and with the same concentrations of ldCS-NC and hdCS-NC. In Figure 5, the comparison between the grafted nanocapsules and the ungrafted ones is reported in terms of percentage of viability of cell culture after 24 h incubation.
It can be observed from Figure 5 that the grafting significantly improved the safety of the nanocapsules, especially when using high concentrations of nanocapsules (greater than 0.15 mg/mL). Both types of grafted nanocapsules were less toxic to the cells, especially the high density ones (at high concentrations).
Using an inverted microscope, it can be observed that non-sonicated nanocapsules lead to bigger vesicles inside cells than both types of grafted nanocapsules (Figure 6), which could be related with the higher toxicity).
The cytotoxicity of chitosan-based nanocapsules before and after the grafting was tested on Vero cells through MTT spectrophotometric assay. Cells were incubated for 24 h with different concentrations of nsCS-NC, selected as ungrafted nanocapsules over the sonicated ones due to their better stability in water, and with the same concentrations of ldCS-NC and hdCS-NC. In Figure 5, the comparison between the grafted nanocapsules and the ungrafted ones is reported in terms of percentage of viability of cell culture after 24 h incubation. It can be observed from Figure 5 that the grafting significantly improved the safety of the nanocapsules, especially when using high concentrations of nanocapsules (greater than 0.15 mg/mL). Both types of grafted nanocapsules were less toxic to the cells, especially the high density ones (at high concentrations). Using an inverted microscope, it can be observed that non-sonicated nanocapsules lead to bigger vesicles inside cells than both types of grafted nanocapsules (Figure 6), which could be related with the higher toxicity). The test proved not only that aminated PEG was definitively linked to nanocapsule surface, but also that the conformation of polymer chains obtained using the developed method of grafting for ldCS-NC hdCS-NC is effective for significantly improving the cytotoxicity of chitosan-based nanocapsules. Moreover, it should be noted that in the case of PEG-grafted nanocapsules the decrease in the cytotoxicity is still associated with a very high degree of internalization, indicating that the developed material can be considered a very effective carrier for drug delivery applications.
Discussion
Among all kinds of nanocarriers, nanocapsules are frequently the material of choice for biomedical applications since they offer the advantage of providing a good stability of encapsulated drugs and a favorable pharmacokinetic. The aim of the work was to develop nanocapsules to be used in the future as potential drug reservoir and whose composition could allow maximum flexibility of application together with great feasibility for the administration by different routes (intravenous injection, oral administration, and inhalation).
In this work, chitosan-a biocompatible polymer-has been used to obtain a potentially smart nanocarrier whose surface properties could be properly tuned depending on the desired application. A nanoemulsion-based nanocapsule has been used as template to be coated with a chitosan shell. The test proved not only that aminated PEG was definitively linked to nanocapsule surface, but also that the conformation of polymer chains obtained using the developed method of grafting for ldCS-NC hdCS-NC is effective for significantly improving the cytotoxicity of chitosan-based nanocapsules. Moreover, it should be noted that in the case of PEG-grafted nanocapsules the decrease in the cytotoxicity is still associated with a very high degree of internalization, indicating that the developed material can be considered a very effective carrier for drug delivery applications.
Discussion
Among all kinds of nanocarriers, nanocapsules are frequently the material of choice for biomedical applications since they offer the advantage of providing a good stability of encapsulated drugs and a favorable pharmacokinetic. The aim of the work was to develop nanocapsules to be used in the future as potential drug reservoir and whose composition could allow maximum flexibility of application together with great feasibility for the administration by different routes (intravenous injection, oral administration, and inhalation).
In this work, chitosan-a biocompatible polymer-has been used to obtain a potentially smart nanocarrier whose surface properties could be properly tuned depending on the desired application. A nanoemulsion-based nanocapsule has been used as template to be coated with a chitosan shell.
The process of chitosan hydrogel coating of a nanoemulsion template was studied in terms of exposure of amino groups on the surface depending on the interaction produced during the sonication process. It could be hypothesized that the sonication promoted interactions and produced stronger hydrophobic interaction between polymer chains, leading to a condensation of the structure that would lead to a higher exposure of amino groups and so to a higher availability for the interaction with the dye. On the other hand, it was also supposed that a different amount of polymer could be incorporated on the surface of the nanoemulsion template when sonication is used during the synthesis, leading to a different total amount of glucosamine units (and so amines) in the shell. The characterization of the material and the comparison between the sonicated and not sonicated one alloowed finally to confirm that the sonication process lead to a higher amount of amino groups exposed on nanocapsule surface.
Chitosan shell offers the possibility of an easy functionalization through amino groups on its surface so chitosan-based nanocapsules have been further coated with PEG since this is known to be a very effective strategy to stabilize surfaces in biologically relevant media. The possibility to tune the surface properties by varying the PEG density coverage has been explored and results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their aminated terminal.
It is reported in literature that the attempt to produce high-density coverage of surfaces with PEG can result in a loss of efficiency of grafting. In fact, similarly to what reported for the grafting of other surfaces, it is reasonable to suppose that not all PEG molecules react immediately with BS 3 linker on nanocapsule surface. Once they are linked, their tendency is to assume a mushroom-like conformation until the density of PEG molecules on the surface does not reach a value high enough to produce a change into a brush-like conformation [41]. In the case of high-density PEG-covered nanocapsules, the decrease in amino groups on the surface was found to be not proportional to the reactants, differently to what happened in the case of low-density ones, indicating that eventually a conformation of PEG molecules laying adsorbed on nanoparticle surface was hampering the grafting of masked amino groups.
Despite the fact that the obtained grafting degree was slightly lower than the one expected, a highly improved stability in physiological medium (resistance to salting out effect) was observed with both low and high PEG-covered nanocapsules (although at different degrees) indicating that the presence of PEG on the surface will allow the use of the grafted capsules in physiological media for biomedical applications.
We also demonstrated that the presence of PEG on chitosan surface of nanocapsules was effective in avoiding the unspecific adsorption of proteins. This issue is of the utmost importance as, following intravenous injection, non-passivated nanoparticles tend to adsorb plasma proteins (protein corona), changing the charge and size of them. This binding of plasma components will be responsible of the final fate of the nanocapsules, and can greatly influence the biodistribution and therapeutic efficacy [42]. To date, PEG is the most widely used polymer to prevent this protein corona formation, although the final effect depends on different parameters such as the molecular weight or grafting density [43].
Following the encouraging results obtained in physiological media and protein-rich media, preliminary tests with cell cultures have been carried out showing very interesting results.
The obtained coating demonstrated effectiveness in tuning the cytotoxicity of chitosan-coated nanocapsules. Moreover, the nanocapsules showed a high degree of internalization in Vero cells, a useful property for the potential application as drug reservoirs directed to cytoplasm release. Finally, the tunability of all the properties of the synthesized chitosan-based nanocapsules was obtained by a very easy and reproducible chemical grafting, indicating that the developed nanocarrier was very promising for further studies oriented toward drug encapsulation for biomedical application.
Vero For the preparation of chitosan-based nanocapsules an organic solution containing 400 mg oleic acid and 86 mg Span ® 85 in 40 mL of absolute ethanol was added to the aqueous one, containing 136 mg Tween ® 20 solved in 80 mL water, under magnetic stirring during 15 min for the formation of the nanoemulsion. Then 25 mg from a 5 mg/mL chitosan solution in acetic acid 1% (v/v) were added and again the mixture was left under stirring 15 min. Finally, the chitosan-coated nanoemulsion was added to 200 mL of 50 mM Na 2 SO 4 under sonication (or stirring in the case of optimized nanocapsules). Capsules were separated from Na 2 SO 4 through ultracentrifugation (30 min, 69673 G, 10 • C), washed with 100 mL of water, centrifuged again, and resuspended in water. The concentration of the nanocapsules in water suspension was obtained by measuring the weight of 1 mL of sample after freeze-drying.
For the grafting of nanocapsule surface suspensions, 20 mg of nanocapsules at a concentration of 2 mg/mL in borate buffer 10 mM pH 8.3 were added with different amounts of the linker bis(sulfosuccinimidyl) suberate (BS3) (20-100 nmol/mgNC) and they were kept under stirring for 30 min. Then a double amount of α-methoxy-ω-amino poly (ethylene glycol) (MeO-PEG-NH 2 ) was added and the mixture was kept under stirring for 2 h at 37 • C. Finally, 20 mL of Tris-HCl buffer 10 mM pH 8.0 was added to quench the linker that eventually did not react with PEG. Grafted nanocapsules were filtered using an Amicon Ultrafiltration unit using Millipore Biomax 300 kDa Ultrafiltration Discs to separate them from unreacted PEG. After a washing with fresh water nanocapsules, they were concentrated to a final volume of 2 mL.
Several techniques have been used for the characterization of chitosan nanocapsules. DLS analysis has been carried out using a Brookhaven 90Plus DLS instrument, by means of the Photo-Correlation Spectroscopy (PCS) technique. Nanoparticle hydrodynamic diameter and polydispersity index (PDI) have been measured in water at the concentration of 0.05 mg/mL.
Electrophoretic mobility (Z Potential) of nanoparticles at different pH values has been determined by measuring the potential of a 0.05 mg/mL nanoparticle suspension in 10 mM KCl with a Plus Particle Size Analyzer (Brookhaven Instruments Corporation).
Nanocapsule composition was analyzed by Fourier Transform Infrared Spectroscopy analysis in a JASCO FT/IR-4100 Fourier transform infrared spectrometer in a frequency range of 600-4000 cm −1 with a resolution of 2 cm −1 and a scanning number of 32.
Thermogravimetric analysis was performed in a TASTD 2960 thermogravimetric analyzer, by heating the sample at 10 • C/min under air atmosphere.
Environmental Scanning Electron Microscopy (ESEM) images were obtained using a QUANTA-FEG 250 microscope in Scanning Transmission Electron Microscopy (STEM) mode. Bright Field Transmission Electron Microscopy (BF-TEM) analysis was carried out in a FEI Tecnai T20 microscope operating at 200 kV. Due to the sensitive nature of the sample, previous fixation, dehydration, and epoxy resin embedding were necessary for both techniques. This process can be briefly achieved as follows. A fresh sample was synthesized and it was fixed with glutaraldehyde 0.25% in phosphate buffer 10 mM at pH 7.4 for 2 h. It was washed three times with buffer and incubated with 1% osmium tetroxide in PBS for further fixation and staining. Finally, the sample was accurately washed with water and resuspended in 5% gelatin. The sample was centrifuged to obtain a pellet and was incubated overnight at 4 • C. The obtained solid sample was cut in very small pieces before undergoing the subsequent steps. The dehydration of the samples was carried out using the following steps: incubation in ethanol 30%, ethanol 50%, and incubation overnight in ethanol 70%; incubation in ethanol 90% for 1 h; and finally incubation three times in absolute ethanol. After that, samples were incubated overnight in a 1:1 mixture absolute ethanol/epoxy resin (r.t.). The mixture was then removed, changed with absolute epoxy resin and samples were left for impregnation for 8 h at room temperature. After another change of the medium, the final incubation in epoxy resin was carried out overnight at 60 • C to obtain the polymerization.
From different ESEM images (Figures S1 and S2 of Supplementary Materials), an estimation of the diameter distribution has been obtained using Digital Micrograph ® (Gatan Inc., Pleasanton, TX, USA) and OriginLab ® (OriginLab, Northampton, MA, USA) softwares to measure the diameters of more than 100 nanocapsules and for the frequency count statistical analysis respectively.
Nanocapsule amino content was measured by the Orange II spectrophotometric assay [34,35]. 0.2 mg of nanocapsules were put in contact with 1 mL of 2 mM Orange II sodium salt acidic solution (pH 3) and kept under stirring for 30 min at 37 • C. Capsule suspension was passed through a syringe membrane filter (Millex syringe-driven filter unit, PVDF filter with 0.22 µm pores, purchased from Merck Millipore) to adsorb nanocapsules in the membrane and keep them retained in order to separate them from the Orange II solution. After that, an acidic solution (pH 3) was passed several times through the same filter until all the unbound dye was removed from the nanocapsules (verified by measuring spectrophotometrically the supernatant content). Then they were washed with an alkaline solution (pH 12) to desorb the bound dye from the amino groups on nanocapsules. The washing fractions were collected, the pH was adjusted at 3 and the amount of unadsorbed and desorbed dye was measured at a wave length of 480 nm with a Varian Cary 50 UV/V is spectrophotometer after carrying out a calibration curve.
Resistance of nanocapsules to aggregation has been determined by incubating nanocapsules (3 mL of a 0.15 mg/mL suspension) at different concentrations of albumin from bovine serum (BSA). The hydrodynamic diameter of the capsules under the incubation conditions was measured after 10 min using a Brookhaven 90Plus DLS instrument.
In vitro cell viability test was carried out to determine the cytotoxicity of nanocapsules using 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Vero cells were grown at 37 • C in a 5% CO 2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL), and glutamine (2 mM). 7500 cells were seeded using a standard 96-well plate (five replicates per sample). After 24 h of incubation in a humidified atmosphere containing 5% CO 2 , the medium was replaced with new medium containing five different concentrations of nanocapsules and a negative control containing no capsules (non-treated cells). After 24 h of incubation, the medium was replaced with fresh medium containing MTT dye solution (0.5 mg/mL in DMEM). After 2 h of incubation at 37 • C and 5% CO 2 , the medium was removed and the formed crystals were dissolved in 200 µL of DMSO. The absorbance was read on a ThermoScientificMultiskan GO TM microplate reader at 570 nm. The relative cell viability (%) related to control cells without nanocapsules was calculated using the percentage ratio between absorbance of the sample and the absorbance of the control. Experiments were carried out in triplicate.
To perform optical microscopy analysis, 3 × 10 4 cells were seeded on glass coverslips in a 24-well plate at 37 • C. 24 h later, nanocapsules were added at 50 µg/mL in DMEM and incubated for 24 h at 37 • C. Non-internalized nanocapsules were removed, washing with DPBS twice. Cells were fixed with 4% paraformaldehyde for 20 min at 4 • C, washed twice with DPBS, and incubated for 10 min at room temperature with 4 ,6-diamidino-2-fenilindolphenylindole (DAPI) for nucleus labeling. The coverslips were mounted on glass microscope slides using ProLong ® (Thermo Fisher Scientific Inc., Waltham, MA, USA) Gold Antifade. Optical microscopy was performed using an inverted microscope (Nikon Eclipse Ti-E), and images were analyzed using NIS-Elements Advanced Research software. | v3-fos-license |
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} | pes2o/s2orc | Modulation of Insulin Resistance and the Adipocyte-Skeletal Muscle Cell Cross-Talk by LCn-3PUFA
The cross-talk between skeletal muscle and adipose tissue is involved in the development of insulin resistance (IR) in skeletal muscle, leading to the decrease in the anabolic effect of insulin. We investigated if the long chain polyunsaturated n-3 fatty acids (LCn-3PUFA), eicosapentaenoic and docosapentaenoic acids (EPA and DPA, respectively) could (1) regulate the development of IR in 3T3-L1 adipocytes and C2C12 muscle cells and (2) inhibit IR in muscle cells exposed to conditioned media (CM) from insulin-resistant adipocytes. Chronic insulin (CI) treatment of adipocytes and palmitic acid (PAL) exposure of myotubes were used to induce IR in the presence, or not, of LCn-3PUFA. EPA (50 µM) and DPA (10 µM) improved PAL-induced IR in myotubes, but had only a partial effect in adipocytes. CM from adipocytes exposed to CI induced IR in C2C12 myotubes. Although DPA increased the mRNA levels of genes involved in fatty acid (FA) beta-oxidation and insulin signaling in adipocytes, it was not sufficient to reduce the secretion of inflammatory cytokines and prevent the induction of IR in myotubes exposed to adipocyte’s CM. Treatment with DPA was able to increase the release of adiponectin by adipocytes into CM. In conclusion, DPA is able to protect myotubes from PAL-induced IR, but not from IR induced by CM from adipocytes.
Introduction
In 2060, the European population is expected to reach 517 million and one third of people will be more than 65 years old [1]. This represents a significant public health challenge with a major economic cost, notably because of several associated chronic diseases. Variations in lean and fat masses have common pathophysiological mechanisms, including insulin-resistance (IR), a low-grade inflammation [2], and specific dysfunctions of adipose tissue and skeletal muscle metabolisms. Altogether, these abnormalities are involved in the development of anabolic resistance of skeletal muscle cells and the progressive muscle atrophy observed during aging.
It has been well described that obesity induces changes in the secretory activity of adipose tissue. Chronic inflammation of adipose tissue decreases adiponectin (an adipokine with insulin-sensitizing and anti-inflammatory effects) production whereas the release of pro-inflammatory cytokines (interleukine-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α) is increased [3]. The contribution of IL-6 and TNF-α production by muscle cells [4] to systemic inflammation is unclear but it was shown that chronically increased plasma levels of IL-6 and TNF-α could mediate insulin and anabolic resistance of skeletal muscle's cells [5,6].
In addition, combination of a high energy intake and insulin resistance (IR) in adipose tissue increase the net efflux of free fatty acids (FA) from adipose tissue into the blood circulation [7]. Free FA will be caught up by the liver and skeletal muscles, leading to ectopic fat deposition. In insulin-sensitive tissues, the increase of intracellular triglycerides (TG) is associated with the accumulation of lipid intermediates, such as diglycerides (DG) and ceramides. These molecules can activate protein kinase C (PKC) [8,9], which inhibits protein kinase B (PKB, also known as Akt), a crucial effector of insulin signaling. Therefore, these lipotoxic events could participate in the development of resistance to the anabolic action of insulin and the loss of skeletal muscle's function.
Nutritional strategies are currently developed to prevent IR and metabolic disturbances. Recently, we have shown that long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) could protect skeletal muscle cells from IR. Eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) supplementations improved Akt activation and glucose uptake in vitro by the inhibition of PKC activation and the accumulation of intracellular DG and ceramides that are induced by an exposure to palmitic acid (PAL) [10]. Furthermore, it has also been shown that DHA could improve the growth of myotubes in a model of PAL-induced atrophy [11].
Among the different LCn-3PUFA found in human tissues, less attention was given to docosapentaenoic acid (DPA, 22:5n-3) which is produced during the conversion of EPA into DHA. Nevertheless, some evidences showed that DPA was as efficient as EPA and DHA in the prevention of metabolic disorders. Its plasma level was associated with fewer coronary heart disease deaths [12]. DPA could also inhibit cyclooxygenase and the production of pro-inflammatory prostaglandins by macrophages [13]. Interestingly, we recently observed in mice fed with a high fat, high sucrose diet supplemented with EPA an increase in DPA content in different tissues concomitantly to an improvement in insulin sensitivity as compared to non-supplemented animals [14].
LCn-3PUFA are quite prone to beta-oxidation as well as incorporation into phospholipids (PL). It could be then hypothesized that their effects are, at least partially, mediated by the modulation of the membrane's PL composition. Indeed, the FA composition of PL regulates the fluidity of cytoplasm or intracellular membranes and could be involved in insulin signaling efficiency [15].
Considering all these elements, the present study aimed at evaluating the effect of DPA on the interrelation between adipose and muscle cells in the context of IR. Our objectives were (1) to explore the reversion of IR by EPA and DPA in adipocytes and muscle cells; (2) to determine the impact of adipocyte IR on the induction of muscle IR; and (3) to evaluate the potential mechanisms and mediators involved in the cross-talk between adipocytes and myotubes.
Effects of LCn-3PUFA Treatments on Membrane Phosphatidylcholine Content and Fatty Acid Profile
In order to validate the effectiveness of LCn-3PUFA treatments on the modification of the profile of membrane's PL, the FA composition of phosphatidylcholine (PC) has been evaluated (Table 1). In 3T3-L1 adipocytes, the amount of PC was not significantly affected by CI or FA treatment (data not shown). DPA relative abundance in PC was enhanced in adipocytes exposed to DPA compared to those treated with chronic insulin (CI) only (p = 0.013 vs. CI). Similarly, EPA treatment significantly increased EPA relative abundance in PC compared to CI condition alone (p < 0.0001 vs. CI). In C2C12 muscle cells treated directly with FA, DPA and EPA also exhibited a significant incorporation into PC compared to cells treated with PAL alone. As shown in Figure 1, treatment with PAL significantly increased the PC relative amount compared to control (CTRL) (+ 6%, p = 0.005, PAL vs. CTRL). Treatment with LCn-3PUFA restored the PC relative amount to the CTRL value. Cells were harvested for lipid extraction and isolation of phosphatidylcholine (PC). PC collected were characterized after methylation and analyzed by gas chromatography. They were obtained from 3T3-L1 mature adipocyte after a 48-h treatment with 10 µM docosapentaenoic acid (DPA) or 50 µM eicosapentaenoic acid (EPA) with the last 16 h with or without chronic insulin (CI) or from C2C12 myotubes after 16 h of incubation with 500 µM palmitic acid (PAL) with or without 50 µM EPA or 10 µM DPA. Data are mean ± SEM expressed in % of total fatty acids (n = 3-4 obtained after two experiments). In each cell type and line, different letters mean significant differences between groups obtained by ANOVA followed by the Tukey post-hoc test. n.d.: not detected. . Cells were harvested for lipid extraction and phospholipids were separated by high performance liquid chromatography (HPLC). Phospholipid classes were quantified and expressed as the percent of total phospholipids. Data are mean ± SEM (n = 4-6 obtained after three experiments). Different letters mean significant differences (p < 0.05) between groups obtained by ANOVA and the Tukey post-hoc test.
Effect of Fatty Acid Treatments on Insulin Resistance in C2C12 Muscle Cells
As PAL can induce IR in skeletal muscle cells, we investigated the effects of DPA on PAL-induced IR in C2C12 muscle cells ( Figure 2). Akt protein is almost not phosphorylated in the absence of insulin stimulation and FA alone have no significant effect ( Figure S1). The effect of FA was then investigated in cells exposed to insulin for 10 min. PAL treatment for 16 h significantly decreased Akt ser473/4 phosphorylation after insulin stimulation. Similar observations could be observed on thr308/9 residue [10]. When medium was supplemented with LCn-3PUFA, Akt phosphorylation tended to be improved by EPA (p = 0.18, EPA vs. PAL) and was restored by DPA (p = 0.015, DPA vs. PAL).
Chronic Insulin Induced Insulin Resistance in 3T3-L1 Adipocytes
We investigated the effect of chronic insulin on 3T3-L1 adipocytes previously exposed to LCn-3PUFA or not ( Figure 3). It has been previously shown that serum starved adipocytes exhibited a very poor basal Akt protein activation [16]. We then exposed 3T3-L1 adipocytes to insulin for 10 min after serum and insulin deprivation to explore the phosphorylation of Akt protein as an index of insulin sensitivity. We observed that CI treatment induced a strong decrease in insulin-dependent Akt phosphorylation on ser473/4 residue (p < 0.001 vs. CTRL). Although the improvement did not restore Akt activation to the control value, pre-incubation of adipocytes with EPA tended to partially restore, while DPA partially and significantly restored, Akt phosphorylation (p = 0.1 and p = 0.031 vs. CI condition, respectively). 3T3-L1 preadipocytes were differentiated for eight days before a 48 hour-treatment with 10 µM docosapentaenoic acid (DPA) or 50 µM eicosapentaenoic acid (EPA). Insulin resistance was induced by adding insulin (10 µM) during the last 16 h of treatment (chronic insulin, CI). Cells were then starved for 6 h. Before harvesting, cells were stimulated with insulin (100 nM) to evaluate insulin sensitivity by the quantification of Akt protein phosphorylation. Data are mean ± SEM (n = 10 in CI and control (CTRL) groups and n = 20 in LCn-3PUFA groups obtained after four experiments.) * p < 0.05 vs. CI and *** p < 0.001 vs. CI were obtained by ANOVA followed by the Dunnett post-hoc test.
Effects of Chronic Insulin and LCn-3PUFA Treatments on Gene Expression in 3T3-L1 Adipocytes
The mRNA levels of genes related to lipid metabolism, insulin signaling, encoding key transcription factors involved in lipid or FA metabolisms and regulators of energy metabolism were evaluated in 3T3-L1 adipocytes exposed to CI and LCn-3PUFA ( Figure 4). The mRNA amount of genes encoding for proteins involved in lipid mitochondrial oxidation (ACADVL, ACAT1, HADHB) were significantly enhanced by DPA (p = 0.004, p = 0.02 and p = 0.032 vs. CI, respectively) ( Figure 4A). EPA tended to increase ACAT1 mRNA expression (p = 0.055 vs. CI). The mRNA level of CD36 was enhanced by DPA and EPA, but only cells treated with DPA exhibited a significant difference with the CI group (p < 0.05 vs. CI and p = 0.071 vs. CI, respectively). FABP4 and LPL mRNA expression was enhanced by DPA treatment compared to CI group (p = 0.078 and p = 0.014 vs. CI, respectively). PLIN2 mRNA expression was also slightly enhanced by DPA and EPA treatments (p = 0.109 and p = 0.065 vs. CI, respectively). As shown in Figure 4B, IRS2 mRNA level strongly tended to be reduced in CI group vs. CTRL group (p = 0.062). Supplementation for 48 h with DPA significantly enhanced both IRS1 and IRS2 gene expression compared with CI alone (p = 0.019 and p = 0.034 vs. CI, respectively). PPARG, NR1H3 (coding for LXRα1), and PPARA mRNA expressions were all enhanced by DPA supplementation compared to CI treatment alone (p = 0.002, p = 0.003 and p = 0.035 vs. CI, respectively) ( Figure 4C). CTRL and EPA+CI groups exhibited a lower expression of SREBF2 mRNA compared to CI (p = 0.004 and p = 0.06 vs. CI, respectively). No effect of DPA supplementation was observed on SREBF2 gene expression. ADIPOQ (adiponectin) gene expression strongly tended to be enhanced by DPA supplementation vs CI treatment alone (p = 0.078 vs. CI) ( Figure 4D). Finally, APLN gene expression was reduced in the CTRL group (p = 0.02 vs. CI), whereas mRNA level was increased by DPA treatment compared to CI (p = 0.046 vs. CI). EPA treatment had no effect on APLN gene expression compared to CI.
Effects of Chronic Insulin and LCn-3PUFA Treatments on Mature Adipocyte Secretion
Adipokine and chemokine secretions were analyzed in conditioned media (CM) collected after starvation of adipocytes for 6 h ( Figure 5). As compared to CI treatment, IL-6 secretion was lower in control adipocytes compared to insulin-resistant adipocytes (CI). EPA and DPA treatment of CI adipocytes had no effect ( Figure 5A). MCP-1 secretion was increased by EPA supplementation (p < 0.01 vs. CI) and C-C motif chemokine ligand 5 (CCL5) secretion was increased by DPA and EPA supplementation compared to CI condition ( Figure 5B,C). Adiponectin secretion ( Figure 5D) was increased by DPA supplementation (p < 0.05 vs. CI).
Effect of Adipocyte-Conditioned Media on Akt Phosphorylation in C2C12 Muscle Cells
CM collected after starvation for 6 h did not contain FA (data not shown) or insulin allowing the characterization of the effects of adipocyte secretions. In C2C12, CM from insulin-resistant adipocytes significantly decreased the response to insulin as demonstrated by the lower Akt phosphorylation on ser473/4 residue compared to control C2C12 (p < 0.01 vs. CTRL) ( Figure 6). CM from adipocyte supplemented with LCn-3PUFA also induced IR as well, without any preventive effect.
Discussion
Several studies have previously demonstrated the beneficial effects of EPA or DHA, two LCn-3PUFA on IR in skeletal muscle cells exposed to high concentrations of PAL [10,17,18]. The alteration in the activation of Akt could be considered as a first step in the development of IR. We previously showed that LCn-3PUFA can normalize Akt activation by insulin on both serine and threonine residues in C2C12 myotubes exposed to PAL [10]. DPA, an intermediate of DHA production from EPA, remains poorly studied. Here we showed that DPA which is less abundant in plasma membranes than EPA or DHA, is as efficient as other LCn-3PUFA to improve muscle insulin sensitivity in vitro, even when used at a lower concentration (10 µM compared to 30-50 µM of EPA or DHA). This effect is due to a reversion of the lipotoxic effect of PAL. Hence, LCn-3PUFA without PAL, are not able to enhance the effect of insulin on Akt phosphorylation ( Figure S1). The enrichment of membrane's PL with LCn-3PUFA could facilitate protein interactions in cellular membranes and then play a role in the modulation of insulin signaling [15]. A net increase of DPA and EPA concentration was observed in PC from both muscle and adipose cells after 16 to 48 h of incubation. It might partially explain the beneficial effects of LCn-3PUFA. In adipocytes, only a slight improvement of insulin-induced Akt activation was observed when cells were supplemented with LCn-3PUFA before the induction of IR. The model of insulin-resistant adipocytes was first described by Kozka et al. in 1991 [19] who demonstrated a decrease of cell surface glucose transporter 4 in 3T3-L1 adipocytes after chronic exposure to insulin. To further explore the effect of LCn-3PUFA on the cellular effects of insulin downstream of Akt, the exploration of Akt substrate of 160 kDa (AS160) activation, and glucose transporter 4 translocation to plasma membrane will be of particular interest.
In our study, LCn-3PUFA only partially improved IR induced by CI in adipocytes but induced some metabolic adaptations, especially on lipid metabolism. We observed an increase of FAT/CD36 expression with DPA and EPA, DPA having a stronger effect. FAT/CD36 can also modulate insulin sensitivity and the incorporation of essential FA in 3T3-L1 adipocytes [20]. DPA significantly enhanced the expression of lipid mitochondrial oxidation markers (ACAT1, ACADVL, HADHB). This is in perfect agreement with previous results published by Madsen et al. showing that the mitochondrial oxidation of FA was enhanced by EPA and DHA in fully differentiated 3T3-L1 adipocytes [21]. DPA also induced some changes in the expression level of genes involved in insulin signaling. Expression of IRS1 and IRS2 mRNA was enhanced in DPA-treated adipocytes. IRS1 and IRS2 were identified as crucial regulators of adipocyte differentiation as simultaneous knock out of these genes completely blocked differentiation in mice [22]. In our study, we incubated fully-differentiated adipocytes with LCn-3PUFA. We could then hypothesize that DPA may affect the maintenance of these cells, suggesting a preserved capacity for FA and glucose buffering during IR and with over-nutrition. Supporting this idea, the mRNA levels of PPARG, PPARA, and NR1H3, which are crucial transcription factors involved in differentiation and lipid metabolism, were increased in adipocytes exposed to DPA. The exact time-window of the treatment of adipocytes with LCn-3PUFA might be critical as it has been shown that only DHA treatment during the differentiation process had inhibitory effects [23], whereas EPA or DHA incubation before and during the induction of differentiation increased adipogenesis [24].
Although we found marked changes in the mRNA level of key metabolic genes following DPA treatment, it was not sufficient to reduce the ability of adipocyte-CM to induce IR in skeletal muscle cells. Induction of IR in mature adipocytes resulted in a higher secretion of adipokines involved in inflammation and macrophage recruitment (MCP-1 and CCL5). These increases were not reversed by treatment with LCn-3PUFA. Surprisingly, DPA and EPA further increased CCL5 secretion and EPA also had an enhancing effect on MCP-1 secretion by adipocytes. MCP-1, CCL5 (also known as regulated upon activation normal T cell expressed and secreted, RANTES) and IL-6 secretions are known to be enhanced in mature adipocytes compared to undifferentiated adipocytes [25,26]. Nevertheless, incubation of muscle cells with IL-6 for less than 96 h is known to have an insulin sensitizing effect [27], whereas MCP-1 is able to impair insulin signaling [28]. DPA also significantly improved adiponectin secretion and tended to increase its gene expression. Adiponectin has anti-inflammatory and insulin-sensitizing properties [29], which could then have a protective role against metabolic abnormalities in other tissues. However, the beneficial effects of this adipokine could be masked by an opposite action of pro-inflammatory chemokines secreted by adipocytes.
The factors involved in the induction of IR from adipocytes to skeletal muscle cells remains unknown. Literature on the role of extracellular vesicles is increasing and might be useful to identify new mediators. These vesicles secreted by all types of tissues contain proteins, RNA, and lipids, and are involved in the paracrine or endocrine communications between cells and tissues [30]. For example, it has been shown that exosomes (one category of extracellular vesicles) from lipid-induced insulin-resistant muscles are able to modulate gene expression and proliferation of beta cells in mice [31]. Another study showed that exosomes from adipose tissue macrophages of obese mice were able to induce IR in L6 muscle cells [32]. The miRNA 155 seemed to be implicated in these effects. We cannot rule out that exosomes from adipocytes had a role in our study and could modulate IR and metabolism of skeletal muscle cells.
Some hypotheses could be proposed to explain the regulation of intracellular insulin action and lipid metabolism by LCn-3PUFA in muscle cells. LCn-3PUFA have a strong protective effect against lipotoxicity when they are directly added to muscle cells, allowing their incorporation in cell's PL. Consequently, the incorporation of LCn-3PUFA in cellular membranes and an improvement in the membrane's fluidity could facilitate the protein-receptor interactions and, thus, insulin signaling [10]. On the contrary, an excess of saturated FA could induce an elevation of PC content as we observed in moytubes. It remains to be determined if it has a significant impact on insulin sensitivity.
Our experiments in adipocytes suggested that the co-activation of transcriptional regulators, such as PPARs and LXR by LCn-3PUFA, might also play a role, as we observe a regulation of the mRNA level of several genes (ADIPOQ, FABP4, CD36, etc.) that are under the control of these proteins [33,34]. It remains to be determined if similar transcriptional adaptations are involved in the improvement of FA oxidation and detoxification of lipotoxic metabolites in skeletal muscle [35]. The effect of LCn-3PUFA on apelin secretion by adipocytes should be investigated in future studies. Apelin is an adipokine that is known to be insulin-mimetic, which could then modulate the cross-talk between adipose and muscle cells. It was shown to increase glucose utilization in insulin-resistant obese mice [36]. In our study, APLN mRNA levels were increased by DPA treatment. Unfortunately, we did not measure its concentration in CM. It was shown that EPA (200 µM) can activate the secretion and gene expression of apelin in the same model of adipocytes [37]. It remains to be determined if such an effect could be observed in situation of IR with a lower and more physiological concentration of LCn-3PUFA.
In conclusion, DPA has similar effects than EPA on insulin sensitivity in PAL-induced IR in skeletal muscle cells. DPA and EPA partially reversed IR in adipocytes. DPA improved gene expression of key molecules in lipid metabolism and insulin signaling. However, these changes are not sufficient to improve insulin sensitivity of skeletal muscle cells exposed to the CM from insulin-resistant adipocytes. Further studies are necessary to clearly identify which molecules could be responsible for IR transmission from adipocytes to skeletal muscle cells.
Induction of Insulin Resistance in 3T3-L1 Adipocytes
IR was induced by treating mature adipocytes with 10 µM of insulin (chronic insulin, CI) for the last 16 h of day 10. On day 11, cells were washed twice with Phosphate Buffered Saline (PBS), fatty acid-and insulin-deprived for 6 h before the collection of conditioned media (CM). Once CM were collected, fresh DMEM media was added with 100 nM insulin for 10 min for the analysis of insulin sensitivity.
C2C12 Muscle Cell Culture.
C2C12 myoblasts from ATCC (Molsheim, France) were seeded in 100 mm dishes in proliferation medium composed of DMEM with 4.5 g/L glucose, 2.4 g/L sodium bicarbonate, 10% FBS, and 1% of 100× penicillin and streptomycin mix (100 UI/mL and 100 µg/mL respectively). Cells were kept in a humidified, 37 • C and 5% CO 2 atmosphere. The medium was changed every 48 h to ensure growth until reaching 80-90% of confluence and the induction of differentiation into myotubes using differentiation medium (2% horse serum instead of 10% FBS in proliferation medium) for five days before cell treatment.
BSA-Bound PAL Solution for Muscle Cell Treatment with Fatty Acids
A solution of 50 mM PAL (Sigma-Aldrich, Saint-Quentin Fallavier, France) in EtOH was prepared and sterilized by filtration before a dilution (1:25) in BSA-enriched DMEM (2% of FA-free BSA) containing 1% penicillin/streptomycin 100×. This 2 mM PAL solution was sonicated 4 min and heated for 15 min at 55 • C.
Fatty Acid Treatment
The BSA-bound PAL solution was diluted four times with BSA-enriched DMEM (0.5 mM final concentration). LCn-3PUFA-supplemented media were prepared by adding stock solution of EPA or DPA (d = 1:600) to reach a final concentration of 50 and 10 µM, respectively. All media were kept at 37 • C to ensure BSA-binding before treatment. Differentiated C2C12 muscle cells were washed three times with PBS before exposure to BSA-bound 0.5 mM PAL solution (PAL) with or without 50 µM EPA (PAL+EPA) or 10 µM DPA (PAL + DPA) for 16 h. Control cells were challenged with a FA-free BSA-enriched (2%) DMEM containing 2% of EtOH, without FA. After treatments, cells were stimulated with insulin (100 nM) for 10 min, washed twice with cold PBS, and harvested for protein isolation.
Treatment of Myotubes with Conditioned Media
One volume of CM were first diluted with one volume of fresh DMEM not supplemented with BSA or serum. Then, differentiated C2C12 myotubes were exposed to adipocyte-conditioned media for 16 h before insulin stimulation and harvesting.
Reverse Transcriptase Polymerase Chain Reaction
RT-PCR was performed as previously described [14]. RNA extraction from cells was performed using TRIzol ® (Invitrogen, 1 mL/10 cm 2 ) according to the manufacturer's instructions. Chloroform-isoamylalcohol was added (0.2 mL/mL of TRIzol ® ) and samples were mixed and centrifuged 15 min at 12,000× g and 4 • C. Aqueous phase containing ribonucleic acid (RNA) was collected, mixed with isopropanol to precipitate RNA and centrifuged (12,000× g, 4 • C, 15 min). After centrifugation, the pellet was washed with ethanol 70% (v/v), dried, and suspended in water. RNA quantification and integrity were evaluated by measuring the ratio of optical density at 260 nm and 280 nm and by agarose gel migration, respectively. Reverse transcription of messenger RNA was performed from 2 µg of total RNA using the High Capacity RNA-to-cDNA Master Mix from Applied Biosystems (Thermo Scientific). A TaqMan low-density array was used for 3T3-L1 adipocyte samples using a 7900HT Fast Real Time PCR system (Applied Biosystems). The entire list of genes investigated and the corresponding relative expression are supplied in the Supplementary Table S1.
Phospholipid Extraction
Lipids were extracted by adding 5 mL chloroform: methanol (1:1, v/v) to cell extracts. Samples were shaken for 5 min and centrifuged for 10 min at 125× g. A total of 2.5 mL of chloroform and 1.5 mL of 0.9% sodium chloride were then added. After shaking for 5 min and centrifugation at 250× g for 10 min, the chloroform phase was kept and dried under nitrogen flux. Lipids were suspended in 50 µL of chloroform. Lipid extracts were kept at −20 • C until analysis.
Phospholipid Separation and Collection by HPLC
Phospholipid extracts were analyzed in a Thermo Scientific Ultimate 3000 HPLC associated with a charged aerosol detector (Corona, Thermo Scientific) to analyze phospholipid profile and identify the retention time of the different phospholipid classes for collection. Separation was performed using the hydrophilic interaction liquid chromatography (HILIC) method for polar compounds. Briefly, 5-10 µL of phospholipid extracts were injected and separated with an Accucore TM HILIC column (length 1500 mm × diameter 2.1 mm × particle size 2.6 µm, Thermo Scientific). An isocratic inverse phase protocol was used with the following parameters: the mobile phase was (A) acetonitrile/H 2 O (%) 95/5 containing 5 mM ammonium acetate, (B) acetonitrile/H 2 O (%) 50/50; the mobile phase flow and the temperature column were maintained at 0.8 mL/min and 30 • C, respectively. During the run, percentages of A/B were 65/35 at 0 min, 70/30 at 1 min, 85/15 at 20 min, 100/0 at 23 min and 65/35 at 24 min and until the end of the run. Phospholipid collection was performed using an automated fraction collector (Thermo Scientific). Phosphatidylcholine (PC) were collected between 10 min and 12 min and 30 s after injection.
Fatty Acid Profile of PC Fraction
PC fractions were dried under nitrogen and dissolved in boron trifluoride 7% in methanol (Sigma-Aldrich) for FA methylation at 90 • C for 2 h. Analysis of fatty acid methyl-esters (FAMEs) was performed on a gas chromatograph (Thermo Electron Corporation, Waltham, MA, USA) coupled with a flame ionization detector using a select FAME (Agilent Technologies, Les Ulis, France) column (0.25 mm inner diameter, 100 m., 0.25 µm film thickness) and helium as the carrier gas (2.6 bar, constant pressure, inlet temperature of 250 • C). Data were expressed as percent of total fatty acids (% TFA) for EPA (20:5n-3) and DPA (22:5n-3). In the cases of pic area below the detection limit, value was replaced by the mean of the corresponding group.
Quantification of Adipokines and Non-Esterified Fatty Acids in Conditioned Media from Mature Adipocytes
IL-6, adiponectin, MCP-1 and CCL5 were quantified using enzyme-linked immunosorbent assay (ELISA) kit from Sigma-aldrich, Assaypro, Thermo Fisher Scientific and R&D Systems, respectively. Non-esterified fatty acids (NEFA) were quantified using a colorimetric kit from Diasys (Grabels, France) according to the manufacturer's instructions operated on Konelab TM 20 analyzer (Thermo Electron SA, Cergy-Pontoise, France). Regardless of the treatment, every NEFA concentrations found were under the detection limit (10 µM).
Statistical Analyses
All data presented are mean ± standard error about the mean (SEM). For statistical tests, one-way analysis of variance (ANOVA) was performed, followed by a Dunnett post-hoc test taking CI group as reference group, or followed by a Tukey post-hoc test for experiments using palmitic acid on muscle cells and for fatty acid profiles. The number of experiments and samples were indicated in each legend of figures. Analysis have been performed using R software (R Foundation for Statistical Computing, Vienna, Austria), version 3.1.2 and the multcomp package. | v3-fos-license |
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} | pes2o/s2orc | Solution Structure of-Conotoxin PIIIA , a Preferential Inhibitor of Persistent Tetrodotoxin-sensitive Sodium Channels *
-Conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of -conotoxins PIIIA and PIIIA-(2–22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2–22), and -conotoxin GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. H NMR studies found that PIIIA adopts two conformations in solution due to cis/ trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of -conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective -conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by -conotoxins.
VSSCs are inhibited by local anesthetics and modulated by toxins that act at one inhibitory site (site 1) and at least four other sites that result in excitatory actions. -Conotoxins from the venom of marine cone snails act selectively to occlude the pore of the VSSC by competing with TTX and saxitoxin (STX) for binding to site 1 in the P-loop region of the ␣ subunit. To date, sequences for four members of the three-loop -conotoxin class have been published (Table I). GIIIAϪGIIIC from Conus geographus venom are potent blockers of skeletal muscle but not neuronal VSSCs. The three-dimensional structures of selected -conotoxins (19,20) have been used to describe the architecture of the outer vestibule of the VSSC (21)(22)(23)(24)(25). The most recently described member of this class is -conotoxin PIIIA (26) from C. purpurescens (Fig. 1). PIIIA is notable for its ability to inhibit neuronal as well as muscle TTX-S sodium channels (26) and to discriminate among VSSCs in rat brain (27). Thus, PIIIA is the first peptide toxin for investigating the architecture of site 1 of neuronal VSSCs.
Previous studies on GIIIA (21,22) have revealed that the cationic residues, particularly Arg 13 , are important for the high potency of this peptide at Na v 1.4 (see Fig. 1). The high sequence identity and similarities in the three-dimensional structures of GIIIA and GIIIB (19,20) provide a rational basis for comparison with PIIIA, which also contains a number of conserved residues and the same disulfide connectivities as GIIIA and GIIIB (and GIIIC). However, a number of primary structural differences are apparent between PIIIA and other -conotoxins, which may affect the relative position and orientation of backbone loops and their projecting side chains and thus allow PIIIA to interact with both neuronal and muscle forms of TTX-sensitive VSSCs.
To further investigate the potential of PIIIA as a probe of VSSCs, we determined its structure by 1 H NMR spectroscopy and characterized its mode of action on native tissues using electrophysiological and ligand binding approaches. These studies revealed that PIIIA and PIIIA- preferentially inhibited the persistent TTX-S currents in rat hippocampal neurons, whereas in rat DRG the TTX-R current was spared. Comparisons of the three-dimensional structures of PIIIA, GIIIA, and GIIIB revealed important structural differences, including an alternative major conformation accessed by PIIIA, which had not been identified previously in -conotoxins.
Peptide Synthesis
Peptides were prepared by Boc chemistry (28) using methods described for -conotoxins (29). The side chain protection chosen was Arg(tos), Asp(OcHex), Lys(CIZ), Ser(Bzl), and Cys(p-MeBzl). The crude reduced peptides were purified by preparative chromatography, using a 1% gradient (100% A to 80% B, 80 min) and UV detection at 230 nm. The reduced peptides were oxidized at a concentration of 0.02 mM in either aqueous 0.33 M NH 4 OAc, 0.5 M guanidine HCl or aqueous 2 M NH 4 OH. The solution was stirred for 3-5 days at pH 8.1. Purification of oxidized peptide was completed using preparative reversed phase high pressure liquid chromatography.
Electrophysiological Experiments
Electrophysiological experiments were conducted to further investigate the effect of PIIIA on TTX-S and TTX-R sodium channels in native tissue.
Dissociation of Nodose and DRG Neurons-Sensory neurons from rat nodose ganglia and dorsal root ganglia (DRG) were isolated as previously described (32,33). Briefly, young rats (10Ϫ21 days) were killed by cervical dislocation, and the nodose and DRG were carefully removed. The ganglia were placed in physiological saline solution containing collagenase (ϳ1.0 mg/ml type 2; Worthington-Biochemical) and incubated for 1 h at 37°C in 95% air and 5% CO 2 for 24 -48 h. Neurons from the nodose ganglia that were clear and round were selected for experiments. Small diameter cells (ϳ20 m) from the DRG were used, since these have previously been reported to predominantly express TTXresistant Na ϩ currents (34).
Dissociation of Hippocampal CA1 Neurons-Young rats (14Ϫ21 days) were anesthetized under CO 2 and decapitated with an animal guillotine. The brain was removed and transferred to ice-cold artificial cerebrospinal fluid (containing 124 mM NaCl, 26 mM NaH 2 CO 3 , 3 mM KCl, 1.3 mM MgSO 4 , 2.5 mM NaH 2 PO 4 , and 20 mM glucose). The brain was mounted in a vibratome and bathed in ice-cold artificial cerebrospinal fluid equilibrated with 95% O 2 and 5% CO 2 while the 500-mthick slices were prepared. Brain slices were incubated for 30 min with 200 units/ml papain (Worthington), 1.1 mM cysteine (Sigma), 0.2 mM EDTA, and 13.4 mM mercaptoethanol at 35°C. Following incubation, the CA1 region was located, removed, and gently triturated using a fire-polished Pasteur pipette. Neurons of 10 -15 m were used, and cells that were flat, swollen, or grainy in appearance were avoided.
Electrophysiological Recordings-Whole cell Na ϩ currents were recorded using the patch clamp technique. Patch pipettes (GC150F; Harvard Apparatus Ltd., Edenbridge, Kent, UK) were prepared that had resistances of between 1 and 2 megaohms (nodose and DRG neurons) and between 6 and 10 megaohms (CA1 neurons) when filled with pipette solution. Whole cell Na ϩ currents from nodose and DRG neurons were made using a List EPC 7 amplifier (List Medical). Voltage steps were generated by a PC (Dell Pentium) running pClamp (Axon Instruments Inc., Union City, CA). Whole cell Na ϩ currents from CA1 neurons were made using a Axopatch 1D amplifier (Axon Instruments) with voltage steps generated using a PC (Osborne 486-SX) running custom software (14,15,35).
Solution and Toxins-To record Na ϩ currents from DRG and nodose neurons, patch pipettes were filled with the following solution: 135 mM CsF, 10 mM NaCl, 5 mM HEPES, with pH adjusted to 7.2 with CsOH. The bath solution contained 50 mM NaCl, 3 mM KCl, 90 mM tetraethylammonium chloride, 0.1 mM CdCl 2 , 7.7 mM glucose, 10 mM HEPES, with pH adjusted to 7.4 with TEA-OH. To record Na ϩ currents from CA1 neurons, the patch pipette solution contained the following solution: 125 mM CsF, 5 mM NaF, 10 mM KCl, 10 mM TES, with pH adjusted to 7.4 with KOH. The bath solution contained 135 mM NaCl, 5 mM KCl, 3 mM MgCl 2 , 1 mM CaCl 2 , 5 mM CoCl 2 , 5 mM CsCl, 10 mM TES, with pH adjusted to 7.4 with NaOH.
Data Analysis-Three distinct Na ϩ currents were measured: a transient TTX sensitive Na ϩ current (TTX-S I NaT ), a transient TTX-resistant Na ϩ current (TTX-R I NaT ), and a persistent TTX-sensitive Na ϩ current (TTX-S I NaP ). The amplitude of evoked TTX-S I NaT was measured at its peak after subtraction of the current evoked in the presence of TTX (0.5Ϫ1 M). The amplitude of the TTX-R I NaT was measured at least 2 min following the addition of 0.5Ϫ1 M TTX. The amplitude of TTX-S I NaP was measured at the end of a 400-ms voltage step after subtraction of the current evoked in the presence of TTX (0.5Ϫ1 M). All values are expressed as means Ϯ S.E. with n indicating the number of cells in a given series of experiments. Comparisons of two means were made using Student's two-tailed unpaired t test.
H NMR Spectroscopy
All NMR experiments were recorded on a Bruker ARX 500 spectrometer equipped with a z-gradient unit or on a Bruker DMX 750 spectrometer equipped with an x,y,z-gradient unit. Peptide concentrations were ϳ2 mM. PIIIA was examined in 95% H 2 O, 5% D 2 O (pH 3.0 and 5.5; 275-298 K) and in 50% aqueous CD 3 CN (260 -293 K). 1 H NMR experiments recorded were NOESY (36,37) with mixing times of 150, 200, and 400 ms, TOCSY (38) with a mixing time of 80 ms, DQF-COSY (39), and E-COSY in 100% D 2 O (40). All spectra were run over 6024 Hz (500 MHz) or 8192 Hz (750 MHz) with 4 K data points, 400 -512 free induction decays, 16 -64 scans, and a recycle delay of 1 s. The solvent was suppressed using the WATERGATE sequence (41). Spectra were processed using UXNMR as described previously (29) and using Aurelia; subtraction of background was used to minimize T 1 noise. Chemical shift values were referenced internally to 4,4-dimethyl-4-silapentane-1-sulfonate at 0.00 ppm. Secondary H␣ shifts were measured using random coil shift values of Wishart et al. (42). 3 J NH-H␣ coupling constants were measured as previously described (29).
Distance Restraints and Structure Calculations
Peak volumes in NOESY spectra were classified as strong, medium, weak, and very weak, corresponding to upper bounds on interproton distance of 2.7, 3.5, 5.0, and 6.0 Å, respectively. Lower distance bounds were set to 1.8 Å. Appropriate pseudoatom corrections were made (43), and distances of 0.5 and 2.0 Å were added to the upper limits of restraints involving methyl and phenyl protons, respectively. 3 J NH-H␣ coupling constants were used to determine dihedral angle restraints (44), and in cases where 3 J NH-H␣ was 6Ϫ8 Hz and it was clear that a positive dihedral angle was not present, was restrained to Ϫ100 Ϯ 70°. 3 J H␣-H coupling constants, together with relevant NOESY peak strengths, were used to determine x1 dihedral angle restraints (45). Where there was no diastereospecific assignment for a prochiral pair of protons, the largest upper bound for the two restraints was used. Where stereospecific assignments were established, these distances were specified explicitly.
Structures were calculated using the torsion angle dynamics/simulated annealing protocol in X-PLOR (46) version 3.8 using a modified geometric force field based on parhdg.pro. Structure refinements were performed using energy minimization (200 steps) under the influence of a full force field derived from Charmm (47) parameters. Structure modeling, visualization, and superimpositions were done using In- sightII (MSI). Surface calculations, r.m.s. deviations, and hydrogen bond analysis were done using MOLMOL (48). The quality of the structures was analyzed using procheck-NMR (49).
Effects of PIIIA and PIIIA-(2-22) on Neuronal Whole Cell
Na ϩ Currents-The effects of -conotoxin PIIIA and a truncated analogue, PIIIA-(2-22), were investigated on three distinct VSSCs found in neurons of the peripheral and central nervous system. Rat nodose ganglion neurons were used to investigate the transient TTX-sensitive voltage-dependent Na ϩ current (TTX-S I NaT ), DRG neurons were used to investigate the transient TTX-resistant sodium current (TTX-R I NaT ) (34), and rat hippocampal neurons in the CA1 region were used to investigate the persistent TTX-sensitive sodium current (TTX-S I NaP ) (15,50,51).
PIIIA-(2-22) caused a concentration-dependent reduction in the peak amplitude of the TTX-S I NaT in rat nodose ganglia neurons (Fig. 2). In contrast, in rat DRG neurons (n ϭ 12), PIIIA- produced only a small reduction in the peak amplitude of the TTX-R I NaT (Fig. 2). High frequency stimulation can modify the degree of block by some neurotoxins that act in a use-dependent manner. Compared with control, 1 M PIIIA- failed to produce any use-dependent inhibition of peak TTX-S I NaT during 20 depolarizing pulses from a holding potential of Ϫ80 mV to a test potential of Ϫ30 mV for 25 ms delivered at a frequency of 20 Hz (n ϭ 6). In rat hippocampal CA1 neurons, the addition of PIIIA- to the bathing solution caused a concentration-dependent reduction in the peak amplitude of the TTX-S I NaT and the TTX-S I NaP (Fig. 3). Interestingly, PIIIA-(2-22) had a greater effect on the TTX-S I NaP than on the TTX-S I NaT (Fig. 3, inset). At 1 M, the peak amplitude of the TTX-S I NaT was unaffected, whereas the amplitude of the TTX-S I NaP was reduced by ϳ70%.
The native -conotoxin, PIIIA, also reduced the TTX-S I NaT in rat nodose (n ϭ 12), DRG (n ϭ 3), and CA1 (n ϭ 3) neurons (data not shown) with a similar potency to PIIIA- . PIIIA had a preferential effect on the persistent compared with the transient sodium current, being slightly more potent at reducing the amplitude of the TTX-S I NaT current in CA1 neurons than PIIIA- . In preliminary experiments, 10-min bath application of GIIIB (1-10 M) had no effect on either the TTX-S or TTX-R I NaT in rat nodose or DRG neurons, respectively (n ϭ 3; data not shown).
Radioligand Binding Studies--The ability of -conotoxins to displace [ 3 H]STX from VSSCs in human and rat brain and rat skeletal muscle is shown in Fig. 4. All peptides were more potent at the rat skeletal muscle than rat brain VSSCs, with GIIIA and GIIIC showing most selectivity and PIIIA least selectivity. The pIC 50 values and percentage inhibition for these peptides are given in Table I. The data show that PIIIA and PIIIA- have greatest potency at rat and human brain VSSCs, GIIIB has intermediate potency, and GIIIA and GIIIC have least potency. These peptides were less potent than TTX, with none able to fully displace [ 3 H]STX from rat or human brain (relative to TTX displacement). PIIIA and PIIIA- produced the largest displacement of [ 3 H]STX, and GIIIA and GIIIC produced the least displacement. GIIIB was more effective at displacing [ 3 H]STX from rat compared with human brain (Fig. 4, A and B). All displacement curves were best fitted with a Hill slope of Ϫ1.
1 H NMR Spectroscopy--PIIIA was examined by 1 H NMR spectroscopy in a range of different solvent conditions. In aqueous solution at pH 2.5Ϫ5.5 over 275Ϫ298 K, it was apparent that two conformations of PIIIA were present in a ϳ3:1 ratio. In aqueous solution at low pH over 283Ϫ298 K, the NH resonances of several residues, including 4Ϫ7, 10Ϫ12, 20, and 22, were broad, and that of Cys 21 was not observable. At higher pH values and lower temperatures (275 K), these peaks sharpened (residues 4 and 5) or separated into two distinct sets of peaks (residues 6 and 7, 10Ϫ12, 20, and 22) so that complete assignment of the major and a partial assignment of the minor conformations was possible. The assignment of PIIIA was improved by the addition of up to 50% CD 3 CN, where the set of peaks arising from the minor conformation was less evident, and all resonances from the major conformation were present. Chemical shift assignments for PIIIA are given in Table II. The two hydroxyproline (Hyp) residues in PIIIA are assigned as trans from the observation of strong H␦-H␣ i Ϫ 1 NOEs in the case of Hyp 8 and weak to medium H␦-H␣ i Ϫ 1 , together with the stronger H␦-H␣ i Ϫ 1 NOEs in the case of Hyp 18 (52). The minor conformation of PIIIA results from a cis conformation of Hyp 8 , indicated by a H␦-H␣ i Ϫ 1 NOE to the preceding residue. PIIIA- was also examined under similar conditions and found to have almost identical chemical shifts and to adopt two conformations in proportions similar to those observed for PIIIA. The remainder of this paper describes the major conformation observed for both PIIIA and PIIIA- , unless otherwise specified.
Secondary H␣ shifts were used to examine the effects of solvent conditions on the backbone structure of PIIIA (Fig. 5). The shifts of PIIIA- are also shown. These results clearly indicate that the backbone conformation is the same over a range of pH and solvent conditions for the native and truncated sequences. Similarly, the differences in H shifts for AMXbearing side chains, the H␦ shifts of the two Hyp residues, and the H␣ shifts of Gly 6 remain largely unchanged over these conditions for PIIIA and PIIIA-(2-22) (data not shown), indicating that the conformations of the side chains are not significantly affected by changes in the solution environment. One exception is the H protons of Cys 4 , where the chemical shift differences between the H2/H3 protons increase with pH from 0.22 ppm at pH 3 to 0.66 ppm at pH 5. This is likely to arise from the ring current effect of an aromatic ring in proximity to the side chain of Cys 4 at higher pH.
Comparison of the H␣ shifts of the minor conformation of PIIIA show significant differences from residues 7Ϫ11, indicating differences in backbone conformation in these regions. This is supported by differences in H shifts that are evident from residues 6Ϫ11, and also at Cys 16 . Due to low signal intensities, it was not possible to observe peaks for both H and protons of Cys 4 , Cys 5 , and Cys 21 . The ring current effects observed for H protons of the major conformations of PIIIA were not present in the minor conformations, indicating a difference in the positions of either Phe 7 or His 19 relative to Cys 4 . Fig. 5 also compares the secondary H␣ shifts of PIIIA with those of GIIIB, which adopts the same structure in solution as GIIIA (20). Overall, the trends are similar, indicating that the global fold of PIIIA and GIIIB are similar, as may be expected based on their identical disulfide pairings and loop sizes (Fig. 1). However, differences observed at residues 5Ϫ11 and 19Ϫ20 indicate that in some regions significant structural divergence exists. To directly address potential structural differences, we determined the three-dimensional structure of the major conformation PIIIA (see below). Interestingly, the secondary shifts of the minor conformation of PIIIA at residue 10 are more like those of GIIIB than the major form of PIIIA (Fig. 5), suggesting that the structure of the minor conformation of PIIIA is similar to the major conformation of GIIIB and GIIIA.
The local medium range NMR data that provide information on the secondary structure of PIIIA are given in Fig. 6. The presence of several H␣-NH i ϩ 2 , NH-NH i ϩ 2 , and H␣-NH i ϩ 3 NOEs are indicative of the presence of several turns over the entire peptide and perhaps helix over residues 13Ϫ17. Although several long range NOEs are present, these did not correspond to the long range NOEs prescribing the -hairpin of GIIIB (20). In fact, a number of long range NOEs were present that preclude a corresponding -hairpin in the major conformation of PIIIA.
At higher pH values (Ͼ4.0 at 293 K) in aqueous solution or in 50% aqueous CD 3 CN, the hydroxyl proton of Ser 13 side chain was observed. This resonance sharpened considerably with the lowering of temperature (275 K in H 2 O; 260 K in CD 3 CN) to reveal several medium range NOEs to residues 15 and 16, indicative of a hydrogen bond involving the side chain of Ser 13 . These flanking residues apparently stabilize the position of Arg 14 , which has been shown to be crucial to the potency of PIIIA (26). No equivalent interaction has been observed previously for either GIIIA or GIIIB, although an Asp in the equivalent position could conceivably stabilize the crucial Arg 13 through the formation of a hydrogen bond with Gln 14 (GIIIA) or through a salt bridge with Arg 14 (GIIIB).
Three-dimensional Structure of PIIIA-A total of 372 NOEderived distance restrained (149 intraresidual, 98 sequential, 125 long/medium range) and 27 dihedral (16 and 11 1) were used to generate a set of 50 structures of PIIIA. Of these, 46 converged to a similar fold with no NOE violations greater than 0.2 Å and no dihedral violations greater than 3°. The structural analysis and data indicating the quality of the structures are summarized in Table III. From this, it is apparent that the backbone structure is highly defined over residues 3Ϫ22, a conclusion that is supported by high average angular order parameters (S ϭ 0.96) over this region for the and backbone dihedral angles and low backbone r.m.s. deviations (Fig. 7A). Fig. 8A shows an overlay of the 20 lowest energy structures, which indicate that PIIIA is dominated by a series of turns over the N-terminal part of the molecule. From Ser 13 to the C terminus, the structure adopts a distorted helix, with devia-tions from ideality at residues 18 and 19. Examination of the structures of PIIIA reveals that the positions of some side chains are less precisely defined than others. Although there has been no quantitative investigation of the correlation between surface exposure and geometric precision within families of NMR-derived structures, it might be expected that more surface-exposed residues are less conformationally constrained than buried residues. To investigate this correlation for PIIIA, the surface area of each residue is compared with the heavy atom r.m.s. deviations for each residue in Fig. 7B. From this plot, it is evident that surface exposure correlates with r.m.s. deviation values (r 2 ϭ 0.83 for all residues, r 2 ϭ 0.90 for residues 3Ϫ22). This comparison pro- vides an additional means of checking and comparing NMRderived structures, beyond a comparison of r.m.s. deviation values alone.
DISCUSSION
The present study confirms that -conotoxins PIIIA and PIIIA- are potent blockers of neuronal VSSCs. It has been previously shown in radioligand binding studies that PIIIA and GIIIA discriminate among subtypes of the TTXsensitive VSSC found in rat brain (26). This discrimination now extends to PIIIA- and GIIIB in rat brain and to all -conotoxins except GIIIA in human brain. These differences in potency and extent of inhibition of rat and human brain VSSCs arise from relatively small sequence differences, with positions 14 and 18 influencing neuronal activity among the muscleselective -conotoxins. Despite differential effects on neuronal TTX-S sodium channels in brain, GIIIA, GIIIB, and GIIIC have similar potency at skeletal muscle VSSCs. In the peripheral nervous system, PIIIA-(2-22) and PIIIA inhibit TTX-S VSSCs without significantly affecting the TTX-R sodium current. Since -conotoxins have been shown to bind higher in the pore of Nav1.4 than TTX (53), it would appear that in addition to residue differences deep within the pore of the VSSC that render the channel TTX-R (54), additional changes occur further out in the pore to render TTX-R VSSCs insensitive to block by -conotoxins.
PIIIA and its analogue PIIIA- are the first -conotoxins shown to distinguish between transient and persistent TTX-sensitive subtypes. Selective inhibition of persistent over transient VSSCs may control seizures, where the accompanying slow persistent sodium currents might be blocked without affecting the transient action potentials (7). It has been postulated that the persistent sodium channels are the same as those that generate transient sodium currents and that a small fraction of these channels enter a noninactivating mode to generate the persistent sodium current (55,56). This type of persistent current has been observed in cell lines transfected with cDNA for Na v 1.6 (11), Nav1.3 (13), or Nav1.2 (57). The persistent Na ϩ current is also thought to play an important role in pacemaking currents and setting rythmicity in central neurons (58). During hypoxia or in the presence of free radicals (oxidative stress), these channels become more active (15,17,35) and could thus serve as a prominent pathway for Na ϩ influx, triggering a cascade of damaging events that eventually cause cell damage and cell death (59). Hence, specific inhibitors of persistent Na ϩ current may have neuroprotective effects. TTX, lidocaine, and quinidine can also inhibit persistent Na ϩ channels without blocking transient Na ϩ channels (14,15). The -conotoxins extend the list of blockers able to discriminate between persistent and transient sodium currents.
Insights into the structure of the outer vestibule of the Nav1.4 channel have been obtained using the three-dimensional structure GIIIA and GIIIB as molecular calipers (21)(22)(23).
The fact that PIIIA is also able to block Nav1.4 indicates that many of the structural features found in GIIIA and GIIIB might also be conserved in PIIIA. However, additional structural differences must also exist to account for the high affinity of PIIIA and the structurally equivalent PIIIA- at both neuronal and muscle forms of TTX-sensitive VSSCs. The threedimensional structures of PIIIA are compared with those of GIIIA in Fig. 8, C-F. Although the positions of the C-terminal regions overlap and the positions of the functionally important Arg 14 (Arg 13 in GIIIA and GIIIB) are exposed in a similar manner, further comparison indicates marked differences in the orientation of the N-terminal region to the end of loop 1 at Cys 11. In GIIIB, this loop was described as forming a distorted -hairpin that was suggested to exist also in GIIIA (20). This structural feature is not present in the major conformation of 15 (purple), and Cys residues (orange) indicated. A comparison is shown of the positions of surface residues in PIIIA (C) and their counterparts in GIIIA (D). Note that the same surface-exposed residues are found in PIIIA and GIIIA and are all considered important for the potency of GIIIA to 1 VSSCs (Nav1.4). E and F, comparison of core residues in PIIIA and GIIIA, respectively. Core residues differ between these two peptides and thus may contribute to selectivity differences at muscle and neuronal sodium channels. Side chains shown are Leu/Thr (yellow), Hyp/Ser (pink), Arg/Lys (dark blue), His (light blue), Gln (purple), Phe (brown), and Asp (red). PIIIA, where instead a series of loops exist. The structural difference between the major and minor forms arises from a difference at Hyp 8 (Hyp 7 in GIIIA), which adopts a predominately trans conformation in PIIIA but a cis conformation in GIIIA and GIIIB (19,20). Importantly, residues including Lys 8 / Lys 9 and Arg 1 /Arg 2 , which have been shown in GIIIA to be of moderate importance to binding, are placed in an entirely different position in the major conformation of PIIIA (Fig. 8, C and D). However, the effects of the cis/trans isomerization on the C-terminal region of PIIIA are minimal, with the conformation of the putatively important binding residues Arg 14 , Arg 20 , and Lys 17 not being significantly different from their GIIIA counterparts. The structural difference between the major forms of PIIIA, GIIIA, and GIIIB are unexpected, given that these peptides share the same disulfide connectivity and loop sizes and have considerable sequence homology. In contrast, the minor conformation of PIIIA, like GIIIA and GIIIB, arises from the cis form of Hyp 8 /Hyp 7 , indicating that it adopts a three-dimensional structure more closely resembling GIIIA and GIIIB.
Comparison of the major conformation of PIIIA to a model of the minor conformation of PIIIA derived from the three-dimensional structure of GIIIA ( Fig. 9) reveals that the positions of several side chains differ markedly between the two forms. Apart from the aforementioned structural differences at Arg 2 and Lys 9 , the hydrophobic residues Leu 3 and Phe 7 are exposed to the solvent in the cis form yet hug the surface in the trans form, providing a different surface profile. In addition, the side chain of Cys 4 lies above the plane of the His 19 ring in the trans conformation (accounting for the ring current effects mentioned previously) but lies away from His 19 in the cis form, despite the fact that the position of His 19 is unchanged in either conformation. Thus, a simple cis/trans isomerization not only affects the surface of this peptide but somewhat surprisingly also alters the shape of part of the cysteine framework.
Conformational flexibility was proposed as a possible reason for the broadness of resonances associated with residues in the loop 2 of GIIIB (20). The present study shows that there are differing relative proportions and different rates of interconversion between the cis/trans forms. In GIIIA and GIIIB, it is apparent that the cis form predominates, with the trans form being masked by broadening associated with intermediate exchange occurring on the NMR time scale. In PIIIA, the trans form predominates, but the minor form is detectable because the two forms are in slow exchange. It is possible that the bulky Phe residue adjacent to Hyp 8 in PIIIA acts to slow the rate of Hyp isomerization. Two questions arise from the conformational heterogeneity found in PIIIA. First, which of the possible -conotoxin conformations binds to the VSSC? Second, what role is played by these conformational differences in determining VSSC selectivity among -conotoxins? Given that the broadened lines observed in GIIIA and GIIIB are indicative of alternative conformations, it is possible that a minor conformation of these muscle-selective -conotoxins binds to the VSSCs. If this is indeed correct, it could impact on studies investigating the structure of the outer vestibule of the VSSC using the currently available structures of -conotoxins.
Apart from Arg 14 , which has been shown to be important for the activity of PIIIA, it is not known which other residues in this peptide are involved in VSSC binding. An examination of the three-dimensional structures and the surface profile (Fig. 7B) of PIIIA reveals residues that are on the surface (Fig. 8C) and are hence potentially available for interactions with the sodium channel. Along with Arg 14 , these include Lys 17 , Hyp 18 , and Arg 20 , which parallel residues Lys 16 , Hyp 17 , and Arg 19 in GIIIA (Fig. 8D), thus defining a common pharmacophore, as previously suggested (26). Note that Ser 13 is buried, consistent with it playing a structural role that ensures the exposure of Arg 14 . In GIIIA, the residue Asp 12 , which corresponds to Ser 13 in PIIIA, may also play a structural role. The other exposed residues, Lys 9 and Arg 2 , have structural counterparts in GIIIA (Lys 8 and Arg 1 ) but adopt quite different positions in the predominant conformations of these two peptides.
It is interesting that the residues that differ between PIIIA and GIIIA cluster on one face of the peptide, perhaps forming a functionally significant pocket or cavity (Fig. 8, E and F). It is possible that one or more of these mostly hydrophobic and polar residues contribute to binding to the neuronal VSSCs and thus confer broader specificity to PIIIA (and PIIIA-(2Ϫ22)). Thus, core residues and the positioning of exposed residues that differ FIG. 9. Comparison of the major conformation of PIIIA (A) with a modeled structure of its minor conformation (B). The minor conformation was modeled from the structure of the major conformation of GIIIB (20). Structures are superimposed for Arg 12 to Cys 22 . Side chains are labeled as in Fig. 8. between the -conotoxins may contribute to selectivity differences of -conotoxins at VSSCs. The results from this study show that the -conotoxin framework is less conformationally conserved than previously suspected and illustrate the need for careful analysis of the range of structures this class of conotoxins can access. The structure of PIIIA described here provides a new molecular caliper for neuronal and muscle VSSCs. | v3-fos-license |
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} | pes2o/s2orc | Anaerobic Conversion of Saline Phenol-Containing Wastewater Under Thermophilic Conditions in a Membrane Bioreactor
Closing water loops in chemical industries result in hot and highly saline residual streams, often characterized by high strength and the presence of refractory or toxic compounds. These streams are attractive for anaerobic technologies, provided the chemical compounds are biodegradable. However, under such harsh conditions, effective biomass immobilization is difficult, limiting the use of the commonly applied sludge bed reactors. In this study, we assessed the long-term phenol conversion capacity of a lab-scale anaerobic membrane bioreactor (AnMBR) operated at 55°C, and high salinity (18 gNa+.L–1). Over 388 days, bioreactor performance and microbial community dynamics were monitored using specific methanogenic activity (SMA) assays, phenol conversion rate assays, volatile fatty acids permeate characterization and Illumina MiSeq analysis of 16S rRNA gene sequences. Phenol accumulation to concentrations exceeding 600 mgPh.L–1 in the reactor significantly reduced methanogenesis at different phases of operation, while applying a phenol volumetric loading rate of 0.12 gPh.L–1.d–1. Stable AnMBR reactor performance could be attained by applying a sludge phenol loading rate of about 20 mgPh.gVSS–1.d–1. In situ maximum phenol conversion rates of 21.3 mgPh.gVSS–1.d–1 were achieved, whereas conversion rates of 32.8 mgPh.gVSS–1.d–1 were assessed in ex situ batch tests at the end of the operation. The absence of caproate as intermediate inferred that the phenol conversion pathway likely occurred via carboxylation to benzoate. Strikingly, the hydrogenotrophic SMA of 0.34 gCOD-CH4.gVSS–1.d–1 of the AnMBR biomass significantly exceeded the acetotrophic SMA, which only reached 0.15 gCOD-CH4.gVSS–1.d–1. Our results indicated that during the course of the experiment, acetate conversion gradually changed from acetoclastic methanogenesis to acetate oxidation coupled to hydrogenotrophic methanogenesis. Correspondingly, hydrogenotrophic methanogens of the class Methanomicrobia, together with Synergistia, Thermotogae, and Clostridia classes, dominated the microbial community and were enriched during the three phases of operation, while the aceticlastic Methanosaeta species remarkably decreased. Our findings clearly showed that highly saline phenolic wastewaters could be satisfactorily treated in a thermophilic AnMBR and that the specific phenol conversion capacity was limiting the treatment process. The possibility of efficient chemical wastewater treatment under the challenging studied conditions would represent a major breakthrough for the widespread application of AnMBR technology.
INTRODUCTION
Phenols are major contaminants found in wastewaters of several chemical industries, which are often discharged at high temperatures (Busca et al., 2008;Rosenkranz et al., 2013;Wang et al., 2017). Additionally, closing water loops in the chemical sector often result in concentrated, highly saline wastewaters, which increases the complexity of the produced wastewater . Despite the existing physicochemical processes applied for phenol removal, i.e., membrane distillation, pervaporation, adsorption, extraction, nanofiltration, reverse osmosis, and oxidation processes (wet air, electrochemical, ozonation, UV/H 2 O 2 , Fenton) (Villegas et al., 2016;Raza et al., 2019); biological treatment processes are preferred due to its cost-effectiveness. In this regard, anaerobic treatment offers the advantages of minimal energy requirement, low sludge production, and the conversion of organic pollutants into energy-rich biogas. Under both saline and high-temperature conditions, effective biomass immobilization becomes cumbersome, constraining the application of anaerobic sludge bed systems to treat these wastewaters (Dereli et al., 2012;van Lier et al., 2015). Moreover, the phenol degrading capacity of methanogenic consortia is generally very low and is expected to develop only slowly. Therefore, combining anaerobic treatment with membrane assisted biomass separation is an attractive option when phenol conversion is required at high salinity, and thermophilic conditions (Lin et al., 2013;Muñoz Sierra et al., 2018b).
When chemical wastewaters are at high temperatures, direct thermophilic treatment becomes of interest because it reduces the need for cooling the wastewater. Particularly when process water reclamation is envisaged, maintaining the temperature reduces the overall energy requirement. Despite its potentials (van Lier, 2008), thus far, full-scale anaerobic membrane bioreactors (AnMBRs) are not applied for high-temperature chemical wastewater treatment (Duncan et al., 2017). Only a few previous studies have shown the potential of thermophilic conditions for treating phenolic compounds in continuous reactors (Wang et al., 2011;Ramakrishnan and Surampalli, 2013). Wang et al. (2011) compared UASB reactors under mesophilic and thermophilic conditions and concluded that thermophilic anaerobic digestion improves about 30% the degradation of phenolic compounds. Likewise, Ramakrishnan and Surampalli (2013) suggested that thermophilic conversion of phenolic wastewater in an anaerobic hybrid reactor is superior to mesophilic in terms of methane yield, effluent quality, and stability.
Conversely, other studies have also found drawbacks of treating phenol containing wastewater under thermophilic conditions. Fang et al. (2006) indicated that the phenol conversion rate at 55 • C in a UASB reactor was significantly lower than under mesophilic conditions. Furthermore, Levén and Schnürer (2005) concluded that phenolics are mineralized to methane and carbon dioxide under mesophilic conditions, whereas under thermophilic conditions, only benzoic acid is degraded. Muñoz Sierra et al. (2018b) suggested that under mesophilic and hyper-mesophilic conditions (42-45 • C), the phenol conversion capacity of an AnMBR at high salinity is more stable compared to thermophilic conditions. However, because the operation at 55 • C in that study was carried out only during a short-term, it remains unclear whether or not a stable phenol degrading methanogenic consortium may develop. Therefore, this study aims to determine the maximum conversion capacity of a laboratory-scale AnMBR during a long-term operation at 55 • C, treating phenol-containing wastewater at 18 gNa +. L −1 . Moreover, the microbial community activity and structure in response to increasing phenol loading rates along with three phases of operation were evaluated.
Experimental Set-Up and Operation
The experiments were performed by using a 6.5 L laboratoryscale AnMBR reactor, equipped with an ultra-filtration (UF) sidestream membrane module (Figure 1). A tubular polyvinylidene fluoride membrane (X-flow, Pentair, Netherlands) with 5.2 mm inner diameter, 0.64 m length and 30 nm nominal pore size was used. The reactor was equipped with feed, recycle, and effluent pumps with 4-20 mA variable speed (120U/DV, 220Du, Watson-Marlow, Netherlands), pH, and temperature sensors (Memosens, Endress & Hauser, Germany), and a biogas meter (Milligas Counter MGC-1 PMMA, Ritter, Germany). Transmembrane pressure (TMP) was measured by using three pressure sensors (AE Sensors ATM, Netherlands). The temperature of the jacketed reactor was controlled under thermophilic conditions by a thermostatic water bath (Tamson Instruments, Netherlands). The entire set-up was controlled by a programmable logic controller (PLC) connected to a PC with LabVIEW software (version 15.0.1f1, National Instruments, United States).
The AnMBR was operated at 55.0 ± 0.8 • C for 388 days. During this time, the reactor was fed with synthetic phenolic wastewater with phenol concentration between 50 and 800 mgPh . L −1 and a sodium concentration of 18 gNa +. L −1 . The experiment was divided into three phases (I, II, and III). In all of the phases, the phenol concentration was increased step-wise to determine the maximum conversion capacity and the attainable phenol loading rate of the AnMBR. The applied total organic loading rates (OLRs) were between 2.0 and 4.0 gCOD . L −1. d −1 during all phases ( Table 1). The OLRs were calculated as OLR = influent COD (g.L −1 ) * Flow rate (L.d −1 )/AnMBR volume (L). The applied flow rate was 1.0 L.d −1 . An average solids retention time (SRT) of about 120 ± 13 days was maintained. The average SRT was calculated periodically as SRT av = X (g VSS in the AnMBR) av /X removed net (gVSS.d −1 ) , where X removed net resulted from the biomass removed.day −1 (sampling for tests) and the biomass returned.day −1 (after some tests).
The AnMBR was completely mixed applying a turnover of 170 times.d −1 .The membrane unit was operated at a cross-flow velocity of 0.65 m s −1 . A cyclic membrane filtration operation was carried out, consisting of 500 s filtration and 30 s backwash. Backwash was done by reversing the permeate pump flow. An operational flux of 4.0 L . m −2. h −1 was applied as a result of the experimental settings. The permeate flow was controlled with the variable speed of the effluent and influent and pumps, and it was regularly double-checked manually. An average TMP of 177 ± 92 mbar and a total membrane filtration resistance of 9.77 × 10 12 [1/m] could be maintained during the AnMBR long-term operation.
Volatile Fatty Acids
Prior analysis, 10 mL of the AnMBR permeate samples was filtrated over 0.45 µm filter paper. The filtrated liquid was diluted with pentanol (300 mg.L −1 ). 10 µL of formic acid (purity > 99%) was added into the final 1.5 mL vials. Volatile fatty acids (VFAs) were measured by gas chromatography (GC) using an Agilent 19091F-112, 25 m × 320 µm × 0.5 µm column and an FID detector (Agilent 7890A, United States). The sample injection volume was 1 µL. Helium was used as carrier gas with a total flow rate of 67 mL/min and a split ratio of 25:1. The GC oven temperature was programmed to increase from 80 to 180 • C in 10.5 min. The temperatures of the injector and detector were 80 and 240 • C, respectively.
Permeate Characterization
Phenol concentrations were measured using high-performance liquid chromatography HPLC LC-20AT (Shimadzu, Japan) equipped with a 4.6 mm reversed-phase C18 column (Phenomenex, Netherlands) and a UV detector at a wavelength of 280 nm. The mobile phase used was 25% (v/v) acetonitrile at a flow rate of 0.95 mL . min −1 . The column oven was set at 30 • C. Fast phenol measurements were also carried out by Merck -Spectroquant Phenol cell kits by using a spectrophotometer NOVA60 (Merck, Germany). Hach Lange kits were used to measure chemical oxygen demand (COD). The COD was measured using a VIS -spectrophotometer (DR3900, Hach Lange, Germany) making proper dilutions to minimize interference by high chloride concentrations, without compromising the accuracy of the measurement.
Anaerobic Phenol Conversion Rates
Batch tests were conducted in triplicate at the end of phase II to assess the phenol conversion recovery after AnMBR performance perturbation. The volatile suspended solid (VSS) were analyzed according to standard protocols using the lowest possible sample volume (APHA, 2005). Biomass samples were taken with a 150 mL syringe and transferred to 500 mL Schott glass bottles. The bottles were filled to a volume of 400 mL with AnMBR biomass (0.68 g VSS), and a medium containing acetate (3.1-4.6 g . L −1 ), phenol (60-109 mgPh . L −1 ), 6 mL . L −1 macro-and 0.6 mL . L −1 micronutrients solution, 10 mM phosphate buffer solution, and 18 gNa +. L −1 . Three consecutive feedings of the medium were applied. Initial COD and phenol concentrations varied between 3.4-5.1 gCOD . L −1 and 60-109 mgPh . L −1 , respectively. Temperature and mixing were controlled in an orbital incubator shaker (New Brunswick Biological Shakers Innova 44/44R, United States) at 55 • C and 120 rpm respectively. Periodically, liquid samples were taken, and phenol and COD concentrations were measured. Phenol conversion rates [mgPh . gVSS −1. d −1 ] were calculated by using the slope of the phenol concentration vs time curve in each bottle. After the batch tests were finished, the supernatant was removed and biomass was returned to the AnMBR. Similarly, at the end of the AnMBR operation at day 388, biomass samples were taken, and phenol conversion batch tests were carried out with initial phenol concentrations of 40 and 60 mgPh . L −1 .
Specific Acetotrophic and Hydrogenotrophic Methanogenic Activity
Specific acetotrophic methanogenic activity (SMA) tests were performed in triplicate using an automated methane potential test system (AMPTS, Bioprocess Control, Sweden). All the SMA tests were carried out at 55 • C, following the method described by Spanjers and Vanrolleghem (2016). For the hydrogenotrophic methanogenic activity, 250 mL Schott glass bottles were filled with biomass (0.57 g VSS) and medium (6 and 0.6 mL . L −1 macro-and micro-nutrients solution, respectively and 10 mM phosphate buffer solution at pH 7.0) to a liquid volume of 200 mL. The gas-phase of the bottles was exchanged by using a gas exchange board (G.R Instruments B.V, Netherlands) with a gas mixture of 80% CO 2 and 20% H 2 to an end pressure of 0.5 bar during 5 continuous automated cycles to ensure the complete absence of oxygen. Bottles were incubated in an orbital shaker (New Brunswick Biological Shakers Innova 44/44R, United States) at 55 • C and 120 rpm for 10 days, and biogas production was calculated using the exact headspace volume and the drop in headspace pressure versus time. The headspace pressure was measured as described by Coates et al. (1996) using a pressure transducer. The methane content of the biogas was analyzed by using a gas chromatograph 7890A (GC) (Agilent Technologies, United States) equipped with a front thermal conductivity detector (TCD). The temperature of the oven was 45 • C for 6 min, then 25 • C/min to 100 • C. The temperatures of the front inlet, and a front detector were both 200 • C.
Microbial Community Analysis
Biomass samples were taken from the AnMBR on days 88, 241, and 376 to evaluate the microbial community. The DNA extraction was performed from 0.5 g of biomass by using the DNeasy UltraClean Microbial Kit (Qiagen, Hilden, Germany). Agarose gel electrophoresis and Qubit3.0 DNA detection (Qubit dsDNA HS Assay Kit, Life Technologies, United States) were used for quality and quantity control of the DNA. The amplification of the 16S rRNA gene (V3-V4 region) was performed and followed by high throughput sequencing using the MiSeq Illumina platform (BaseClear, Leiden, Netherlands) using the primers 341F (5 -CCTACGGGNGGCWGCAG-3 ) and 785R (5 -GACTACHVGGGTATCTAATCC-3 ). The Illumina fastq reads (2 × 250) were processed in the QIIME2 pipeline (2018.7) (Bolyen et al., 2019). Reads were quality filtered, denoised, and the amplicon sequences variants (ASVs) were resolved with the DADA2 plugin (Callahan et al., 2016), removing chimeras with the "consensus" method. The taxonomic classification of the representative sequences of ASVs was performed with the "classify-consensus-vsearch" plugin (Rognes et al., 2016) using the SILVA (132) database as a reference. The representative sequences were aligned with the MAFFT algorithm (Katoh and Standley, 2013), and a phylogenetic tree was constructed with FastTree (Price et al., 2010). The feature table and tree were exported to the R environment. Differential abundance analyses between reactor operation phases were performed with the DESeq2 library (Love et al., 2014). The abundance and the tree were visualized with the phyloseq library (McMurdie and Holmes, 2013). ASVs with differential abundances within the operational phases were analyzed with BLAST against the refseq RNA database to identify the closest related species. The sequences reported in this paper have been deposited at ENA under the study accession number PRJEB38467.
Thermophilic AnMBR Performance
Influent and effluent phenol and COD concentrations during the long-term thermophilic AnMBR operation are shown in Figure 2. During days 0-96 in phase I, the effluent COD concentrations were in the range of 2.0-10.0 gCOD . L −1 , and the corresponding removal efficiencies were between 59.0 and 92.3% ( Figure 2B) at an average OLR of 3.8 gCOD . L −1. d −1 . The increase in the phenol loading rate ( Table 1) from 0.01 to 0.09 gPh L −1 d −1 concomitantly increased the phenol removal efficiency from 54 to 95% (Figure 2A). At the end of phase I, the phenol concentration in the reactor reached about 738 mgPh . L −1 (Figure 2A).
Volatile Fatty Acids (VFAs) Spectrum
Throughout the entire thermophilic operation, VFAs were detected in the AnMBR permeate (Figure 3), indicating a limiting methanogenic conversion capacity. The effluent COD mainly consisted of acetate (0.02-9.6 g . L −1 ), which peaked at almost 9.6 g . L −1 between 133 and 144 days when phenol accumulation occurred. Concomitantly, the butyrate concentration increased to 616 mg . L −1 in this period. In phase II, high concentrations of acetate (5.2 g . L −1 ) and to a lesser extent butyrate (295 mg . L −1 ) were found at day 260 when phenol again accumulated after an increase in the phenol loading rate. Propionate was most of the time present in the reactor effluent with an average concentration of 129 ± 57 mg . L −1 . The valerate concentration increased to 254 and 156 mg . L −1 when the reactor performance deteriorated in phases I and II, respectively. In phase III, on day 350, an increase in all VFAs was observed when reactor phenol concentration increased to 124 mgPh . L −1 .
Conversion Rates and Methanogenic Activity
Specific Methanogenic Activity and Phenol Conversion Rates SMA tests using acetate as the substrate showed a drop in the methanogenic activity of the phenol-degrading biomass in phase I, meanwhile, an increase in the specific phenol conversion rates was observed ( Table 2). During phase II the lowest observed SMA was 0.04 ± 0.02 gCOD-CH 4 . gVSS −1. d −1 , following phenol accumulation at the end of phase I. In phase III when the influent phenol concentration was decreased to 0.2 gPh . L −1 , the SMA of the phenol-degrading biomass increased to 0.13 gCOD-CH 4 . gVSS −1. d −1 , which was similar to the value observed at the beginning of phase I. Table 2 also shows the calculated maximum in-reactor phenol conversion rates in the different phases. At the end of phase I, the phenol conversion rate had increased from an initial value of 1.3 to 21.3 ± 0.2 mgPh . gVSS −1. d −1 . During the recovery periods of perturbation, i.e., days 156-198, the phenol conversion rate was 5.9 ± 0.3 mgPh . gVSS −1. d −1 . From days 220 to 262, the phenol loading rate increased until an average of 16.9 ± 0.6 mgPh . gVSS −1. d −1 . In phase III, the phenol conversion rate decreased to the range 2.6 ± 0.1-7.6 ± 1.6 mgPh . gVSS −1. d −1 .
Ex situ Phenol Conversion Rate After Reactor Perturbation
After reactor perturbation at the end of phase II, the phenol conversion rate was assessed in a batch test. Three different feedings were applied with different initial phenol concentrations (see Table 3). After the first feed, the phenol conversion rate was calculated as 4.0 ± 1.4 mgPh . gVSS −1. d −1 after 7 days of incubation. The phenol conversion rate increased to 9.6 ± 2.6 and 10.5 ± 3.3 mgPh . gVSS −1. d −1 after the second and third consecutive feeding respectively.
Specific Methanogenic Activity and Phenol Conversion Rates at the End of the Long-Term Operation
Since low acetate-fed SMA values at the end of the long-term thermophilic operation period were observed of 0.13 ± 0.10 gCOD-CH 4 . gVSS −1. d −1 , SMA tests were performed with both acetate and hydrogen as electron donor. Likewise, the phenol conversion rate was measured in batch test with an initial phenol concentration of 40 and 60 mgPh . L −1 ( phenol conversion rate of 32.8 ± 0.5 mgPh . gVSS −1. d −1 was found, applying an initial phenol concentration of 60 mgPh . L −1 .
Microbial Community Structure Analysis
The microbial community analysis revealed a total of 141 ASVs with differential abundance across samples. Figure 4 shows the genera from both bacteria and archaea domains with main population changes in relative abundance at the different phases of the thermophilic AnMBR at 18 gNa + L −1 . Petrotoga (Thermotogae class) was enriched along with the long-term operation up to 21.1% in phase III. Thermovirga (Synergisitia class) increased from 8.0 to 14.1% from phase I to II and then decreased to 7.95% relative abundance in phase III. Acetomicrobium also belonging to phylum Synergistetes FIGURE 4 | Heatmap of the genera from bacteria and archaea domains in the thermophilic AnMBR that were positive after differential abundance analysis (DESeq2) among the three phases of reactor operation. The color scale ranges from 0 to 22% relative abundance.
(see Supplementary Figure 1) increased from 0.35% during phase I to 3.93% during phase III. A 100% similarity was found with the specie Acetomicrobium hydrogeniformans sp. (see Supplementary Table 1). Syntrophobacter affiliated to the class Deltaproteobacteria increased from 0.1 to 0.5% in relative abundance from phase I to phase II; however it was not detected in phase III. A hit of 100% similarity was found for the halophilic bacteria Syntrophobacter sulfatireducens sp. Similarly, the relative abundance of WS6 bacterium decreased during the long-term operation. In the case of the Clostridia class, the major abundance decrease was observed with the genera Caldicoprobacter and Tepidimicrobium, whereas an increase was observed for Syntrophaceticus (3.3%), Pelotomaculum (1.2%), and Proteiniclasticum (2.4%). Also, Corynebacterium and Enterococcus genera belonging to Actinobacteria and Bacilli class increased significantly to 6.6 and 1.8%, respectively, in phase III.
Methanosaeta was about 19.7% in relative abundance in phase I decreasing to 11.4% in phase II, while it almost disappeared in phase III. The halotolerant Methanosaeta harundinacea sp. obtained a 100% similarity on this genus. Concomitantly, Methanobacterium increased from 3.5% in phase I to 11.5% in phase II, but significantly dropped in phase III. In phase III, the hydrogenotrophic methanogens Methanoculleus increased to 6.1% in relative abundance becoming the dominant archaea while Methanolinea decreased significantly. Figure 4 also illustrates that the most prominent amplicon sequence variants belong to 15 and 2 different classes in the bacterial and archaeal domain, respectively.
For the bacteria microbial community, the abundance of microorganisms of the class Actinobacteria, Bacilli, Synergistia, Gammaproteobacteria, and Thermotogae increased correspondingly to 6.8, 5.9, 22.9, 12.6, and 26.6% at the end of the long-term thermophilic operation.
Thermophilic AnMBR Performance and Volatile Fatty Acids Spectrum
The COD removal efficiency started to decrease at the end of the applied phenol loading rate of 0.09 gPh . L −1. d −1 (Figure 2B), i.e., before day 96 in phase I, and subsequently acetate concentration increased (Figure 3). The observed deterioration possibly can be ascribed to an accumulation of a phenol conversion intermediate, such as benzoate (Wang and Barlaz, 1998;Tay et al., 2001), impacting acetoclastic methanogens or syntrophic acetate oxidizers. After the phenol loading rate was further increased to 0.12 gPh . L −1. d −1 , equivalent to a sludge phenol loading rate of 22.6 mgPh . gVSS −1. d −1 , the OLR was decreased to 2.3 gCOD . L −1. d −1 to avoid high volatile fatty acids concentrations in the reactor. The increase in phenol loading rate at the end of phase I, severely impacted COD and phenol conversion, resulting in removal efficiencies below 10%. The phenol concentration in the reactor broth reached 738 mgPh . L −1 (Figure 2A), which apparently inhibited both phenol conversion and methanogenesis. Similarly, Madigou et al. (2016) reported inhibition of phenol conversion at a concentration of 600 mgPh . L −1 , and biogas production nearly stopped when phenol reached 895 mgPh . L −1 . In order to reduce the phenol concentration in the AnMBR, the phenol loading rate was decreased to 0.02 gPh . L −1. d −1 (3.8 mgPh . gVSS −1. d −1 ) on day 133 to prevent further intoxication, while the OLR was reduced to 2.0 gCOD . L −1. d −1 . In phase II, the reactor performance again deteriorated. Consequently, biogas production almost ceased on day 260 when the reactor phenol concentration reached 616 mgPh . L −1 , inhibiting the methanogenic consortium. Surprisingly in phase III, by applying a phenol loading rate of 0.04 gPh . L −1. d −1 (9.3 mgPh . gVSS −1. d −1 ) on day 352, phenol and COD removal efficiencies decreased to 50 and 80%, respectively. The latter indicates an increased sensitivity of the biomass to phenol compared to phase I.
Volatile fatty acid concentrations in the permeate, mainly acetate, indicated a limiting methanogenic conversion capacity for the OLR applied. The high observed acetate concentration reaching 9.6 g . L −1 (Figure 3) when phenol accumulation occurred, could have caused a secondary inhibitory effect to the anaerobic microorganisms such as propionate-oxidizing bacteria (Van Lier et al., 1993). Despite the peaks of 620 and 300 mg . L −1 of butyrate during the phenol accumulation events, all VFA concentrations (propionate, butyrate, and valerate) remained at a relatively low level, commonly observed in anaerobic reactors under anaerobic thermophilic conditions (Van Lier et al., 1993). Caproate always remained below detection level throughout the entire thermophilic AnMBR operation. Previous studies inferred that at 55 • C, phenol-degrading biomass might degrade phenol via n-caproate (Evans and Fuchs, 1988;Fang et al., 2006). On the other hand, Hoyos-Hernandez et al. (2014) have demonstrated that thermophilic phenol degradation is possible via the benzoate conversion route, similar to mesophilic conditions, contrasting these previous studies. Even though benzoate was not determined analytically in the AnMBR, the absence of caproate in any of the VFA analyses strongly suggests that the prevailing phenol conversion pathway was likely via benzoate carboxylation at 55 • C. Following the proposed pathway, benzoate is subsequently de-aromatized to form cyclohexane carboxylic acid, which is cleaved to heptanoate, degraded further through β-oxidation to form valerate, propionate, and acetate, or directly to propionate and butyrate, which are further degraded to acetate (Liang and Fang, 2010).
Conversion Rates and Methanogenic Activity
The assessed SMA values found are similar to those reported elsewhere for phenol degrading biomass under thermophilic conditions, i.e., 0.1 gCOD-CH 4 . gVSS −1. d −1 (Fang et al., 2006). The phenol conversion rate assessed in ex situ batch tests increased to 10.5 ± 3.3 mgPh . gVSS −1. d −1 , inferring a 62% recovery of the conversion capacity after about 17 days of incubation. Interestingly, also Chen et al. (2008) found a maximum phenol conversion rate after 10 to 14 days of incubation. The ex situ assessed phenol conversion rate at the end of the batch tests was about 61% of the AnMBR conversion rate before phenol accumulation occurred (see Table 3). Apparently, the phenol-degrading biomass could recover from the phenol shocks after a relatively short recovery period, while being exposed to low phenol concentrations in the reactor (<100 mgPh . L −1 ). The observed maximum phenol conversion rate of 32.8 ± 0.5 mgPh . gVSS −1. d −1 in batch test at the end of the experiment is higher than the maximum observed phenol conversion rate in the AnMBR during phase I, which likely can be attributed to longterm adaptation of the biomass to phenol after three phases. Comparing our present results with the different studies summarized in Table 5, which are performed in a broad range of temperatures, the observed maximum phenol conversion rates are comparable to those obtained under both mesophilic (Rosenkranz et al., 2013;Franchi et al., 2020) and psychrophilic (Collins et al., 2005;Scully et al., 2006) conditions, using other anaerobic high-rate reactors configurations. Nonetheless, it should be recalled that our present results were obtained under extreme salinity conditions applying sodium concentrations of 18 gNa +. L −1 .
The acetate-fed SMA obtained at the end of the longterm operation was 0.15 ± 0.04 gCOD-CH 4 . gVSS −1. d −1 similar to what was observed at the beginning of phase I and at the end of phase III. Remarkably, the hydrogenotrophic methanogenic activity was a factor 2.3 higher (0.34 ± 0.08 gCOD-CH 4 . gVSS −1. d −1 ), which made us hypothesize that methanogenesis of acetate proceeded via syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis, rather than aceticlastic methanogenesis. Of interest is the relatively high hydrogenotrophic SMA, which indicates that acetate is possibly syntrophically methanised via oxidation to hydrogen and carbon dioxide. Note that syntrophic acetate oxidation is energetically more favorable at elevated temperature and high acetate concentration and is more often observed as the dominant pathway in a large number of thermophilic anaerobic reactors (van Lier, 1996;Westerholm et al., 2016;Li et al., 2020). Moreover, hydrogenotrophic methanogenesis is commonly observed at elevated salt concentrations (De Vrieze et al., 2016). Based on our present results, follow-up research in a thermophilic AnMBR should reveal the minimum acetate concentration that is required to enhance phenol conversion and to avoid high VFA concentrations in the permeate. In such research, concentrations in the range of 0.3-1.0 gCOD . L −1 acetate, and 500-800 mgPh . L −1 phenol are recommended. Applying similar salinity conditions, we propose an OLR of 2.0-2.2 gCOD . L −1. d −1 and phenol loading rates of 15-20 mgPh . gVSS −1. d −1 in order to maximize the phenol conversion capacity, without compromising the methanogenic activity.
Microbial Community Structure
Petrotoga was enriched to 21.1% during the AnMBR long-term operation. This bacteria has been enriched under anaerobic thermophilic conditions from an oil reservoir containing mostly halophilic species (Dellagnezze et al., 2016). Similarly, high salinity conditions will enrich for salt-tolerant and halophilic Thermovirga and Clostridium species (Muñoz Sierra et al., 2018a). Acetomicrobium hydrogeniformans sp., which also increased during phase III, is found in oil production water and is capable of producing hydrogen. Some species required NaCl for growth (Cook et al., 2018). In the case of the Clostridia class, the genera found Syntrophaceticus are known for their capability of syntrophic acetate oxidation, and Pelotomaculum play an important role in the conversion of phenol and benzoate under methanogenic conditions (Chen et al., 2008).
The abundance of bacteria of the classes Actinobacteria, Bacilli, Synergistia, Gammaproteobacteria, and Thermotogae increased in phase III. Microorganisms corresponding to these classes have been found in thermophilic saline environments. Clostridia class remained in a comparable relative abundance along with the phases, but with changes at genus level. Especially, microorganisms that belong to this class have been reported as the most essential fermentative bacteria and syntrophic phenol degraders as Pelotomaculum (Chen et al., 2009;Muñoz Sierra et al., 2019).
Our results showed a high relative abundance of uncultured microorganisms in the AnMBR. Bacteria belonging to the class JS1, and the candidate phylum Atribacteria (see Supplementary Figure 1) were dominant during phases I (9.15%) and II (9.41%). Microorganisms belonging to Atribacteria, mostly have been found in deep-sea methane-rich sediments (Carr et al., 2015). Lee et al. (2018) suggested a fermentative role of these microorganisms, capable of using various substrates, and syntrophic acetate oxidation coupled with hydrogen scavenging methanogens. Recently, the first culturable representative strain of this phylum was isolated and it was confirmed that it plays a role in hydrogenogenic fermentative metabolism (Katayama et al., 2019). In our case, the highest species similarity found was Bacillus alkalitolerans strain T3-209, with only 83%.
In the archaea domain, the microbial dynamics indicated a clear switch from acetotrophic to hydrogenotrophic methanogens in the class Methanomicrobia. Both the microbial community structure and the observation that at the end of the experiment the biomass hydrogenotrophic methanogenic activity was substantially higher than the acetate-fed methanogenic activity, support our hypothesis that acetate conversion switched from aceticlastic methanogenesis to acetate oxidation coupled to hydrogenotrophic methanogenesis.
Perspectives and Further Research
The observed AnMBR performance perturbation following phenol accumulation indicated that the cultivated thermophilic phenol-degrading biomass was dependent on the presence of active methanogens. A drop in the hydrogenotrophic methanogenic activity may have led to the observed reduced phenol conversion capacity. It should be noted that the entire experiment was performed under high salinity (18 gNa +. L −1 ) conditions. Further research will focus on the role of syntrophic acetate oxidation and phenol degradation intermediates (e.g., benzoate). Our present study showed that highly saline phenolic wastewaters indeed could be treated in a thermophilic AnMBR. However, the achievable phenol conversion capacity was restricted to 20 mgPh . gVSS −1. d −1 , determining an applicable phenol loading rate of about 0.12 gPh . L −1. d −1 . Although thermophilic operation will bring operational energy benefits when treating high-temperature industrial wastewaters with the target of process water reuse, the phenol conversion capacity of the reactor will be lower than when opting for mesophilic operation.
CONCLUSION
Maximum COD and phenol removal efficiencies of about 95% were achieved during the long-term thermophilic AnMBR operation at 18 gNa +. L −1 . However, severe perturbations occurred following relatively small increments in the phenol loading rate from 0.01 to 0.12 gPh . L −1. d −1 . Moreover, by exceeding a sludge phenol loading rate of 20 mgPh . gVSS −1. d −1 the system immediately responded in phenol build-up to concentrations higher than 600 mgPh . L −1 leading to significant deterioration of methanogenesis. The observed maximum phenol conversion rates were 21.3 ± 0.2 and 32.8 ± 0.5 mgPh . gVSS −1. d −1 in the AnMBR, and in ex situ batch test at the end of the reactor operation, respectively. The absence of caproate in the VFAs spectrum inferred that the phenol conversion pathway was likely via benzoate carboxylation. The assessed hydrogenotrophic SMA was a factor 2.3 higher than the acetate-fed SMA. Correspondingly, microbial population dynamics indicated that hydrogenotrophic methanogens were enriched during the long-term operation and Clostridia class was dominant. Overall, thermophilic AnMBR operation under high salinity seemed to be susceptible to sudden increase in phenol loading rate or phenol shocks, indicating that the specific phenol conversion capacity under the studied conditions was limiting the treatment process.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ebi.ac.uk/ ena, PRJEB38467. | v3-fos-license |
2021-09-24T05:23:37.619Z | 2021-07-28T00:00:00.000 | 237606094 | {
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} | pes2o/s2orc | Evaluation by MALDI-TOF MS and PCA of the diversity of biosurfactants and their producing bacteria, as adaption to weathered oil components
Highlights • MALDI-TOF MS MS and PCA allowed the identification and categorization of newly isolated strains from highly weathered oily soils.• The isolated strains exhibited high diversity in terms of emulsification and solubilization activities.• FTIR analysis combined with PCA revealed further diversity level of the produced biosurfactants.
Introduction
Microbial biosurfactants are surface-active molecules, which are produced by microorganisms, especially bacteria, to increase the bioavailability of hydrophobic substrates [1]. If appropriately produced and formulated, they can be applied in different fields ranging from hydrocarbons remediation to the food industry [2]. The surface-active biomolecules have unique properties such as low toxicity and a relatively simple preparation process [3]. Therefore, these characteristics have increased their demand in a wide range of industries of agrochemicals, fertilizers, petrochemicals, pharmaceuticals, cosmetics, and beverages as well as in petroleum and mining industries [4]. Biosurfactants have gained huge interest from the oil industry due to their property of reducing surface tension, which enables their use in bioremediation of oil pollution and oil recovery. In bioremediation, the major roles performed by biosurfactants include the increase of solubilization and desorption of the hydrophobic pollutants, increasing thus their bioavailability [5]. The future of bioremediation of soils contaminated by hydrocarbons is expected to be based on the surfactant-enhanced bioavailability of the contaminants. Biosurfactants have hydrophobic and hydrophilic sides, which lead to the formation of an interface region between fluids with different polarities such as water and oil hydrocarbons [6]. In comparison with other synthetic or chemical surfactants, biosurfactants have various advantages including their biodegradability, digestibility, and biocompatibility making them more effective in cleaning the environment and in the bioremediation of contaminated soils [7].
Biosurfactants are categorized based on their composition and origin. The molecules are grouped based on their molecular weight, namely high molecular weight (HMW) and low molecular weight (LMW Glycolipid biosurfactants are characterized by the property of a polysaccharide on their head groups. When the head group is affected by changes in pH or by electrolytes, the micellar structure of these biosurfactants changes [8]. Among the glycolipid biosurfactants, various hydrophobic fatty acids have the ability to reduce Kraft temperature at which the solubility of a surfactant matches the surfactant's critical micelle concentration (CMC) [1]. Then, fatty acids ensure that their structure is maintained. On the other hand, rhamnolipids are composed of rhamnoses and hydroxyl fatty acids [9]. Sophorolipids are made of sophorose, which forms the hydrophilic part of the surfactant [10]. The hydrophobic part is made of fatty acids that contain a long chain of carbon atoms [11]. Surfactin, a lipopeptide biosurfactant is composed of surfactin molecules that consist of seven amino acids and fatty acids that form the hydrophobic part. High molecular weight biosurfactants majorly function in the emulsification of hydrophobic molecules and they have a wide diversity in their structures and functions [8]. Biosurfactants are generated from microorganisms like Bacillus, Pseudomonas, and Acenetobacter genera among others. This is achieved through the process of fermentation and the enzyme-substrate reaction. Synthesis of biosurfactants is not only achieved intracellularly but can also be carried out extracellularly by the use of biocatalysts, which are enzymes. The hydrophilic and hydrophobic moieties can either be synthesized independently following different pathways and they can both be dependent on a substrate or one can be induced by the substrate and the others are synthesized a new [12]. In the amphiphilic structure of the biosurfactants, the hydrophobic moiety can be either a hydroxyl-fatty acid or a long-chained fatty acid. The hydrophilic moiety can be either a carboxylic acid, amino acid, carbohydrate, phosphate, alcohol, or cyclic peptide. The synthesis of these moieties involves the carbohydrate metabolic pathway, which synthesizes the hydrophilic moiety, and the hydrocarbon metabolic pathway, which synthesizes the hydrophobic moiety. In most cases, the first enzymes used in the process of precursor synthesis are regulatory enzymes [13]. Various factors influence biosurfactants synthesis and affect the rate of production and their properties. Some of the factors that influence the optimum production of biosurfactants include the carbon and nitrogen sources, temperature, pH, oxygen availability, carbon-nitrogen ratio, and agitation [14]. Consequently, all the reported parameters affect production, composition, and activity. However, in bioremediation approaches, the recalcitrance of certain petroleum compounds to biodegradation results from their strong adsorption on soil particles. Here, adsorption is understood as the retention of a solute in solution by the surface of a solid material, whereas adsorption refers to the retention of the solute within the mass of the solid [15]. The strength of this adsorption depends on the contaminant and the matrix on which it is adsorbed [16]. It is important to note that the climate of the gulf region is characterized by harsh conditions, resulting in accelerated weathering of oil components. Under such conditions, it is anticipated that indigenous microorganisms have adapted to synthesize specific biosurfactants that are effective for these weathered oils, both to mobilize the hydrocarbons and to enhance their bioavailability and biodegradation. Indeed, many failures of bioremediation applications in regions characterized by harsh weather and soils can be attributed to the use of un-acclimated bacteria and their associated biosurfactants [17]. The novelty of this work resides in the ability of the indigenous Qatari strains to produce biosurfactants that have the potential to enhance the biodegradation of weathered hydrocarbons or washing of weathered soil. These biosurfactants may be more active under the harsh physical and chemical conditions with Qatari soils, making them appropriate for use in enhanced oil recovery and bioremediation. However, it is necessary to demonstrate the biodiversity of the producing bacteria and strains, and their adaptation to harsh conditions and weathered hydrocarbons by adapting their biosurfactants structures and activities. A collection of bacteria was formed, and most of the isolates have been identified and differentiated, and their potential to produce biosurfactants has been evaluated. The isolates from the highly weathered oily sites would lead to selecting interesting strains more appropriate in applications in areas characterized by harsh weather. Their adapted biosurfactants were investigated by Fourier-transform infrared spectroscopy (FTIR) and categorized by principal component analysis (PCA). Indigenous bacteria would be highly adapted and when re-introduced or stimulated would conduct to the remediation of these sites.
Soil samples collection
Soil samples were collected from different areas in Qatar, characterized by aged pollution with petroleum hydrocarbons. Two dumpsites in Dukhan industrial area were selected because solid and liquid wastes from the oil industry were discharged and left for self-purification for more than three years in the open air. One sampling site was chosen away from the sea line and another in the intertidal zone of Dukhan. All these selected sites are characterized by pollution with weathered oil as previously demonstrated [18]. An automotive workshop in Doha industrial area was also selected with recent and aged pollution with diesel and lubricants. All samples were collected from the surface soil layer of 10 cm, homogenized in sterile 50 mL tubes and preserved at 4 ∘C until use ( Table 1). The pH of the samples was ranging from 6.85 ± 0.05 to 7.15 ± 0.06. The moisture was of 8.6 ± 0.3 to 13.6 ± 0.5. Based on results of our previous work, the total petroleum hydrocarbons (TPH) and the oil range organics (ORO) contents of the samples were ranging were from 190 mg/kg to 325 mg/kg, and the TPH diesel range organic (DRO) contents were always less than 1 mg/kg g/kg (Alkaabi et al., 2020). Nitrate, ammonia and phosphate contents were below 0.140 mg/g, 0.007 mg/g and 0.350 mg/g respectively.
Enrichment cultures with native bacteria
Hydrocarbon-degrading bacteria were enriched from soil samples using a standard enrichment method [18,19,20] as shown in Fig. 1. In 50 mL sterile tube, 1 g of soil sample was added to 25 mL Minimal Salt Medium (MSM) that contains (w/v): 0.1% NH 4 Cl, 0.1% KH 2 PO 4, 0.4% Na 2 HPO 4 .2H 2 O, 0.006% KCl and 0.04% MgSO 4 .7H 2 O with pH set at 7.0 before sterilization. Before inoculation, the medium was supplemented with 1% (v/v) of a trace element solution. The trace element solution was composed of (g/100 mL): EDTA, 0.1; ZnSO 4 , 0.042; MnSO 4 , 0.178; H 3 BO 3 , 0.05; NiCl 2 , 0.1. All media were autoclaved for 20 min at 121 • C. Solid MSM was obtained by adding 15 g/L agar. 5% (v/v) dieselthe sole carbon source-was then added in a final culture volume of 20 mL. Diesel stock was kindly provided by Mesaieed Refinery (Qatar) with a complete analysis, indicating hydrocarbons composition ranging from n-C 12 to n-C 25 . It contained 750 g/L carbon. All cultures were incubated at 30 • C for two weeks in a shaker set at 200 rpm. After one week of incubation, 2 mL of each culture was used to inoculate a fresh medium, as performed in the first culture. Three subsequent sub-culturing were then performed, before proceeding to the isolation of the enriched hydrocarbon-degrading bacteria.
Table 1
Isolated hydrocarbon-degrading bacterial strains from the oily-soils sampled from different locations in Qatar.
Isolation and purification of bacterial strains from the enrichment cultures
100 µL of the enriched cultures were plated on MSM solid media and then coated with 100 µL diesel, spread on the surface of the plate which was then sealed with PARAFILM® and incubated at 30 • C. Colonies of different aspects (form, color, shining, and size) were spread on LB solid agar plates. The isolated colonies were separately displaced by streaking in LB solid agar plates. Purification of the isolated strains was performed by six successive subculturing steps using separated colonies at each step. Strains were preserved at -80 • C in LB medium containing 30% glycerol.
Sample processing and protein extraction for MALDI-TOF MS analysis
In order to produce the most reliable results, two techniques were used to prepare the samples for matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [21,22,16] . The extraction was performed using equivalent volumes of ethanol and formic acid [23] (Wang et al., 2012). A separate colony of a strain from an LB plate was suspended in 300 µL sterile water and then re-suspended in 900 µL absolute ethanol. After centrifugation at 10,000 rpm for 5 min, the pellet was supplemented with 1 mL of formic acid (70%) and then 1 mL acetonitrile (100%). After centrifugation, 1 µL of the supernatant was introduced into the biotarget of 48 sample spots. Then, 1 µL of alfa-cyano-4-hydroxycinnamic acid (HCCA) matrix solution containing (50% acetonitrile and 2.5% trifluoroacetic acid in ultra-pure water) was added for protein extraction. Triplicates were performed by spotting separate colonies in three wells. In parallel, a second method was performed using the whole bacterial cells. Here, one fresh and separate cell colony was transferred using a sterile loop into the well of the target plate. Subsequently, mass fingerprints were directly generated by the apparatus, allowing obtention of a MALDI-TOF MS score and a protein profile for each strain.
Identification of the bacterial isolates
The MALDI-TOF MS analysis was performed using a Brucker apparatus (Model: microflex LT/SH from Bruker Daltonics, Germany). The identification of the bacterial isolate was performed by the Bruker Biotyper software.
The mass spectra generated by MALDI-TOF MS were analyzed for similarities to the database entries.
Proteins with an m/z between 2,000 m/z and 20,000 m/z z are used to identify bacterial strains based on individual mass peaks corresponding to specific ribosomal proteins of distinct types of microorganisms, compared to a database available in the software. The mass peaks corresponding to specific ribosomal proteins are used for the identification of the isolates by similarities establishment. The results were in form of log(scores) as generated by default by the Biotyper software. A log scale from 0.000 to 3.000 was obtained for each strain. If the score ranges between 2.300 and 3.000, the identification is at the highly probable species level with high confidence. The scores ranging between 2.000 and 2.299 provide high accurate identification at the genus and probable correct at species level. The scores in the range between 1.700 -1.999 provide probable genus-level identification. The scoring system was provided by Bruker Daltonics / Germany, using the software MBT Compass version 4.1.80.24 (Data base version: MBT Compass Library Build 9.0.0.0, Revision: F). It is the most up to date scoring system as per the manufacturer guideline.
Data processing
The protein profiles were generated by the Bruker Flex Control software as mass spectra (linear and positive mode, at 60 Hz laser frequency and 35% intensity) using an acceleration and a source voltage set at 20 kV and 18.7 kV, respectively. For each spectrum, 240 laser shots in 40-shot steps were generated from different areas of the sample spot and analyzed using the default settings. The obtained protein profiles were analyzed by using a Flex Analysis and a Biotyper RTC 3 software. Therefore, the mass spectra, which were generated by MALDI-TOF MS, are multivariate data. Each mass signal is from a distinct molecular dimension. The multivariate statistical processes are applied to differentiate between bacterial strains. The principal component analysis (PCA) was used to reduce the dimensionality and keep the original information. The peaks of each MALDI-TOF MS spectrum were the basis of the PCA analysis. The peaks can be of proteins or peptides. The PCA led to the formation of assembled groups of spectra exhibiting similar characteristics and differences. A 2D or a 3D coordinate system can be generated with the data. However, the 2D system is more recommended since it plots PC1 against PC2 and offers in most cases, more than 80% of the total variance between the studied spectra. Besides, the hierarchical relationship between the isolates was investigated by establishing the dendrogram using the MALDI Biotyper Compass Explorer software that adopts default settings as per the manufacturer's instructions. The analysis by PCA and dendrogram was performed according to the standard operating procedure of the instrument and software. The spectra generated using triplicates were processed for smoothing and subtracting the baseline. Spectra with a strong background noise or high/low intensities were not used and then excluded. A main spectral projection (MSP) was created with the automated MSP creation functionality within the MALDI Biotyper 3.0 software by using the considered spectra. Indeed, the MSP provides information on the means of the peak frequencies, peak masses, and peak intensities. The generated MSPs for each isolate were fed to the functionality of PCA or dendrogram for analysis and generating graphs.
Biosurfactants production
5% (v/v) diesel was used as the carbon source to produce the biosurfactants in MSM liquid medium. The production medium consisted of 19 mL MSM liquid medium supplemented with 1 ml diesel in a 50 mL falcon tube, tightly sealed with PARAFILM® foil and incubated at 30 • C, in a shaker set at 200 rpm for one week or two in dark, as specified with results. The cultures were inoculated with a suspension of cells from colonies of each isolate, formed overnight on LB plates. The initial cell density at the inoculation time corresponded to an optical density (OD) at 600 nm of 0.15. The cell density and the growth of each strain was evaluated by the determination of the colony-forming unit (CFU).
Colony-forming Unit (CFU) determination
CFU was determined by plating 100 µL of serial dilutions of the cultures on LB plates. The dilution corresponding to a number of colonies between 30 and 100 was considered. Then the CFUs were calculated for each ml of the corresponding culture.
Determination of the diesel solubilization activity
The procedure employed to determine the solubilization activity produced by each strain was based on the method described by Mnif et al. (2013) [24] using diesel. Biosurfactants solution was prepared by centrifugation of 1 mL of the cultured MSM (10,000 rpm for 5 min.). The produced supernatant was separated (0.9 mL) and added to 10 mL of Tris-HCl 20 mM (pH 7.0). Then, 0.2 mL of diesel was added to the tube to have a final 2% (v/v) diesel. Control Number 1 corresponded to a similar mixture except that 0.2 mL Tris-HCl buffer was used instead of diesel to determine the quantity of diesel solubilized by the strains in the culture medium. Control Number 2 was performed using 0.9 ml Tris-HCl instead of the culture's supernatant (without biosurfactants) and 0.2 mL diesel to determine the spontaneous solubilization of diesel in the aqueous phase. All tubes were incubated overnight at a vertical position in a shaker set at 30 • C and 300 rpm, in the dark. After incubation, tubes were left out for 0.5 h to separate the diesel top layer. Then, 4 mL of the aqueous phase were mixed with an equal amount of pure hexane for extraction of diesel with vigorous vortexing during 2 min and then centrifugation at 4,500 rpm for 15 min. The optical density of the hexane phase was measured at 295 nm. Hexane was used as a blank. The concentration of diesel in the hexane phase was calculated from the slope of a calibration curve of different concentrations of diesel in hexane, ranging from 0.3 to 1.25 µL of diesel/mL. The amount of solubilized diesel and percent of solubilization were calculated using equations 1 and 2: The OD values were obtained from the calibration curve.
The initial concentration of diesel was 18.349 µL/mL. The solubilization activity of the freeze-dried biosurfactants was determined in 1 mg solubilized in 0.9 mL Tris-HCl and a similar method was employed.
Determination of the emulsification activity
MSM cultured broth of each strain was centrifuged (10,000 rpm for 15 min). 1 mL of the supernatant was supplemented with 0.15 mL diesel. It was vortexed for 2 min and left for 1 h to separate the diesel from the aqueous phase [25]. The aqueous phase was used to measure the optical density at 400 nm. Control Number 1 was performed with the fresh MSM instead of culture broth. Control Number 2 was performed with the culture broth and 0.15 distilled water instead of 0.15 ml Diesel. The emulsification activity (EA) was calculated as units of emulsification per milliliter (EU/mL), where each 0.01 absorbance is considered as one activity unit according to Patil Emulsification Activity (EU/mL) = (OD both -OD control 1 -OD control 2 / 0.01) × dilution factor (3) The emulsification activity of the freeze-dried biosurfactants was determined in 1 mg solubilized in 1 mL Tris-HCl and a similar method was employed.
Extraction of biosurfactants from the cultures
Biosurfactants were extracted from each culture broth after centrifugation at 10,000 rpm for 15 min at 5 • C. The employed method was that described by [29]. The pH of the supernatants was adjusted to 2.0 using 6.0 M HCl, and the solution was incubated for 24 h at 4 • C. Then, CHCl 3 /CH 3 OH (2:1) was added to an equal volume, vigorously mixed, and incubated overnight at room temperature. After centrifugation for 15 min at 10,000 rpm, the pellet was suspended in Milli-Q water. The concentrate was neutralized to pH 7.0 with 1 M NaOH solution, then freeze-dried.
Analysis of the freeze-dried biosurfactants by Fourier transform infrared (FTIR)
The dried extracts of biosurfactants of each culture were analyzed by FTIR using an FTIR Perkin Elmer 400 FT-IR/FT-NIR spectrometer. The spectra were recorded in the range of 400-4000 cm − 1 .
Isolation of hydrocarbon-degrading bacterial strains from weathered hydrocarbons-samples
Since the objective of this research was to establish a collection of bacterial strains isolated from highly polluted soils with weathered hydrocarbons in Qatar, we developed the strategy of isolation and screening shown in Fig. 1. It is expected that the diversity of the hydrocarbon-degrading bacterial strains is affected by the nature and composition of the carbon source. The weathering status of the hydrocarbons and their adsorption to the soil matrix should affect their availability, and thus the produced biosurfactants by the adapted bacterial community [30]. A total of 19 bacterial strains were isolated from the different locations (Table 1). All these strains are expected to be highly adapted to weathered hydrocarbons and tolerant to high toxicity exhibited by the 5% diesel employed in the enrichment and isolation steps.
Identification of the isolated strains by using the MALDI-TOF MS technique
The 19 isolated strains were identified by MALDI-TOF MS protein profiling, using the available database in the used apparatus. Indeed, a MALDI score and a reproducible protein profile were obtained for each strain by MALDI TOF MS profiling ( Table 2). Fig. 2 shows an example of protein profiles obtained with different strains. Proteins with an m/z in the range between 2000 and 20 000 m/z are used to identify bacterial strains based on individual mass peaks corresponding to specific ribosomal proteins of distinct types of microorganisms, compared to a database available in the software. It is interesting to notice that 15 isolates were identified at the level of Bacillus genus (five Bacillus subtilis, four Bacillus cereus, two Bacillus atrophaeus, one Bacillus licheniformis, two Bacillus sonorensis, and one Bacillus mojavensis). Three isolates belong to the Lysinibacillus genus (two Lysinibacillus boronitolerans, and one Lysinibacillus fusiformis). One isolate was Enterococcus faecium.
Bacillus mojavensis is known for forming biofilms and producing biosurfactants [34]. Surfactants produced by Bacillus atrophaeus were reported by Rodriguez et al., (2018) [35]. Bacillus sonorensis is known as a hydrocarbon-degrading bacterium [31] and biosurfactant producer [36]. The genus Lysinibacillus is distinguished but relatively close to Bacillus as phylogeny, composition of peptidoglycan, and physiology [37]. Lysinibacillus fusiformis and Lysinibacillus boronitolerans were originally known as Bacillus fusiformis and Bacillus boronitolerans before 2007 [37]. Lin et al., (2010) [38] reported the potential of Bacillus fusiformis in the biodegradation of naphthalene. Recently, Li et al. (2020) [39] showed the survival of Lysinibacillus fusiformis in petroleum environments. While Lysinibacillus sphaericus and Geobacillus sp are also shown activity in the biodegradation of petroleum hydrocarbons and biosurfactants production [40], however, the involvement of Lysinibacillus (Bacillus) boronitolerans in oil hydrocarbons degradation was never reported. It was only associated with tolerance to boron [37]. Ozyurek and Bilkay (2017) [41] reported the isolation of Enterococcus faecium as a hydrocarbon-biodegrading bacterium in crude oil, waste mud pit, and drilling fluid.
These results also show that some well-known bacteria with their high degradation activity of oil hydrocarbons were not isolated from the highly weathered oil-soils used in this work. The bacterium Pseudomonas aeruginosa was not found within this new collection of hydrocarbondegrading bacterial strains. By considering the potential of biosurfactants to be applied to oil degradation and recovery, Rhamnolipids produced by Alcaligenes eutrophus have been shown to be effective in increasing the solubility of polychlorinated biphenyls (PCBs) and their mineralization [42]. Commercialized Rhamnolipids of Pseudomonas aeruginosa (P. aeruginosa) enhanced extraction of hexadecane residues in a sand column, compared to sodium dodecyl sulfate (SDS) and sorbitan monooleate, both are synthetic surfactants [43]. Rhodococcus ST-5 and Badus AB-2 surfactants allowed recovery of 95% of the crude oil residues. Biosurfactants of Bacillus subtilis were reported but not in the field of oil remediation or recovery [44]. B. licheniformis is also able to produce biosurfactants. Crude biosurfactant production by B. licheniformis can reach 1 g/L, with emulsification power increased up to 96% [45]. Bacillus flexus was also able to produce biosurfactants, even with a low emulsification index.
Differentiation of the isolated strains using PCA and dendrogram analysis
Further information on the relationships between the closely related isolates can be obtained upon combining the MALDI-TOF MS analysis and PCA. By reducing the dimensions of objects being studied, linear combinations could be created for variables, representing the studied objects. The PCA results are shown in Fig. 3A. The PCA clustering revealed large biodiversity between the studied strains at the protein level. The total variance of the 10 principal components is shown in (Fig. 3B). PC1 (34%), PC2 (25.5%) and PC3 (10.5%) combine to show a total of 70% variability in the data. Using the first three principal components, five clusters were obtained. The distances between the clusters indicate the variations at a group level, while the distance between the strains (within each cluster) shows the differences in protein profiles at the strain level. Clusters I and II include B. subtilis strains (S5, S27, SA16, SA6, and SA28). The strain B. mojavensis SA29 falls withincluster I, indicating high similarity in their protein profiles. Similarly, B. licheniformis (S33) falls within Cluster III that includes the two B. sonorensis (SA9 and SA11). Whereas, cluster IV includes three B. cereus (SA17, S24, and SA31). Cluster V includes the three Lysinibacillus strains (SA3, SA4, and SA10). Interestingly, the strain B. cereus S32 is located at a huge distance from Cluster IV, demonstrating a large variation in its protein profile in comparison to the other B. cereus strains in cluster IV. Similarly, the Enterococcus faecium SA12 is distinct from any of the five clusters.
The hierarchical relationship between the isolates was investigated by establishing the class dendrogram (Fig. 4). The dendrogram revealed two major clusters (I, II). Cluster I is formed of 9 strains and cluster II of 10 strains. In each cluster, two distinct clades are distinguished. Cluster I includes clades Ia formed with two strains of Bacillus subtilis (SA6 and SA28) and the strains Enterococcus faecium (SA12). Clade Ib is further subdivided into the sub-clades Ib1 formed with the three Lysinibacillus strains (SA3, SA4, and SA10), and sub-clade Ib2 formed with the three Bacillus cereus strains (SA17, S24, SA31). Cluster II includes 2 clades (IIa, IIb). Clade IIa is formed with the strain Bacillus licheniformis (S33) and clade IIa1 is formed with the two Bacillus sonorensis strains (SA9 and SA11). Clade IIb2 includes one Bacillus cereus strain (SA32). Clade IIb1 is further divided into sub-clade IIb1a formed with the two bacillus subtilis strains (S5 and S27) and sub-clade IIb1b formed with the strains Bacillus subtilis (SA16), Bacillus mojavensis (SA2), and Bacillus atrophaeus (SA29). The phyloproteomic classification of the studied strains allowed the differentiation and separation of strains belonging to the same Bacillus species. Two B. subtilis strains (SA6 and SA28) were classified in clade Ia while other strains (SA5, S27, and SA16) were classified in IIbIa. Similarly, three strains belonging to Bacillus cereus (SA17, S24, and SA31) belong to cluster Ia, while the strain Bacillus cereus SA32 is under cluster IIb. The approach of using protein profiles may be informative in differentiating strains belonging to the same species [46]. Indeed, MALDI-TOF MS could be used to characterize isolates with much higher precision than that of 16S rRNA sequencing. The resolving capability of MALDI-TOF MS is higher than that of 16S rRNA sequencing because it covers a wider range of proteins than the 16S ribosomal subunit. The ability to accurately characterize differences on the strain level is valuable in understanding differences among redundantly isolated strains identified as the same species, while isolated at different occurrences, such as those isolated from different contaminated soils. MALDI-TOF MS is useful for the identification and grouping of isolates based on the strain-level variations [47]. Here, it allowed differentiation and categorization of newly isolated strains from highly weathered oily soils. It also demonstrates the high diversity of the strains as a consequence of their adaptation to harsh conditions (chemical and physical).
Diesel solubilization and emulsification activities of the isolated strains
All the isolated strains were cultured in the MSM medium containing 5% (v/v) diesel as the sole carbon source. After one week and 2 weeks of incubation at 30 • C, the supernatants were collected and used to assess the diesel solubilization (SA) and emulsification (EA) activities of each strain (Table 3). These results show that SA and EA activities are fluctuating in a wide range of 1.4 U/ml to 42.1 U/ml for EA and 1.17 (%) to 8.6 (%) for SA. Moreover, some strains exhibited high EA activity and low SA or vice versa. They confirm the diversity of the strains. The growth of the strains was also varying from 11 to 99 10 8 CFU/mL, which was almost the same or strongly reduced after 2 weeks of incubation. The specific activity for EA and SA activity would be the characteristics of each strain. The strains Bacillus subtilis S5 exhibited the highest growth (99 10 8 CFU/mL) and SA (8.6%) and the second-highest EA (31.7 U/mL). The strain Lysinibacillus fusiformis SA4 exhibited the highest EA (42.1 U/mL) but low SA activity (1.17%) and low growth (11 10 8 CFU/ml). However, the strain Lysinibacillus boronitolerance SA10 produced 2.6 (%) as SA and 23.2 U/mL as EA with 33 10 8 CFU/mL. In contrast, Lysinibacillus boronitolerance SA3 providing a similar growth (30 10 8 CFU/mL) produced 6.7 U/mL of EA and 4.6 % as SA. All these results confirm the high diversity among the collection of the isolated strains from weathered oily-soils. The diversity of the biological activities of the strains, as growth and diesel solubilization and emulsification does not reflect their ordering shown in the dendrogram of Fig. 3. Indeed, each of these activities can modify the succession of the strains differently. Besides the diversity based on the protein profiles, a diversity based on the function can be established. Bacteria employ biosurfactants as one of multiple adaptation mechanisms to use hydrocarbons as substrates. The adaptations largely express specific physiological responses to specific microenvironments of the cell and its nutritional requirements [48]. Indeed, some bacteria developed a strategy of pseudo-solubilization to increase the solubility of poorly soluble hydrocarbons. Therefore, they produce a high capability of self-assembly in micelles, hemi-micelles or aggregates, by using highly dynamic low-molecular-mass molecules of biosurfactants. However, other bacteria develop a direct interaction with hydrocarbons by a different tool through the wall-bound biosurfactants. Thus, the cell surface becomes appropriately hydrophobic. Indeed, the high molecular mass molecules are called bioemulsifiers, which adsorb tightly to the hydrocarbons and thus increase their apparent solubility by covering them in the aqueous phase. Biosurfactants share few traits, although their wide variety of specialization and mechanisms to deal with hydrocarbons. All mechanisms are around the interactions between the three phases (cell physiology, cell surface, hydrocarbons that are the substrates for the cell). To achieve the goal of passing the hydrocarbons across the wall, the cell develops reversible and temporary modifications of the membrane adapted to the nature, composition, and type of hydrophobicity of the available substrates, in addition to making the hydrocarbons more soluble [48]. It is not excluded that a "substrate effect" can be developed during the growth of the cell. However, the synthesis pathways of most of the biosurfactants are not yet elucidated. In contrast, the hydrophobic substrates are known to influence the structural variations of biosurfactants to make them particularly active on the same substrate. In addition, it is now established that biosurfactants stimulate the growth of their producing strains, playing a vital role in the interaction between the microbial communities and their micro-environment [48].
Production and stability of the biosurfactants activity during growth of the strains
The concomitant production of the EA and SA by four selected strains based on the results of Table 3 was investigated. The strain Bacillus subtilis S5 is characterized by high growth and SA and EA activities. The strain Bacillus subtilis SA6 exhibited lower growth and activities than Bacillus subtilis S5. The strain SA28 is Bacillus subtilis strain characterized by a much higher specific activity of SA and EA activities. The strain SA9 is Bacillus sonorensis selected because it is characterized by shining colonies, embedded in the extensive production of exopolymeric substances.
The SA and EA activities of the four strains were evaluated during dynamic growth in 5% diesel-MSM. Results are shown in Figure 5.
The four strains exhibited, during their growth, a maximum of production of the emulsification activity after 5 days incubation, while their solubilization activities were maximal after 7 days incubation. The activities were remarkably unstable with a continuous and rapid decrease after reaching the maximum. The behavior of the 4 Bacillus strains was almost similar.
This result confirms that all the bacterial strains develop reversible and temporary modifications of the membrane adapted to the available substrates while making a more continuous activity of solubilization of the hydrocarbons. The concomitant production of both activities is necessary to achieve the goal of passing the hydrocarbons across the wall. The bio-emulsification touching the cell surface structure and functionality is normal in that it attains its highest activity during the vegetative growth of the cells, after which, the cells enter into sporulation phase with loss of and disintegration of the cell membranes, and thus of the related activities. The solubilization activity can continue to be produced by the sporulating cells or the remaining vegetative ones.
Analysis of the freeze-dried biosurfactants by Fourier transform infrared (FTIR)
The FTIR absorption spectra of the biosurfactants produced by the studied strains revealed the presence of protein, polysaccharide, ester, and carbonyl groups indicating the presence of lipopeptides (Fig. 6 A-D). The strong absorption peak at 3270 cm − 1 corresponds to the stretching vibrations of -NH and -OH groups related to peptides [49]. The absorption peaks in the range from 2960 cm − 1 and 2860 cm − 1 are due to the asymmetric and symmetric stretching of the methylene groups of lipids (-CH 2 ) [50,51]. The absorption peak at 1640 cm − 1 -1630 cm − 1 can be attributed to the CO-NH bend (due to the stretching vibrations of C=O and C-N groups), which confirms the presence of a peptide group in the biosurfactants [52]. The absorption peaks at 1451 cm − 1 and 1360 cm − 1 appear due to the presence of alkyl (-CH 2 and -CH 3 ) groups [53]. The presence of ether moiety is confirmed due to the presence of an absorption peak at 1230 cm − 1 . Hence, the biosurfactants were expected to contain fatty acids and peptide moieties indicating their lipopeptides nature [49].
PCA analysis of the FTIR spectra
To have more insights into the variations in the obtained FTIR spectra for the biosurfactants, PCA analysis was performed (Fig. 7). The FTIR-PCA clustering revealed three main groups. Group 1, the largest one, contained the biosurfactants obtained from nine bacterial strains; The FTIR bands at 2924 cm − 1 , 2850 cm − 1 were preserved in all FTIR spectra of the studied, biosurfactants. However, variations were observed in FTIR bands in the region from 800 cm − 1 to 1640 cm − 1 . Obvious shifts in the amide I peaks from 1645 cm − 1 to 1623 cm − 1 were observed in the FTIR spectra of the biosurfactants assembled in PCA-Group 2 ( Fig. 7 & Fig. 6B). These shifts are attributed to changes in protein secondary structure [54,55]. Moreover, the peaks at 1154 cm − 1 that may be ascribed presence of ester bonds [56] are characteristics of the biosurfactants categorized in PCA-Group 2 ( Fig. 7 & Fig. 6B). The weak bands at 930 cm − 1 which corresponds to phosphorus and oxygen stretching in aliphatic and aromatic molecules [57] were clearly observed in the FTIR spectra of biosurfactants clustered in PCA-Group 3 containing biosurfactant produced by B. subtilis (SA6 & SA28) and L. fusiformis (SA4) (Fig. 6C), which may indicate the presence of phosphate in these biosurfactants. The FTIR peak at 1028 cm − 1 attributed asymmetric and symmetric C-O-C stretching of ester [58,59] was only observed in the FTIR spectra of the biosurfactants of B. lichenifomis S33. The FTIR peak 1003 cm − 1 observed only in the FTIR spectra of B. atrophaeus SA2 (Fig. 6D) may be assigned to O-C-O extend vibrations of carboxylic acids. This is a remarkable indication of the oxidation of the hydroxyl groups in the hydrolysates from the medium peptides [60].
Indeed, even with the same group of isolates clustered based on their protein profiles, the corresponding biosurfactants are clustered in different groups based on their FTIR spectra. This means that each isolate was able to adapt differently its biosurfactant composition in response to the existing weathered oil components and the weather conditions. However, the three strains, Enterococcus faecium (SA12), Bacillus mojavensis (SA2) and B. licheniformis (S33) which were not within any of the isolates' clusters produce biosurfactants with structure that are strongly different from those of all the clustered ones, although several similarities were observed. The strain S32, which is not clustered with the other isolates produces biosurfactants highly similar to those of FTIR-cluster I, grouping 9 out of the 19 surfactants.
Conclusion
The ability of the indigenous Qatari strains to produce biosurfactants with great potential to enhance the biodegradation of weathered hydrocarbons was investigated. The obtained findings showed that two types of adaptations occur with hydrocarbons degrading bacteria in the weathered-oily soils, one related to the bacterial cell composition maintaining the biosurfactants composition and one to the biosurfactants which is the primary tool employed by the bacterial cell to interact with the weathered oil. Indeed, the phyloproteomic classification of the studied strains allowed the differentiation and separation of strains belonging to the same Bacillus species. High diversity of emulsification and solubilization activities were recorded among biosurfactants produced by the studied strains. Moreover, combining FTIR data with PCA analysis resulted in further classification of the biosurfactants produced by the studied bacterial isolates.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | v3-fos-license |
2020-09-09T14:10:19.451Z | 2020-09-09T00:00:00.000 | 221542999 | {
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} | pes2o/s2orc | VDR–SOX2 signaling promotes colorectal cancer stemness and malignancy in an acidic microenvironment
The acidic tumor microenvironment provides an energy source driving malignant tumor progression. Adaptation of cells to an acidic environment leads to the emergence of cancer stem cells. The expression of the vitamin D receptor (VDR) is closely related to the initiation and development of colorectal carcinoma (CRC), but its regulatory mechanism in CRC stem cells is still unclear. Our study revealed that acidosis reduced VDR expression by downregulating peroxisome proliferator-activated receptor delta (PPARD) expression. Overexpression of VDR effectively suppressed the stemness and oxaliplatin resistance of cells in acidosis. The nuclear export signal in VDR was sensitive to acidosis, and VDR was exported from the nucleus. Chromatin immunoprecipitation (ChIP) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) analyses showed that VDR transcriptionally repressed SRY-box 2 (SOX2) by binding to the vitamin D response elements in the promoter of SOX2, impairing tumor growth and drug resistance. We demonstrated that a change in the acidic microenvironment combined with overexpression of VDR substantially restricted the occurrence and development of CRC in vivo. These findings reveal a new mechanism by which acidosis could affect the stemness of CRC cells by regulating the expression of SOX2 and show that abnormal VDR expression leads to ineffective activation of vitamin D signaling, resulting in a lack of efficacy of vitamin D in antineoplastic process.
INTRODUCTION
Colorectal carcinoma (CRC) is a common malignancy whose morbidity and mortality rates rank it among the top five malignancies worldwide. 1 Despite advances in the treatment of CRC, many patients show tumor progression with existing therapies. [2][3][4] Genomic studies have shown that CRC has obvious heterogeneity and is influenced by a variety of epigenetic modifications that are closely related to the existence and heterogeneity of CRC stem cells. Notably, the tumor microenvironment can regulate the functions and fates of tumor cells. Strategies targeting CRC stem cells are urgently needed to prevent CRC recurrence and improve the prognosis of CRC patients.
The tumor microenvironment has an important role in determining the cancer stem cell (CSC) phenotype. 5,6 Low oxygen and low pH are two physicochemical characteristics of the tumor microenvironment. These characteristics lead to a series of changes related to cancer cell biological phenotypes, including an induced CSC phenotype and a metabolic reprogramming phenotype. 7 In addition, low oxygen and low pH change the core cell metabolic phenotype, providing the basic conditions required for cancer cells to optimize their metabolism. 8,9 Our recent studies have investigated the roles of metabolic microenvironment reprogramming in promoting gastrointestinal cancer progression. [10][11][12][13] However, the effects of acidic stress on cancer and the underlying mechanisms need further study. Thus, we sought to investigate the effects of the acidic tumor microenvironment on CRC stem cells.
Vitamin D has long been considered to have potential applications in cancer therapy. The active form of vitamin D (1α,25-(OH)-2-D3) can inhibit tumor growth. 14 Vitamin D regulates the transcription of target genes by binding with the vitamin D receptor (VDR), 15 which has a transcriptional regulatory role through binding of its DNA-binding domains with vitamin D response elements (VDREs) on target genes. 16 Studies have shown that the VDR expression level is related to the degree of differentiation of cancer cells, and we have previously found that an acidic environment can suppress the vitamin D signaling pathway to promote the CSC phenotype among glioma cells. 17 These findings suggest that the expression of VDR is closely related to the initiation and development of cancer, but the VDR regulatory mechanism in CRC stem cells is still unclear. Therefore, clarification of the effects of vitamin D and VDR on the stemness of CRC stem cells and elucidation of the interaction between CRC stem cells and the acidic tumor microenvironment are important.
In this study, we found that the acidic tumor microenvironment can reduce VDR expression via PPARD and prevent the accumulation of VDR in the nucleus. This relieves the inhibitory effect of VDR on the SOX2 promoter, thereby promoting SOX2 expression and leading to tumor growth and drug resistance. Fig. 1h. The Pearson correlation coefficient (r) and P value are shown. Three independent experiments were performed to obtain the data in b, c and f, g. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 Fig. S1d). The number of tumor spheres was maximized under acidic conditions with a pH of 6.8 (Fig. 1a, l Immunoblotting of the stemness markers CD133 and SOX2 in CC tissue-adherent cells treated with control or VDR-targeting shRNA. m The percentages of CD133-positive cells among CC tissue adherent and RKO cells treated with control or VDR-targeting shRNA were evaluated using flow cytometric analysis. Student's t-test. n IC50 of oxaliplatin in CC tissue-adherent cells treated with control or VDR-targeting shRNA and with oxaliplatin. Student's t-test. Three independent experiments were performed to obtain the data in d-g, j, k, m, and n. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 of the stemness markers prominin 1 (CD133), POU class 5 homeobox 1 (OCT4) and SOX2 in non-stem CRC cells ( Fig. 1c;
Acidosis inhibits
Supplementary S1e, f). Thus, the acidic tumor microenvironment could induce and maintain the CSC phenotypes of CRC cells.
To determine the important pathways involved in the regulation of the CSC phenotype in the acidic microenvironment, we analyzed RKO cells cultured under pH 7.4 and 6.8 by RNA sequencing and performed signaling pathway enrichment analysis. We found that the metabolic process of fat-soluble vitamins was significantly changed by the acidic conditions (Fig. 1d). Vitamin D is an important fat-soluble vitamin, and the vitamin D signaling pathway is known to be involved in the regulation of cancer cell differentiation. 14 Therefore, we tested the expression of several proteins that have key roles in the vitamin D signaling pathway (Fig. 1e) under pH 7.4 and 6.8, and found that VDR expression in each cell line was significantly decreased under acidosis (Fig. 1f, g). The mRNA expression of VDR differed markedly between CRC tissues and paired adjacent tissues, while the expression of CYP24A1, CYP27A1, and CYP27B1 did not differ significantly ( Fig. 1h; Supplementary Fig. S1g). Furthermore, VDR expression in recurrent CRC tissues was significantly reduced (Fig. 1i) Kaiser, and Hong databases, VDR expression in CRC tissue was lower than that in normal tissue (Fig. 1j), and VDR showed the lowest expression in stage-IV CRC tissues (Fig. 1k). We also examined the relationship between VDR expression and prognosis, and the results showed that patients with low VDR expression had short survival times (Fig. 1l). The expression of lysosomal-associated membrane protein 2 (LAMP2) is positively correlated with tumor microenvironment acidity, 18 and we found that VDR expression and LAMP2 expression in CRC were negatively correlated (Fig. 1m). These results suggest that the acidic tumor microenvironment can inhibit VDR expression, which is closely related to the degree of malignancy and the recurrence of CRC.
VDR impairs stemness and malignancy in CRC
To investigate the effects of VDR on the phenotypes of CRC stem cells, we first confirmed that VDR expression was lower in CRC stem cells than in non-stem cancer cells (Fig. 2a). VDR expression was also confirmed to be lower in CRC cell lines than in the normal colonic epithelial cell line ( Supplementary Fig. S1h). In addition, VDR expression was decreased in oxaliplatin-resistant HCT116 cells (Fig. 2b). Therefore, we overexpressed VDR in CRC stem cells with low VDR expression (Fig. 2c). Microscopic imaging showed that the tumor spheres adhered to the bottoms of the plates and that the cellular differentiation occurred in the tumor spheres ( (Fig. 2n). All these findings demonstrate that VDR inhibits the CSC phenotype and enhances the sensitivity of CRC stem cells to drugs in the acidic tumor microenvironment.
The acidic tumor microenvironment regulates the stemness and drug resistance of CRC through the VDR-SOX2 axis In order to clarify how VDR affects the CSC phenotype and drug resistance in CRC, we used chromatin immunoprecipitation (ChIP) to analyze the regulatory effects of VDR on the transcription of stemness markers and found that VDR could bind to the promoter regions of SOX2, OCT4, CD44, and NANOG ( Fig. 3a, b). Then, we investigated the chromatin accessibility of the stemness genes in cultured CRC cells under either acidic or alkaline culture condition by analyzing assay transposase-accessible chromatin with highthroughput sequencing (ATAC-seq) signals from upstream of transcription start sites (TSSs) throughout the whole ranges of the SOX2, OCT4, CD44, and NANOG genes. The open chromatin in SOX2 preferentially occurred upstream of the TSS in acidic conditions ( Fig. 3c), which may reflect the possibility of VDR binding at the promoter region of SOX2. We verified that SOX2 protein expression was decreased by overexpression of VDR ( Fig. 3d) and found that the binding effect was enhanced in cells overexpressing VDR (Fig. 3e). Overexpression of VDR inhibited the transcriptional activity of the SOX2 promoter, while knockdown of VDR promoted it (Fig. 3f).
We further deleted the binding sequence in the SOX2 promoter region (Fig. 3g) and found that the inhibitory effect of VDR on the SOX2 promoter was attenuated ( Fig. 3h-j). Through bioinformatics analysis, we found three VDREs in the SOX2 promoter (Fig. 3g). We deleted the three VDRE sites and detected the effects of VDR on the transcriptional activity of the SOX2 promoter. Mutation 1 and mutation 3 significantly weakened the inhibitory effect of VDR (Fig. 3k). These results suggest that VDR may downregulate SOX2 expression by inhibiting SOX2 promoter activity.
To confirm that VDR suppresses the malignant phenotype of CRC cells by downregulating SOX2 expression, we overexpressed SOX2 in CRC stem cells overexpressing VDR ( Fig. 4a; Supplementary Fig. S2a). The self-renewal ability of CRC stem cells increased upon SOX2 overexpression ( Fig. 4b; Supplementary Fig. S2b). Knockdown of SOX2 in CRC cells with low VDR levels reduced the expression of CD133 and CD44 ( Fig. 4c; Supplementary Fig. S2c) and decreased self-renewal ability (Fig. 4d). We also analyzed the SOX2 mRNA in CRC stem cells with and without VDR expression in both acidic and alkaline pHs. Results showed that SOX2 mRNA was increased in cells without VDR expression in acidic pH (Fig. 4e). Consistent with the previous results, knockdown of SOX2 in the acidic tumor microenvironment weakened the self-renewal ability of CRC stem cells ( Fig. 4f; Supplementary Fig. S2d) and strengthened the sensitivity of the cells to oxaliplatin (Fig. 4g). And we detected the mRNA expression of stem cell gene networks at pH 6.8 when silencing SOX2. Results showed that the stem cell gene, OCT4, MYC, andCCND1, which are also target genes of SOX2, were decrease when silencing SOX2. And other stem cell genes were also downregulated when silencing SOX2 (Fig. 4h). Moreover, we found that the active form of vitamin D could reverse the acidic environment-mediated promotion of selfrenewal and CD133, SOX2, and OCT4 expression in CRC stem cells (Fig. 4i, j; Supplementary Figs. S2e and 4k), suggesting that the acidic microenvironment affects the stemness of CRC cells through the vitamin D-VDR signaling pathway. Together, our findings demonstrate that downregulation of VDR in the acidic tumor microenvironment relieves the transcriptional inhibition of SOX2, resulting in increased SOX2 expression. These changes promote the stemness and drug resistance of CRC cells. ChIP was performed to assess VDR binding to the promoters of SOX2, OCT4, CD44, and NANOG in CC tissue adherent and DLD1 cells (a). A polyclonal anti-VDR antibody or a mouse IgG antibody was used. The immunoprecipitated DNA was quantified by qPCR (b). Student's t-test. c ATAC-seq enrichment from 2500 bp upstream of the TSSs throughout the whole ranges of the SOX2, OCT4, CD44, and NANOG genes in CC tissue-adherent cells cultured under pH 7.4 (red) and pH 6.8 (blue). d Immunoblotting of SOX2 in control and VDR-overexpressing CC tissue CSCs. e The extent of VDR binding to the SOX2 promoter in control and VDR-overexpressing CC tissue CSCs was measured by ChIP assay (left). The immunoprecipitated DNA was quantified by qPCR (right). A polyclonal anti-VDR antibody or a mouse IgG antibody was used. Student's t-test. f The transcriptional regulatory activity of VDR on the promoter of SOX2 in control and VDR-overexpressing CC tissue CSCs (left) and CC tissue-adherent cells treated with control or VDR-targeting shRNA (right) was measured by dual-luciferase reporter assay. RLU, relative luciferase unit. g Schematic representation of the SOX2 promoter containing three VDREs. The mutation strategy of the promoter is shown. h-j The transcriptional regulatory activity of VDR on the full-length SOX2 promoter and a mutant promoter with deletion of sites −1109 to −965 in control and VDRoverexpressing CC tissue CSCs (h) and in CC tissue adherent and RKO cells treated with control or VDR-targeting shRNA (i-j) was measured by dual-luciferase reporter assay. △, SOX2 promoter mutant with deletion of sites −1109 to −965. k The transcriptional regulatory activity of VDR on the full-length SOX2 promoter and on a triple VDRE-mutated SOX2 promoter (as described in g) in CC tissue-adherent cells treated with control or VDR-targeting shRNA was measured by dual-luciferase reporter assay. FL, full length. Three independent experiments were performed to obtain the data in b, e, f, and h-k. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 The acidic microenvironment inhibits VDR aggregation in the nucleus and suppresses VDR expression through PPARD VDR regulates target genes in the nucleus. Thus, we detected the subcellular localization of VDR in acidic environments. We found that VDR expression was decreased and that VDR protein accumulation in the nucleus was reduced under acidic conditions (Fig. 5a, b). Further analyses showed that the intracellular pH value was decreased under acidic conditions (Supplementary Fig. S2f) and that VDR contained a nuclear export signal (NES) (Fig. 5c). Nuclear export protein inhibitor leptomycin B (LMB) binds to nuclear export receptor (chromosome region maintenance 1, CRM1) and inhibits the binding of other export substrates. 19 We used LMB to treat CRC cells under pH 7.4 and 6.8. LMB prevented the nuclear export of the VDR protein in pH 6.8 culture medium (Fig. 5d), indicating that the nuclear export of the VDR protein in acidic environments is dependent on CRM1. To confirm that the nuclear export of VDR requires NES, we transfected CRC cells with wild-type and NES site-mutant (Fig. 5c) plasmids and found that the VDR NES mutation prevented VDR nuclear export in an acidic environment (Fig. 5e). To investigate whether the expression of nuclear VDR make cells insensitive to pH-driven reprogramming, we transfected CC tissue CSCs with wild-type and NES site-mutant plasmids, and subjected them to acidic conditions. The results showed that the NES mutation of VDR inhibited the self-renewal of colorectal cancer stem cells under acidic conditions ( Fig. 5f; Supplementary Fig. S2g, h). This finding indicates that the nuclear export of VDR in the acidic tumor microenvironment is mediated by the NES and is dependent on CRM1.
Using the Qiagen database, we predicted that peroxisome proliferator-activated receptor (PPAR) family members, which are closely related to tumor occurrence, were associated with the VDR promoter. We first analyzed the correlations among PPARA, PPARD, PPARG, and VDR in CRC data in the TCGA database ( Fig. 5g; Supplementary Fig. S3a, b). PPARA and PPARD expression was positively correlated with VDR expression (Supplementary Fig. S3b; Fig. 5g). We also found that PPARD mRNA and protein levels in CRC cells were significantly decreased under acidic conditions (Fig. 5h, i), while PPARA and PPARG levels were not significantly changed (Supplementary Fig. S3c). Surprisingly, PPARD could bind to the promoter of VDR (Fig. 5j), and knockdown of PPARD decreased the mRNA and protein expression of VDR and upregulated the expression of stemness markers (Fig. 5k-m; Supplementary Fig. S3d). These results suggest that the acidic tumor microenvironment inhibits the expression of VDR through PPARD, inducing nuclear export of the VDR protein and inhibiting the transcriptional regulatory function of VDR.
Normalization of the acidic tumor microenvironment and induction of VDR expression restrain the initiation and development of CRC
To determine whether CRC growth can be attenuated by modification of the acidic tumor microenvironment and VDR expression, we injected CRC cells into nude mice and gave the mice water or a sodium bicarbonate (NaHCO 3 ) solution. 20,21 We found that NaHCO 3 treatment after VDR overexpression significantly inhibited SOX2 expression ( Supplementary Fig. S3e, f) and tumor development (Fig. 6a) and that NaHCO 3 treatment also effectively attenuated SOX2 expression and tumor formation after VDR knockdown (Supplementary Fig. S3e, g; Fig. 6b). These findings suggest that CRC development can be inhibited by decreasing the acidity of the tumor microenvironment and inducing VDR expression. We further found that a combination of vitamin D signaling activation and oxaliplatin treatment could inhibit the tumor growth of the patient-derived xenografts (PDXs) (Fig. 6c). VDR expression was upregulated and SOX2 expression was downregulated in the PDXs (Fig. 6d; Supplementary Fig. S3h). These results provide a new theoretical basis for the clinical treatment of CRC.
Finally, we verified the influences of VDR and SOX2 expression on the tumorigenic ability of CRC stem cells in vivo. The results of the limiting dilution experiment in vivo showed that overexpression of VDR significantly inhibited tumor occurrence, whereas overexpression of SOX2 attenuated the repressive effect of VDR overexpression (Fig. 6e). Overall survival (OS) was significantly different among patients with high/low expression levels of VDR and SOX2 (P < 0.001). Patients with both low expression of VDR and high expression of SOX2, which were unfavorable for survival, had the worst prognoses (P < 0.001) (Fig. 7a, b). The expression levels of VDR and SOX2 were negatively correlated among stage-III and stage-IV specimens (Fig. 7c). Furthermore, we evaluated the expression of VDR and SOX2 in samples from 65 patients with advanced CRC treated with the FOLFOX or XELOX regimens. Only 27.69% of patients with high VDR expression in their primary tumors showed resistance to chemotherapy (progressive disease, PD), whereas 72.31% of patients with high SOX2 expression showed resistance to chemotherapy. The group of patients with both low VDR expression and high SOX2 expression had the highest proportion of chemotherapy resistance (Fig. 7d). These results indicate that both low VDR expression and high SOX2 expression predict resistance to oxaliplatin-based chemotherapy.
DISCUSSION
Acidity is a basic characteristic of the tumor microenvironment, and provides an energy source driving the malignant progression of tumors. Adaptation of cells to acidic environments leads to the emergence of tumor cells with increased aggression, proliferation, and drug resistance. 22,23 Thus, acidic microenvironments are favorable for the survival and growth of tumor cells but unfavorable for the survival and growth of normal cells. Acidic environments directly regulate tumor cell invasion by affecting immune cell function, cell clone evolution, and drug resistance. 24 Researchers have long assumed that acidic environments are associated with hypoxia. However, interestingly, acidic regions are not confined to hypoxic regions in the tumor-stroma interface but rather overlap with regions where cells proliferate and invade. The expression of matrix metalloproteinases is increased in these regions, and the basement membrane is degraded. 25,26 Transcriptome studies have shown that tumor-related stressors, such as hypoxia, nutritional deficiencies, and lactic acid-mediated acidification, can regulate gene expression at the transcriptional and posttranscriptional levels in vitro. 20,27,28 For example, low extracellular pH levels lead to increased histone deacetylation, which affects the expression of certain stress response genes and promotes the normalization of intracellular pH, primarily through enhancement of the release of protons by the monocarboxylate transporter (MCT). 29,30 Smad5 has been shown to positively Fig. 4 SOX2 overexpression reverses the VDR-mediated inhibition of stemness, and the vitamin D-VDR signaling pathway affects the stemness of CRC in acidic environments. a Immunoblotting of VDR and SOX2 in control and VDR-overexpressing CC tissue CSCs with or without SOX2 overexpression. b Tumor sphere formation assays (left) and limiting dilution assays (right) of control and VDR-overexpressing CC tissue CSCs with or without SOX2 overexpression. Student's t-test. c Immunoblotting of SOX2, CD133, and CD44 in control and VDRknockdown CC tissue-adherent cells treated with control or SOX2-targeting shRNA. d Tumor sphere formation assays of control and VDRknockdown CC tissue-adherent cells treated with control or SOX2-targeting shRNA. Student's t-test. e qPCR of VDR and SOX2 in control and VDR-knockdown CC tissue CSCs cultured under pH 7.4 and 6.8. Student's t-test. f Tumor sphere formation assays of control and SOX2knockdown CC tissue CSCs under pH 7.4 and 6.8. Student's t-test. g Cell viability of control and SOX2-knockdown CC tissue CSCs with oxaliplatin treatment under pH 7.4 and 6.8. The IC50 is shown as a dotted line. h qPCR of SOX2, OCT4, MYC, CCND1, CD133, NANOG, CD44, and EPCAM in control and SOX2-knockdown CC tissue CSCs cultured under pH 7.4 and 6.8. Student's t-test. i, j Representative images (i) and quantified data (j) for tumor spheres formed by CC tissue CSCs treated with 0, 50, or 100 nM of the active form of vitamin D (1α,25-(OH)-2-D3, VD 3 ) under pH 7.4 and 6.8. Scale bars: 200 μm. Student's t-test. k Immunoblots (right) and quantified levels (left) of VDR, SOX2, and OCT4 in CC tissue CSCs treated with 0, 50, or 100 nM of the active form of VD 3 under pH 7.4 and 6.8. Student's t-test. Three independent experiments were performed to obtain the data in b, d-h, j, and k. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 respond to changes in intracellular pH (pHi) and to shuttle from the nucleus to cytoplasm. 19 Moreover, an acidic environment can activate eEF2K and enhance the phosphorylation of eEF2. Five histidine residues in eEF2K have been found to have crucial roles in the activation of eEF2K under acidic conditions. 31 Here, we discovered that the NES in VDR is sensitive to acidic conditions and that VDR is exported from the nucleus. These results indicate that the acidic tumor microenvironment can affect VDR-mediated transcriptional regulation of target genes by changing the subcellular localization of VDR. Specifically, the acidic environment may regulate the activity of VDR by influencing leucine residues in it. The acidic environment would change the cellular proteome and cellular metabolism to a large scope; we discovered that VDR could attenuate the acidic tumor microenvironment-mediated promotion of CSC phenotype. Acidic environment affected SOX2 expression through VDR, and it is known that SOX2 affects a wide variety of proteins related to stem cell pluripotency. But the correlation between other changes of cellular proteome and VDR need further investigation. According to previously reported statistics, high levels of circulating 25(OH)D significantly reduce CRC risk in women but not in men. The optimal concentration of 25(OH)D for CRC risk reduction is 75-100 nmol/l, which is higher than the current Institute of Medicine (IOM) recommendations. 32 This finding suggests that low VDR expression may reduce the risk of CRC by increasing the levels of vitamin D. Notably, vitamin D3 supplementation has not been found to significantly reduce the incidence of aggressive tumors. Similarly, compared with placebo treatment, vitamin D treatment does not reduce overall cancer mortality or the incidence of breast cancer, prostate cancer, or CRC. 33 Such clinical findings show that vitamin D is not very effective in reducing the incidence of cancer or improving the prognoses of cancer patients. We hypothesized that cancerrelated abnormalities in VDR, the key factor mediating the function of vitamin D, lead to ineffective activation of the vitamin D signaling pathway, causing vitamin D to be ineffective as a cancer treatment. In our study, we found that overexpression of VDR could effectively suppress the CSC phenotype, decrease invasion, and increase sensitivity to oxaliplatin in CRC cells in acidic environments.
Another study has shown that VDR is expressed in the stroma in human pancreatic cancer. The VDR ligand calcipotriol can significantly decrease the levels of inflammation and fibrosis markers in inflamed pancreas tissue and the tumor stroma. As the major transcriptional regulatory factor of pancreatic stellate cells (PSCs), VDR can restore the quiescent state of cells; thus, compared with chemotherapy alone, VDR induce tumor stromal remodeling, increase gemcitabine levels in tumor tissue, reduce tumor volumes, and improve survival rates. 34 We also demonstrated that VDR can significantly suppress the growth of tumors and that modification of the acidic tumor microenvironment combined with VDR overexpression substantially restricts the occurrence and development of CRC in vivo.
SOX2 is a transcription factor with a high-mobility group domain and sequence-specific DNA-binding activity. 35 This transcription factor is not only necessary for embryonic stem cells but also a key factor for induced pluripotent stem cells. 36 The expression of SOX2 is increased in many cancers and is associated with poor prognosis. 37,38 SOX2 has roles in maintaining tumor-initiating cells, and in determining the selfrenewal ability and tumorigenic potential of various types of cancer cells. 39,40 SOX2 can also regulate other transcription factors; for example, this molecule can interact with OCT3/4 to regulate the transcription of NANOG and other pluripotentrelated genes, such as FGF4, UTF1, and LEFTY1. 35 However, other transcription factors can in turn regulate SOX2. Previous reports have shown that CDK1 binds SOX2 and regulates its phosphorylation, nuclear transport, and transcriptional activity, thus promoting tumorigenesis. CDK1 is therefore a new SOX2 regulator in tumor cells. 41 In addition, SIX2 is a transcription factor with homologous domains. Six2 directly binds the srr2 enhancer of SOX2 to promote the expression of SOX2 in breast cancer, indicating that a SIX2/SOX2 axis is necessary for effective metastatic cloning and highlighting the critical role of the stemness factor SOX2 in tumor growth at metastatic sites. 42 In a previous study, miR-638 was found to inhibit the luciferase activity of a reporter gene connected to the 3′ UTR of SOX2 in CRC. 43 SOX2 has also been confirmed to be a target of miR-200c, as miR-200c inhibits SOX2 expression and blocks PI3K-AKT pathway activity. Moreover, miR-200c and SOX2 mutually control their expression levels through feedback loops. 44 However, the roles of transcription factors in the regulation of SOX2 in CRC remain unclear. Our study shows that there are three VDREs in the promoter region of SOX2. We found that the transcription factor VDR transcriptionally represses SOX2 by binding to the VDREs, consequently reducing SOX2 expression. Overexpression of SOX2 can markedly facilitate CRC growth in vivo. Furthermore, the acidic tumor microenvironment alters SOX2 in a VDR-dependent manner (Fig. 7e). These findings reveal a new mechanism through which the acidic tumor microenvironment can affect the CSC phenotype of CRC cells by regulating the expression of the pluripotent transcription factor SOX2. We suppose that there is antagonism between those factors and VDR, but it needs further investigation. In addition, some factors might bind to the upstream of TSSs in SOX2 gene, and most of them activate the expression of SOX2. Under acidic condition, VDR was exported from the nucleus into cytoplasm, the inhibiting effect of VDR on SOX2 expression was relieved, and those factors could bind to the open chromatin at upstream of TSSs in SOX2 gene. We suppose that there is antagonism between those factors and VDR, and this is an exciting future area of investigation.
MATERIALS AND METHODS
A detailed description of the methods can be found in the Supplementary materials.
Cells and specimens
Human CRC cell lines and immortalized colon epithelial cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured as recommended. All cells tested negative for mycoplasma contamination and were authenticated by short tandem repeat (STR) fingerprinting before use. All CRC specimens were obtained with written informed consent from all patients. Fig. 5 The acidic tumor microenvironment inhibits VDR aggregation in the nucleus and suppresses the expression of VDR through PPARD. a Immunofluorescence staining of VDR (green) and DAPI (blue) in CC tissue-adherent cells under pH 7.4 and 6.8. Scale bars: 10 μm. b Immunoblotting of VDR in the cytoplasm and nucleus in CC tissue-adherent cells cultured under pH 7.4 and 6.8. α-Tubulin is mainly expressed in the cytoplasm. Lamin B is a fibrous protein that exhibits a structural function and performs transcriptional regulation in the cell nucleus. c Schematic representation of the NES site in the LBD domain of VDR and the mutated amino acids in NES mutant. NES, nuclear export signal. LBD, ligand-binding domain. DBD, DNA-binding domain. d Immunofluorescence staining of VDR (green) in CC tissue-adherent cells treated with ethanol or LMB for 1 h at 37°C under pH 7.4 and 6.8. LMB, leptomycin B (a nuclear export protein inhibitor). Scale bars: 10 μm. e Immunofluorescence staining of VDR (green) and DAPI (blue) in CC tissue-adherent cells transfected with pLV-CMV-VDR or pLV-CMV-VDR-mut (the wild-type or mutated VDR shown in c, respectively). Scale bars: 5 μm. f Tumor sphere formation assays of CC tissue CSCs transfected with pLV-CMV-VDR or pLV-CMV-VDR-mut under pH 7.4 and 6.8. Student's t-test. g Correlation of PPARD expression with VDR expression in CRC samples from TCGA. h qPCR of PPARD in CC tissue adherent, HCT8, DLD1, SW480, SW620, and RKO CRCs cultured under pH 7.4 and 6.8. Student's t-test. i Immunofluorescence staining of PPARD (green) and DAPI (blue) in CC tissue-adherent cells under pH 7.4 and 6.8. Scale bars: 10 μm. j ChIP was used to assess PPARD binding to the promoters of VDR in CC tissue-adherent cells (upper). A polyclonal anti-PPARD antibody or a mouse IgG antibody was used. The immunoprecipitated DNA was quantified by qPCR (lower). Student's t-test. k qPCR of PPARD and VDR in CC tissue-adherent cells transfected with siRNA for PPARD. Student's t-test. l, m Immunoblotting of PPARD, VDR, SOX2, and the stemness markers NUMB, CD133, and OCT4 in CC tissue-adherent cells transfected with siRNA for PPARD. NUMB is an endocytic adaptor protein that has a crucial role in asymmetrical cell division. Three independent experiments were performed to obtain the data in f, h, j, and k. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001 VDR-SOX2 signaling promotes colorectal cancer stemness. . . Hu et al. Fig. 7 VDR expression is negatively correlated with SOX2 expression and has the potential to be used for clinical prognosis prediction. a, b Overall survival of patients according to the expression of VDR and SOX2 in 419 CRC tissue samples, as described in Fig. 1k. The Kaplan-Meier method and the log-rank test were used. c Correlation between VDR expression and SOX2 expression in 253 stages III-IV CRC tissue samples from our hospital (SYSUCC). The data are presented as the percentage of total samples. Pearson's Chi correlation analysis was used. d The percentages of specimens with low/high VDR and SOX2 expression relative to the response to FOLFOX or XELOX chemotherapy were analyzed (middle and right). The results for two representative cases are shown (left). Pearson chi square test. PD progressive disease, CR complete response, PR partial response and SD stable disease. Scale bars: 50 μm. e Schematic illustration of VDR-SOX2 signaling in CRC cells under acidosis. There are three VDREs in the promoter region of SOX2. The transcription factor VDR transcriptionally represses SOX2 by binding to the VDRE, reducing SOX2 expression. The acidic tumor microenvironment upregulates SOX2 in a VDR-dependent manner and facilitates the stemness and malignancy of CRC basic fibroblast growth factor, 20 ng/ml epidermal growth factor, 10 μg/ml heparin, and 2% B27).
Number of cells
Acidic culture conditions HEPES (25 mM) and PIPES (25 mM) (Sigma-Aldrich, St. Louis, MO, USA) were added to RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin or to DMEM/F12 stem medium, and the pH value was titrated using 1 M HCl or 1 M NaOH. The medium was incubated for 24 h and then retitrated.
ChIP ChIP was performed as described previously. 45 A MAGnify Chromatin Immunoprecipitation System (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer's instructions. A total of 5-10 × 10 6 cultured cells were used per test, and 4 μg of anti-VDR antibody or 1 μg of isotype-control antibody (mouse IgG) was used per test. PCR and real-time quantitative PCR (qPCR) were used to verify the binding ability between VDR and the promoters of target genes. The primers used are listed in the Supplementary Methods. The data were calculated as the percentage of the input. Three independent experiments were repeated.
ATAC-seq A total of 5 × 10 5 tissue-adherent CC cells cultured under pH 7.4 or 6.8 were processed according to a previously published protocol, 46 and 150 bp paired-end sequencing was performed on Illumina Xten to yield an average of 97 M reads/sample.
Tumor development and NaHCO 3 treatment Subcutaneous xenograft models were established as described previously. 17 Briefly, 1 × 10 4 CC tissue CSCs or tissue-adherent CC cells were injected subcutaneously into nude mice in 0.1 ml of Matrigel and PBS (1:4). After 1 week, 200 mM NaHCO 3 or water was provided to the mice and remained available throughout the course of the experiment. 20 The tumor volumes of the mice in each group (n = 5) were estimated each week using the formula V = ab 2 /2 (V, volume; a, length; b, width). After 35 days, the tumor tissues were dissected, and the weights were measured. The animal protocol was approved by the Animal Ethics Committee of Sun Yat-Sen University.
Statistics
Student's t-test was used for statistical analysis. The data are shown as the mean ± standard deviation and were analyzed by SPSS 20.0 software, and a P < 0.05 was considered to indicate statistical significance. Pearson correlation coefficients were used to analyze the expression correlations of different genes.
Study approval Informed consent was obtained from patients before surgery. The study was approved by the Medical Ethics Committee of Sun Yat-Sen University Cancer Center. The animal protocol was approved by the Animal Ethics Committee of Sun Yat-Sen University.
DATA AVAILABILITY
The original RNA sequencing data and ATAC-seq data have been uploaded to the Genome Sequence Archive (GSA; http://gsa.big.ac.cn/) and are accessible under the GSA numbers CRA001942 (RNA sequencing data) and CRA002255 (ATAC-seq data). | v3-fos-license |
2014-10-01T00:00:00.000Z | 2008-03-19T00:00:00.000 | 9635684 | {
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} | pes2o/s2orc | Structural Elements Regulating Amyloidogenesis: A Cholinesterase Model System
Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE), AChE586-599, through the effect of single point mutations on β-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE586-599 was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE586-599 assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high β-strand propensity, for the conformational transition to β-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to β-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE586-599 surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE586-599 assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-π, SH-aromatic, metal chelation and polar-polar) would maintain the β-sheets together. We also propose that the stacking between the strands in the β-sheets along the fiber axis could be stabilized through π-π interactions and metal chelation. The dissection of the specific molecular recognition driving AChE586-599 amyloid assembly has provided further knowledge on such poorly understood and complicated process, which could be applied to protein folding and the targeting of amyloid diseases.
Introduction
Protein misfolding can be deleterious by triggering aggregation and insolubilization. In turn, the aggregation can lead to toxic conformation during which polymerization by folding and stacking of cross-b sheets result in the formation of amyloid fibrils. Fibril formation is a multiple kinetic event during which an energetically unfavourable nucleated polymerization (characterized by a lag phase) initiates the formation of a minimal selfassembled complex (nucleus or seed) serving as a structural template for a cooperative amyloid elongation [1]. Amyloid fibrilization is proposed to be the molecular basis of and the common link between a variety of pathological conditions and human neurodegenerative syndromes, such as type II diabetes, Alzheimer's, Parkinson's, Huntington's and prion diseases [2]. Although amyloid formation and deposition is a common feature of these diseases, the amyloid fibrils originate from different and distinct proteins or peptides that do not appear to share any sequence homology or function. However, fibrils formed from these amyloid-related sequences possess similar structural, physical and chemical properties, including formation of b-sheets whose strands run perpendicular to the fibril axis, fibril morphology and typical X-ray diffraction pattern, kinetic pattern of fibril formation and staining with dyes such as Congo red and Thioflavin T (ThT) [3,4,1,5,6]. Therefore, amyloid formation involves more than nonspecific aggregation and non-specific hydrophobic interactions and it is recognized that some levels of structural complexity and specific pattern of interactions are important [7,8,9,10]. Indeed, certain types of residues characterized by high b-sheet propensity, and/or fastest kinetics of aggregation, and/or stabilizing and assembling properties have been found to be commonly present in amyloid-related sequences [11,12,13,14,15]. Consequently in recent years, a great deal of attention has focussed on determining the factors and interactions resulting in fibrilization and finding common rules that govern the assembly. Such detailed dissections of the specific molecular recognition and self-assembly during amyloid formation could provide invaluable knowledge for the targeting and control of diseases involving toxic protein aggregation and deposition.
During Alzheimer's disease (AD) pathogenesis, the accumulation in the brain of extracellular amyloid-b-peptide (Ab) in senile plaques and of intracellular hyperphosphorylated Tau in neuro-fibrillary tangles are thought to represent the hallmarks of the disease [16]. However, other proteins have also been implicated in the pathology, with one example being acetylcholinesterase (AChE) [17]. AChE is associated with senile plaques, promotes Ab fibrilisation, and triggers early disease and increases plaque burden in double transgenic mice expressing human amyloid precursor protein (hAPP, from which Ab is proteolytically cleaved) and hAChE when compared to single transgenic hAPP mice [18,19,20]. We have studied a 14 residue peptide named AChE 586-599 , which corresponds to a region within the C-terminal oligomerization domain of human AChE. The region encompassing AChE 586-599 shares homology with Ab and possesses high propensity for conversion to non-native (hidden) b-strand, a property associated with amyloidogenicity [21,22]. Moreover, AChE 586-599 adopts a b-sheet conformation, self-assembles into amyloid fibrils and promotes Ab fibrilization [23,24]. This peptide represents a tractable model for studying amyloid formation because its fibrilization is highly dependent upon pH. This allows a total control of the start of the polymerization process, which is triggered by the addition of physiological buffer to an acid solution. Moreover, AChE 586-599 is an attractive model due to its residue composition with alternating charged, polar and hydrophobic amino acids most of which have been previously shown to be implicated in amyloid formation [11,12,15,25]. Understanding how AChE 586-599 residue composition and chemical nature affect the polymerization process should provide insights and strengthen current knowledge into complex networks of interactions leading to amyloid fibril formation.
In this study, we examined the role of each residue within AChE 586-599 through the effect of single point mutations on the bsheet propensity, conformation, fibrilization, surfactant activity, oligomer formation and fibril morphology of AChE 586-599 . We determined the importance of residues and the potential molecular interactions underlying AChE 586-599 assembly into b-sheets and during the stacking of these sheets. Non-covalent side chain-side chain interactions, such as hydrophobic, cation-p and p-p interactions, were found to be critical for fibrilization and assembly stabilization. This detailed analysis allowed us to propose a model for the amyloid polymerization of AChE 586-599 in which specific interactions between residue side-chains lead to the formation of a steric-zipper maintaining the b-sheets together, and p-p interactions allow the stacking and arrangement of strands within a b-sheet.
Results
To determine the role of each residue of AChE 586-599 in the process of amyloid formation, a library of alanine scanning mutants along with a structurally conserved substitution mutant (Tyr to Phe) and a truncation mutant (missing the last residue) were used.
Identification of the residues important for the b-sheet propensity of AChE 586-599 and its conformational transition from random coil to b-sheet upon neutralization We performed secondary structure prediction in term of high propensity for conversion to non-native (hidden) b-strand, using the method described by Yoon and Welsh [22]. Previously, Yoon and Welsh have predicted the minimal amyloidogenic regions for Ab and a-synuclein, and have also identified AChE 586-599 to be a region of AChE with high non-native (hidden) b-strand propensity [22]. Since the amyloidogenicity of a peptide has been associated with its b-sheet forming propensity, such analysis could provide an insight on the importance of certain residues in the fibrilogenicity of AChE 586-599 . When we applied the algorithm to AChE 586-599 , the whole peptide (with the exceptions of the N-terminal Ala and Glu, and the C-terminal Lys) possessed propensity for conversion to b-strand with the strongest propensity for the sequence YMVH ( Figure 1). Some mutations (E 2 and R 5 ) increased the b-strand propensity. The H 12 /A mutant strengthened the propensity for V 11 albeit decreasing it slightly for Y 9 and M 10 . By contrast, some mutations drastically impaired the b-strand propensity in some part of the sequence: W 6 , Y 9 , M 10 , V 11 and W 13 . The W 6 /A mutant created a break in the continuity of b-strand propensity, whereas the Y 9 /A, M 10 /A, V 11 /A and W 13 /A mutants drastically decreased it in the YMVH region. Four other mutations also impaired the b-strand propensity but moderately: F 3 triggering a stronger random coil propensity for A 1 ; S 7 , S 8 and K 14 slightly decreasing the b-strand propensity in the YMVH region. Only the mutation to Ala did not affect the b-strand propensity. Thus, W 6 , Y 9 , M 10 , V 11 and W 13 appeared to be crucial for maintaining a high b-strand propensity along the entire sequence.
Far-UV circular dichroism (CD) studies were performed to establish the conformation of AChE 586-599 and AChE 586-599 mutants before and after neutralization, allowing us to follow early conformational changes that preceded and occurred during aggregation. AChE 586-599 was previously shown to be random coil when non-aggregated and to switch to a b-sheet structure upon neutralization [23]. Representatives of the conformations and changes in conformation observed are presented in Figure 2A and the conformations found for all the peptides at the different pHs are summarized in Figure 2B. CD spectra typical for a random coil structure (negative molar ellipticity at 200 nm or below) were observed for all peptides at acidic pH, indicating their nonaggregated status under acidic conditions. Nine mutant peptides still displayed random coil spectra after neutralization (e.g. W 6 /A, top left panel in Figure 2A) ( Figure 2B). Therefore, these mutants were not able to adopt a b-sheet conformation under the conditions of the assay, which suggested the importance of the hydrophobicity and/or aromaticity of F 3 , W 6 , S 7 , S 8 , Y 9 , M 10 , V 11 and W 13 in the conformational transition upon neutralization. By Figure 1. Secondary structure propensity of AChE 586-599 mutants as predicted by hidden b-propensity method (available at http://opal.umdnj.edu). Propensities for helices (red squares), b-strands (blue squares) and random coil (green squares) are presented numerically using a 0-1 scale, with low values indicating zero to low propensity and high values indicating high propensity to near certainty. Hydrophobic residues are shown in green and aromatic residues in bold with a bigger font size. doi:10.1371/journal.pone.0001834.g001 Figure 2. Conformation of AChE 586-599 and AChE 586-599 mutants. (A) Far UV spectra (250 to 190 nm) before and after pH neutralization (50 mM NaH 2 PO 4 , pH 7.2) of 4 AChE 586-599 mutants (100 mM). These spectra are representatives of the different structures and different changes in structure observed for the wild-type and mutant peptides. (B) Conformation and changes in conformation after pH neutralization (50 mM NaH 2 PO 4 , pH 7.2) for AChE 586-599 and all AChE 586-599 mutants (100 mM). (C) Near UV spectra (320 to 240 nm) before and after pH neutralization (50 mM NaH 2 PO 4 , pH 7.2) of AChE 586-599 and 4 mutants (100 mM). In all panels, the mutation within AChE 586-599 is indicated in bold and italics. doi:10.1371/journal.pone.0001834.g002 contrast, negative molar ellipticity around 215 nm was found at neutral pH for 6 peptides: AChE 586-599 , E 2 /A, H 4 /A, R 5 /A, K 14 / A and DK 14 mutants (Figure 2A, bottom panels, and Figure 2B). Such a negative ellipticity is typically assigned to b-sheet structures. Some peptides started to adopt a partial (e.g. DK 14 mutant, Figure 2A, top right panel with double negative ellipticities at 200 and 215 nm) or a complete b-sheet structure (e.g. R 5 /A mutant, Figure 2A bottom right panel) after only 10 min at neutral pH. These results indicated that such mutants were faster than AChE 586-599 at adopting a b-sheet conformation, and therefore that the Arg and the Lys residues are not crucially involved in the conformational change during neutralization and may even have a negative effect. After 24 hours at neutral pH, visual inspection of the solutions for the peptides adopting a bsheet structure revealed the presence of insoluble aggregates, indicating tertiary or quaternary arrangements possibly leading to the formation of intermolecular-stacked b-sheets (see below).
We then applied near-UV CD to follow the behavior of the aromatic side-chains during aggregation. AChE 586-599 does not possess Cys residues, therefore any bands in the near-UV spectrum can only be attributed to constraints on the side-chains of aromatic residues. Before neutralization, the AChE 586-599 spectrum did not exhibit any strong positive bands, indicating the absence of conformational restriction of the aromatic sidechains ( Figure 2C, left panel). By contrast, the near-UV spectrum of AChE 586-599 after 24 hours at neutral pH showed three positive bands (below 260 nm, at 287 nm and 297 nm), which were consistent with conformational constraints on the aromatic rings of Phe and Tyr and/or Trp. By contrast the spectra of mutants, in which a single aromatic residue was substituted to Ala (the F 3 /A, W 6 /A, Y 9 /A and W 13 /A mutants), did not display any positive bands ( Figure 2C, right panel). Therefore, the formation of the insoluble aggregates of AChE 586-599 was associated with tertiary or quaternary interactions involving aromatic residues.
Identification of the residues important for the fibrilization properties of AChE 586-599 The ability to form amyloid and the fibrilization kinetics of AChE 586-599 and AChE 586-599 mutants were determined by changes in ThT fluorescence emission in shaking conditions. Shaking was used to accelerate fibrilization, which was necessary at least for some mutants. After a lag phase of 0.09 hours, AChE 586-599 rapidly selfassembled into amyloid aggregates ( Figure 3A). It has to be noted that the kinetics of AChE 586-599 assembly involved 2 phases, with a first rapid assembly (,1 hour) followed by a short plateau and decrease of ThT signal, and a second assembly with a slower rate (between 4.6 and 12 hours) also followed by a short plateau and decrease of ThT signal (Figure 3 A). However, AChE 586-599 assembly performed in quiescent conditions did not display this biphasic behavior (data not shown). Instead, it revealed that the assembly started immediately, without the first assembly, but instead resembling the second assembly observed during the shaking conditions. This assembly in quiescent conditions was also followed by a short plateau and a decrease of ThT signal identical to the ones observed during shaking conditions. Therefore, AChE 586-599 biphasic behavior was triggered by the shaking environment, which might either affect the assembly susceptibility to breakage by increasing shear forces, or affect the peptide surfactant activity by increasing the surface area and peptide recruitment. Collectively, the decay of the ThT signal after plateau in both conditions (shaking or not) suggested that the assembly of AChE 586-599 was not stable.
Most of the substitutions affected both the lag phase and plateau height ( Figure 3A, B and C). The exceptions were the E 2 /A and H 4 /A mutants, which showed a similar lag phase to AChE 586-599 , however their plateau height was affected with a drastic decrease for the E 2 /A mutant and an increase for the H 4 /A mutant. For both mutants, the substitution triggered stability of the assembly. The R 5 /A, S 7 /A, S 8 /A and W 13 /A mutants showed a similar plateau height to AChE 586-599 . However, their lag phases were increased, except for the R 5 /A mutant.
Substitution of H 12 and K 14 reduced the kinetics of fibrilization but did not abolish it completely. Indeed, slight increases of the lag phase were observed (5 and 3 times longer than AChE 586-599 , respectively), however their plateau heights were reduced on average by half ( Figure 3C). Aggregation of the R 5 /A and DK 14 mutants exhibited a significantly shorter lag phase than AChE 586-599 , which indicated a lower kinetic solubility and higher kinetic rate of fibrilization ( Figure 3C). In fact, the R 5 /A and DK 14 mutants immediately aggregated. These results suggested that positive charges from the side-chains of R 5 and K 14 might induce repulsion not favorable to a rapid aggregation and therefore might control assembly through specific interactions. By contrast, substitution of F 3 to Ala and Y 9 to Ala or Phe resulted in a dramatically decreased aggregation rate, with a very long lag phase (respectively 100, 64 or 160 times slower than AChE 586-599 ) and a lower plateau height (except for the Y 9 /A mutant) ( Figure 3A and B). The W 13 /A mutant was also affected with a lag phase 21 times slower than AChE 586-599 ( Figure 3C). These results suggested the importance of aromatic residues in the first stage of aggregation of AChE 586-599 . Interestingly, the substitution of Y 9 to Phe affected the rate of aggregation even more than the substitution to Ala ( Figure 3C). The most dramatic effect was that of the substitution of W 6 and M 10 to Ala, leading to a total loss of the peptide ability to form any aggregates as far as could be detected in the assay ( Figure 3C). Finally, the substitutions to Ala of E 2 , F 3 , H 4 , S 7 and S 8 triggered the AChE 586-599 assembly to be stable ( Figure 3A and C). Some mutants had very low level of fibrilization (very low plateau height) (e.g. the F 3 /A mutant), which may explain the absence of b-sheet conformation by CD for these mutants. Furthermore, the ThT assay done in similar conditions to the CD (no shaking, 24 hours) led to very poor fibrilization (long lag phase and low plateau height) or absence of fibrilization for the S 7 /A, S 8 /A, Y 9 /A and W 13 /A mutants (data not shown), which would also explain the absence of b-sheet conformation by CD.
Effect of electrostatic interactions and ionic strength on the stability of AChE 586-599 aggregates
To examine the effect of charge neutralization on AChE 586-599 fibril formation, we used increasing salt concentrations (NaCl and KCl) in a ThT assay, and the effects on both the E 2 /A and K 14 /A mutants were analysed. The strategy for selecting these mutants was based upon the presence of opposite charges on their sidechain at neutral pH, their position at the AChE 586-599 termini and their fibrilization properties (see above). Moreover, removing the side-chain charge from one of them was potentially leaving the side-chain charge from the other one uncompensated.
Increasing the salt concentration, therefore the ionic strength of the solute, significantly reduced the lag phase and increased the plateau height of the E 2 /A mutant ( Figure 4A and B). Similarly, the salt concentration increase was able to significantly increase the plateau height of the K 14 /A mutant, in a concentrationdependent manner ( Figure 4C). Furthermore, increasing the ionic strength stabilized the assembly of the K 14 /A mutant. Collectively these data strongly suggested that an increase in ionic strength of the solute successfully shielded the uncompensated charges in either mutant. In turn, this would indicate that E 2 and/or K 14 were likely to be involved in electrostatic interactions, such as salt bridges. The very poor fibrilization or the absence of fibrilization of the F 3 / A, W 6 /A and M 10 /A mutants prompted us to investigate the effect of these mutants in co-fibrilization assays. Indeed if F 3 , W 6 and M 10 are crucial, as suggested by the CD and ThT assays (see Figures 2 and 3), for the formation of b-sheets and amyloid aggregation, it is important to determine whether the corresponding mutants could interact with the wild-type peptide AChE 586-599 and if they could affect AChE 586-599 fibrilization. It was previously shown that short sequences of Ab containing Phe residues were able to bind specifically to the full length Ab and to inhibit its fibrilization [26,27]. Such findings could be applied to the prevention of critical interactions occurring during amyloidogenesis. When AChE 586-599 (50 mM) was fibrilized in the presence of various concentrations of the F3/A mutant (ranging from 1.56 mM to equimolar), some of AChE 586-599 fibrilization parameters were affected. First, the F 3 /A mutant was able to stabilize the final aggregation stage of AChE 586-599 ( Figure 5A). Indeed, the assembly of AChE 586-599 on its own was not stable with the plateau reaching a maximum before decreasing ( Figure 5A, black open squares). By contrast, the plateau remained stable when AChE 586-599 was cofibrilized with substoichiometric amounts of the F3/A mutant (from 6.25 to 50 mM). Second, the presence of the F 3 /A mutant (from 6.25 to 50 mM) also shortened the lag phase and increased the plateau height of AChE 586-599 (p,0.005 and p,0.014 respectively) ( Figure 5B and 5C). Thus, AChE 586-599 and the F 3 /A mutant were clearly able to interact with one another.
In contrast to the F 3 /A mutant, the W 6 /A and M 10 /A mutants did not affect AChE 586-599 fibrilization properties. Indeed in the presence of either mutant, both the lag phase and plateau height were similar to the values for AChE 586-599 fibrilized on its own ( Figures 6A, B, D and E). However, both mutants clearly interacted with AChE 586-599 , as indicated by the far-UV CD of the mixture, which was not entirely the arithmetic addition of the two separate spectra (Figures 6 C and F). The spectra of the AChE 586-599 and mutant mixtures after 24 hours under neutral conditions indicated the presence of both random coil (200 nm) and b-sheet (215 nm) structures. The random coil signals for the mixtures were statistically different to the signal of the arithmetic sums of AChE 586-599 with the W 6 /A mutant or with the M 10 /A mutant (p,0.014 and p,0.01 respectively) ( Figures 6C and F, insets).
Identification of the residues important for the surfactant properties of AChE 586-599
Both AChE 586-599 and Ab possess surfactant properties. Similarly to detergents, both peptides reduce the surface tension of water by orientating their hydrophobic moiety away from the aqueous phase and ordering their amphiphilic moiety at the air-water interface [28,29]. The surfactant activity of AChE 586-599 was previously shown to be highly pH dependent [29]. All the substitution mutants were analyzed for surfactant activity by measuring differential absorbance, as described in Material and Methods (Figure 7). AChE 586-599 showed a differential absorbance of 0.2960.02 DOD ( Figure 7A and C). Only one mutant (R 5 /A) displayed surfactant activity similar to AChE 586-599 after 2 min neutralization and only one showed a reduced surfactant effect under these conditions (K 14 / A) ( Figure 7A and C). The remaining mutants showed a larger increase of DOD after 2 min at neutral pH, which demonstrated that their effect on surface tension was bigger than AChE 586-599 and also strongly pH dependent (p,0.035) ( Figure 7C).
When the temporal pattern of the surfactant activity of the mutants was analyzed, it was realized that some mutants were not stably surface active ( Figure 7B and C). Indeed, the surfactant activity of the E 2 /A, F 3 /A, V 11 /A, H 12 /A and W 13 /A mutants, displayed after 2 min at neutral pH, significantly decreased with time (p,0.05). This result indicates that these mutants were able to quickly segregate at the air-water interface after neutralization. The subsequent loss of surfactant effect suggests that these peptides were then either promptly leaving the air-water interface to return to the bulk, or were undergoing a conformational change at the air-water interface to reduce their effect on surface energy. By contrast, the surfactant activity of AChE 586-599 and the other mutants was stable, which suggests that they remained stably associated with the airwater interface during the length of the time course.
Identification of the residues important for the early steps of AChE 586-599 oligomerization The absence of ThT signal during the fibrilization assay did not preclude the presence of small oligomers that could be ThT negative. Therefore, we analyzed the involvement and role of the side-chains of each residue within AChE 586-599 in oligomer formation and oligomer size distribution by performing photoinduced cross-linking of unlabeled AChE 586-599 and AChE 586-599 mutants (PICUP) [30]. It was previously demonstrated that the concentration of monomeric peptide during PICUP had to remain below 15 mM to avoid non-specific collision (Kenneth Baker and David J Vaux, unpublished data). In our assay, it was not possible to detect the cross-linked oligomers via conventional protein stains, presumably because the starting mass of peptide is then distributed across many individually low abundance oligomeric species. Therefore, the oligomers were detected by western-blot using the specific mouse Mab 105A, anti-AChE 586-599 in a b-sheet conformation [23], which also provided additional information about the conformation of the cross-linked products. However, it was impossible to assay the F 3 /A, H 4 /A and R 5 /A mutants since these mutations were shown to abolish 105A immunoreactivity [23].
AChE 586-599 oligomers had a distinct size distribution ranging from ,3 to 16 kDa, with the oligomer intensity decreasing with the increase in size (Figure 8, top panel). These oligomeric forms may range from dimers to nonamers according to their observed molecular weights (AChE 586-599 being 1.86 kDa). The most intense oligomer band was ,5 kDa, which may correspond to trimers. Substitutions of S 7 , S 8 , Y 9 and M 10 to Ala yielded a qualitative distribution of oligomers similar to that of AChE 586-599 . However, the relative total amounts formed were different with S 7 /A much weaker than AChE 586-599 , M 10 /A weaker, S 8 /A similar (except the ,5 kDa oligomers) and Y 9 /A much stronger. Therefore, the side-chains of S 7 , S 8 , Y 9 and M 10 were not essential for normal oligomer distribution. When Y 9 was substituted to Phe, both the oligomer distribution and amount decreased significantly, when compared to the Y 9 /A mutant. The only oligomer bands detected were at ,5 and 6.5 kDa, which could be trimers and tetramers. Phe differs from Tyr by missing the phenolic oxygen, which suggests that the importance of Y 9 in the formation of oligomers bigger than tetramers may reside in the phenolic oxygen rather than the phenolic ring itself.
The substitution of W 6 and W 13 to Ala severely affected both the amount and size distribution of the oligomers, with the oligomers ranging from ,3 to 9 kDa (dimers to pentamers) for W 6 /A and from ,5 to 9 kDa (trimers to pentamers) for W 13 /A, and the oligomer amount being more reduced for W 13 /A. Thus, the side-chains of the W 6 and W 13 appeared to be essential and a driving force in the association into oligomers.
For the V 11 /A, K 14 /A and DK 14 mutants, oligomers at ,78 kDa (circa 42-mers) were detected. For V 11 /A and K 14 /A, the amount of these high molecular weight oligomers was reduced relative to that of the DK 14 mutant. As the abundance of the band at ,78 kDa increased, the low molecular weight oligomers were fewer and less abundant, suggesting a precursor-product relationship. Although, the high molecular weight oligomer band at ,78 kDa was less intense for K 14 /A relative to DK 14 , a prominent smear of oligomers was present between ,12 and 40 kDa. Along with the ,78 kDa oligomers, 2 other types of oligomers ,5 to 6.5 kDa (trimers and tetramers) were prominent for V 11 /A. In addition to the normal oligomer size distribution, substitution of H 12 to Ala also yielded a smear of oligomers ranging from ,12 to 20 kDa (heptamers to dodecamers). These results suggested that the side-chains of V 11 , H 12 and K 14 within AChE 586-599 restrained the formation of high molecular weight oligomers and therefore controlled AChE 586-599 oligomerization.
Mutation of E 2 to Ala abolished the immediate formation of cross-linked oligomers larger than ,3 kDa (dimers), suggesting that E 2 was essential for any oligomer formation above dimers at this low concentration during this short time-course experiment.
Morphology of the amyloid aggregates of AChE 586-599 and AChE 586-599 mutants
The ThT assay has allowed the determination of the aggregation potential and kinetics of the various peptides, and also provided a clue about the stability and quantity of the final amyloid products (plateau height). However, it did not give direct information about the size and morphology of the aggregates formed. Moreover, the analysis of the oligomer formation and oligomer size distribution by PICUP did not allow the study of the F 3 /A, H 4 /A and R 5 /A mutants. Thus, we used negative staining electron microscopy (EM) to examine the ultrastructure of the aggregates for the F 3 /A, H 4 /A and R 5 /A mutants and also to determine whether mutants, which were faster than AChE 586-599 at adopting a b-sheet conformation and/or at fibrilizing (e.g. K 14 / A and DK 14 ), could form different types of amyloid aggregates. Representative images of the morphologies observed are presented in Figure 9.
Appearance of unbranched fibrils was observed for AChE 586-599 and mutants E 2 /A, H 4 /A, R 5 /A, K 14 /A and DK 14 ( Figure 9A). The fibrils formed by the mutants were different to the ones derived from the wild-type peptide. AChE 586-599 fibrils were generally aligned, very long (.2 mm) and broad (10-15 nm) ( Figure 9A, top panel) and showing typical helical twists (inset, black arrows). The periodicity of AChE 586-599 fibrils is 26-34 nm. Some thinner (5-8 nm) and sometimes shorter fibrils were also observed for AChE 586-599 (white arrows). By contrast, the fibrils from the E 2 /A mutant were as broad as the ones from AChE 586-599 (,15 nm), however they were shorter and less abundant ( Figure 9A). The R 5 /A mutant formed large fibrilar aggregates, from which laterally associated (top inset) and tangled (bottom inset) fibrils were emerging ( Figure 9A). Fibrils from H 4 /A mutant were abundant, thin (4-6 nm) and tangled with each other ( Figure 9A), often resulting in structures resembling 'plates' (inset). Similarly, fibrils from DK 14 mutant were more abundant, slightly thinner (8-13 nm) and more tangled than AChE 586-599 ( Figure 9A). DK 14 also displayed some very short fibrils (.50 nm) (inset). The helical twists were also observed for DK 14 mutant (black arrow), however this twisted morphology was less frequent than for the wild-type AChE 586-599 fibrils. The K 14 /A mutant displayed a similar fibril morphology to the DK 14 mutant, albeit the fibrils were thinner (5.5 to 8.5 nm) and fewer ( Figure 9A). Examination of F 3 /A mutant revealed predominantly spherical structures (diameter 10-16 nm), ''rods'' (8-13 nm wide, .50 nm long) (protofibrils) and amorphous aggregates of various sizes (some being over 50 nm wide, inset) ( Figure 9B). The spherical and ''rod'' structures are consistent with the presence of amyloid precursors (oligomers) [31,32,24]. However, all of the ''rods'' appeared branched with the branching not following any particular dimension or orientation.
The morphology of the amyloid aggregates formed by the various peptides was consistent with the results from the fibrilization assay (e.g. the highly tangled and abundant fibrilar structures that are observed for the DK 14 mutant are consistent with the very fast fibrilization kinetics determined in the ThT assay).
Discussion
The goal of this study was to investigate the driving forces involved in the organization of a model peptide, AChE 586-599 , into b-sheet oligomers and subsequently into amyloid fibrils. The choice of AChE 586-599 as a model peptide for amyloid assembly was guided by two criteria, which are the tractability of AChE 586-599 assembly upon neutralization (AChE 586-599 remains monomeric and random coil at acidic pH) and the involvement of AChE or related products in Alzheimer's disease and in the promotion of Ab fibrilization [18,23,19,20,24]. Amyloid fibrils originating from unrelated proteins or peptides possess similar structures and morphologies [1,5]. While overall amino acid composition is an important factor determining amyloid formation, the details of primary sequence also plays an important role. Therefore, a common view is that fibril assembly is governed by properties of the sequence backbone and specific side-chain interactions [7,8,9,33,10]. This would explain that a simple hydrophobic collapse would not be sufficient to drive the aggregation of any polypeptide chains under physiological conditions. On the contrary, a final ordered amyloid assembly would be determined by, and highly dependent on, a number of specific interactions between side-chains at specific positions within the sequence, which is supported by our results.
The effect of the positional scanning mutations suggested that there was a position dependence of AChE 586-599 assembly. Indeed, some positions within the AChE 586-599 sequence were tolerant to alanine substitutions, whereas others were restrictive. The termini of AChE 586-599 appeared to be more permissive to mutations (e.g. E 2 , H 4 , R 5 at the N-terminus, and H 12 and K 14 at the C-terminus) since they did not preclude fibrilization. By contrast, mutations within the core of AChE 586-599 sequence were very restrictive since they sharply abolished or affected fibrilization (e.g. W 6 , S 7 , S 8 , Y 9 , M 10 and V 11 ). The only exceptions were two terminal aromatic residues, F 3 and W 13 , which drastically affected fibrilization. Thus, one could speculate that only the positions within the AChE 586-599 sequence providing maximal and optimal stabilizing interactions, would be affected by mutations. On one hand, the dependence of the observed amyloidogenic potentials on the position of the mutation could correlate with the b-sheet propensity and the hydrophobicity of the side-chain at this position. For example, the side-chains of A 1 and F 3 were optimally fitted with the side-chains of V 11 and W 13 to create a hydrophobic motif at the edges of the assembly (see below and the model in Figure 10B). Similarly, the side-chain of W 6 was optimally fitted with the side-chain of M 10 to create a hydrophobic motif at the core of the structure (see Figure 10B). It was also observed that substitutions within the core of AChE 586-599 , or modifying the hydrophobicity and/or aromaticity, affected to some extend the b-sheet propensity, with the strongest effect for F 3 , W 6 , Y 9 , M 10 , V 11 and W 13 . Since for the H 4 /A and R 5 /A mutants, there was no perturbation of the hydrophobic patch, the stabilizing effect of A 1 -W 13 , F 3 -V 11 and W 6 -M 10 side-chain interactions could still occur. Moreover, substitutions of H 4 and R 5 did not affect the b-sheet propensity within the AChE 586-599 sequence (see Figure 1). This could explain why the fibrilization properties of these mutants were at least as good as those of AChE 586-599 . Although substitution of E 2 and K 14 did not disrupt the hydrophobic patches, the mutations would abolish a putative salt bridge (as discussed below). On the other hand, previous studies have shown that during amyloid formation water molecules barely interact with the hydrophobic patches but instead cluster around the terminal charged residues [34]. This solvent effect could also contribute to the position dependence of AChE 586-599 aggregation.
There are criteria that an amyloid structure must meet to be stable, such as the need to place charged residues outside the core amyloid structure. Moreover, electrostatic interactions, such as salt bridges, may contribute to the orientation and stability of the amyloid assembly [35,36,15,37]. Furthermore, Lys and Glu residues have been often found in neighboring b-strands, and peptides rich in these two residues formed fibrils [38,39,36]. Massi et al. predicted that an equilibrium between electrostatic interactions and hydration determines the stability of amyloidforming peptides, therefore that the fibrilization kinetics would be affected by pH and ionic strength [12]. We have demonstrated that by providing additional ionic strength, we successfully shielded the uncompensated charges of the E 2 /A and K 14 /A mutants resulting in a shorter lag phase for the assembly and an increase of the plateau height. For the K 14 /A mutant, the resulting aggregates were also more stable. Thus, these results suggested that specific Coulombic interactions, such as a salt bridge between E 2 and K 14 , might occur during AChE 586-599 oligomerization. This hypothesis is reinforced by the rapid kinetics of fibrilization and the presence of high molecular weight oligomers upon substitution of the charged K 14 with an uncharged side-chain (Ala), which demonstrated the unequivocal involvement of the positive charge of the K 14 side-chain. A similar effect was observed for Ab, in which the mutation of D 23 to Asn yielded higher oligomers, suggested to be due to the removal of the salt bridge between D 23 and K 28 [14]. Nilsberth et al. also found that for the Ab arctic mutation (E 22 to Gly), the substitution increased the rate of protofibril formation [40]. Furthermore, substitutions of K 14 , by affecting early kinetics and assembly, could destabilize the structure of the final assembly or drive the assembly into a different pathway, which would lead to different morphologies for the aggregates. In accordance with this hypothesis, the fibrils observed for the K 14 /A and DK 14 mutants were shorter, and thinner and more tangled than AChE 586-599 . Similarly, the presence of an Ala instead of the N-terminal Asn, or the absence of Asn, in the peptide NFGAILSS of human islet amyloid polypeptide (IAPP) accelerated the kinetics of aggregation and modified the morphology of the fibrils, which were thinner and more tangled [41,11]. Tenidis et al proposed that Asn directed self-assembly and lateral packing of the filaments [41].
Interestingly, the E 2 /A mutant had a stable plateau height (albeit drastically decreased) and similar lag phase of fibrilization to that of AChE 586-599 . The increase of b-sheet propensity triggered by this mutation (see Figure 1) could compensate the absence of the salt bridge with K 14 during early assembly, which would explain the similarity of lag phase. Moreover, E 2 was found to be essential for the formation of normal amount and normal length fibrils, which could explain the very low plateau height observed in the ThT assay for this mutant. The substitutions of E 2 and K 14 also altered the general properties of AChE 586-599 , such as its net charge and isoelectric point (pI). At acidic pH, the net charge of AChE 586-599 is +4, with the side-chains of H 4 , R 5 , H 12 and K 14 being protonated. This high density of positive charges would prevent interactions between AChE 586-599 molecules, which would explain the random coil conformation. By contrast, at neutral pH the net charge of AChE 586-599 is +1, with the carboxylic group of E 2 side-chain being deprotonated (21), the amino group of R 5 and K 14 side-chains protonated (+2). By substituting E 2 to Ala, the net charge of AChE 586-599 became +2. By substituting R 5 or K 14 to Ala, the net charge of AChE 586-599 became 0. According to Chiti et al., an increase of the net charge would trigger intermolecular repulsion, whereas a low net charge would favor aggregation [42]. Therefore, the E 2 /A mutant would be less prone to fibrilization, and the R 5 /A and K 14 /A and DK 14 mutants would fibrilize more rapidly than AChE 586-599 , just as we observed. The pI of AChE 586-599 is 8.65 and substitution decreasing it could facilitate aggregation at neutral pH. Substitution of E 2 to Ala raises the pI to 10.00, which could explain why the E 2 /A mutant was no faster than AChE 586-599 . By contrast, substitution of R 5 to Ala, or K 14 to Ala or removal of K 14 decreases the pI to 6.96. These observations are in perfect agreement with our CD and fibrilization results, in which each of these mutants was faster than AChE 586-599 by at least one experimental measure.
Single point mutations, which were not affecting the net charge of AChE 586-599 , were found to drastically affect or to completely abolish AChE 586-599 conformational change and fibrilization (e.g. F 3 /A and W 6 /A). Therefore, the minimization of Coulombic repulsion is not the only factor involved in amyloid assembly and interactions between side-chains, particularly of hydrophobic and aromatic nature, could provide additional energy for stabilization. The frequency of aromatic residues is low in proteins in general, however they occur very frequently in amyloid-related sequences [11,43]. Interactions between aromatic ring planes that are parallel to each other, referred as p-p interactions or p-stackings, play a key role in molecular recognition and self-assembly, which could be the function that the aromatic residues play during amyloid formation [44,45,43,46]. Moreover, aromatic residues are characterized by both a high hydrophobicity and a high b-sheet propensity. Aromatic residues are abundant in AChE 586-599 (29% of the total residues) and we assessed their involvement by a complementary approach, including far-and near-UV CD. These assays showed that upon neutralization a conformational transition from random coil to b-sheet occurred for AChE 586-599 and involved strong interactions between aromatic residues for the formation of tertiary or quaternary structures. Indeed, the near-UV CD clearly demonstrated that the aromatic rings had restrained mobility, as when buried, which is consistent with these residues stacking during b-sheet formation. Similar p-stacking was described for several amyloid-forming peptides, such as IAPP and Ab [15,25]. Additionally to the CD studies, the influence of the aromatic residues was also observed in the fibrilization and oligomerization assays. Indeed F 3 , W 6 , Y 9 and W 13 , when mutated, appeared to significantly influence the first stage of aggregation with either no fibrilization observed or a drastically longer lag phase. This result was also confirmed by the study on oligomer formation where the F 3 /A, W 6 /A, Y 9 /F and W 13 /A mutants led to a very poor oligomerization. The effect of the substitutions on the early stage of aggregation might be related to a decrease in hydrophobicity and b-sheet propensity rather than a lack of aromatic ring, as it was proposed for other systems [47,48]. However in later stages of the aggregation, the role of the aromatic rings of F 3 , W 6 , Y 9 and W 13 within AChE 586-599 might involve pstacking to stabilize the cross-b structure. Indeed in a number of models, the rings of Phe residues were proposed to cement together the b-strands in a b-sheet, creating a Phe zipper [49,50,10,15]. Furthermore, the substitution of F 3 or W 13 to Ala may have affected the tight association between strands, at the paired hydrophobic patches within the steric-zipper (see green box in Figure 10B). The smaller side-chain of Ala instead of the bulky side-chain of F 3 or W 13 may permit increased flexibility that could have opposed an ordered assembly. Additionally to an effect on early aggregation, the F 3 mutation to Ala resulted in spherical oligomers, short, wide and branched protofibrils, and amorphous aggregates. Similarly, a Phe to Ala substitution in the peptide NFGAILSS of human IAPP, resulted in the formation of amorphous aggregates [11]. It was previously described that the specificity and the directionality of the amyloid assembly could be provided by the specific orientation of aromatic side-chains [51,52,43]. Without F 3 , AChE 586-599 may be lacking important interactions involved in the directionality of the assembly. This could result in a correct minimal assembly that failed to orientate for further linear stacking, leading to multiple branching and finally aggregation into amorphous structures. Therefore, the branched ''protofibrils'' and amorphous aggregates would be an amyloid dead end and no fibrils would be formed, as our EM results suggested.
The F 3 /A mutant, as mentioned above, would be less hydrophobic at the extremities of the assembly and therefore would be more exposed to the solvent. Thus, the F 3 /A mutant might not segregate at the air-water interface as stably as AChE 586-599 could, due to its amphipathicity. By analyzing the temporal pattern of surfactant activity, we demonstrated that indeed AChE 586-599 stably remained associated with the air-water interface, whereas the F 3 /A mutant quickly left the interface to relocate to the bulk solution. However, the F 3 /A mutant possessed a stronger surfactant activity than AChE 586-599 , after 2 min at neutral pH. This result suggested that soon after neutralization, the F 3 /A mutant was able to relocate and to aggregate faster than AChE 586-599 at the air-water interface. This could explain the shorter lag phase observed when AChE 586-599 was co-fibrilized with the F 3 /A mutant, as compared to AChE 586-599 alone. Indeed if faster at aggregating, only small amount of the F 3 /A mutant would be sufficient to create nuclei to accelerate an assembly. Above a certain threshold of assembly, AChE 586-599 aggregates would eventually relocate to the bulk solution to free the air-water interface for monomer recruitment and further assembly. Once in the bulk, AChE 586-599 assembly would possibly be prone to dissociation or ''shedding''. The F 3 /A mutant formed branched and amorphous aggregates, which by being an amyloid dead-end might be more stable than those from AChE 586-599 . This hypothesis could explain the difference of aggregate stability between the F 3 /A mutant (stable) and AChE 586-599 (unstable), and also the fact that the F 3 /A mutant was able to stabilize AChE 586-599 during co-fibrilization assays (e.g. by capping and slowing dissociation). Furthermore, López de la Paz et al. proposed that water molecules could act as a cement to bring strands and side-chains close enough via water-mediated hydrogen bonds, which would stabilize the amyloidogenic organization [34]. A similar effect of water molecules could stabilize the F 3 /A mutant in the bulk solution. Similarly to the F 3 /A mutant, the assembly stability at the air water-interface of the V 11 /A, H 12 /A and W 13 / A mutants was affected, which correlates with the negative effect of the substitutions on their fibrilization potential. It was previously proposed that surface tension could play an important role in the stabilization of proteins [53]. Collectively, these results suggest that AChE 586-599 amyloid assembly could preferentially occur at an airwater interface rather than in the bulk solution, potentially due to a stabilization effect.
In addition to the non-covalent interactions described above, cation-p interactions have been also found to play a role in molecular association in biological systems [54,55]. A cation-p interaction is a short-range electrostatic interaction between p electrons in an aromatic ring and a positively charged cation, most commonly between Arg and Tyr [56,54]. The formation of a cation-p interaction could lower the cost of desolvating the charge of the cation and could provide a mean for burying the positively charged group within a solvent-excluding domain. Moreover, cation-p interactions are important for specificity and stability during protein association [54,57]. In protein complexes, Arg involved in cation-p interactions were also found to be involved in cation-anion interactions, which provide long range attraction for the guanidium group and ensure the specificity of binding [54]. The importance of cation-p interaction was demonstrated for other amyloidogenic peptide [58,59,48]. Thus, it is possible that a cation-p interaction between R 5 and Y 9 occurred during AChE 586-599 fibrilization (see Figure 10B, pink boxes), allowing the burial of the polar Arg group within the core of AChE 586-599 . Without R 5 , AChE 586-599 rapidly formed large fibrilar aggregates composed of laterally associated and tangled fibrils, which could be due to the absence of specificity and stability provided by an R 5 -Y 9 interaction. In addition to cation-p interaction, aminoaromatic interaction could also contribute to the formation of R 5 -Y 9 side-chain interactions, and Y 9 could be involved in long-range interaction with the negatively charged side-chain of E 2 providing further specificity to the assembly. The substitution of Y 9 with Phe, conserving only the aromatic ring, had a more deleterious effect on fibrilization than the Ala substitution and drastically impaired oligomer formation, suggesting a role of Y 9 OH group during stacking rather than during very early assembly (formation of the nuclei).
Additionally to p-p and cation-p interactions, aromatic residues can also be involved in SH-p interactions. The Met sulfur can favorably and strongly interact with the non-polar surfaces on binding partners (specifically the aromatic face of residues), and can also engage oxygen atoms through S-O interactions and N-H groups through hydrogen bonding. Such interactions would be useful in the association between different sub-units in oligomeric proteins and could be a stabilizing force in holding two b-strands together [60]. Met has been found to preferentially associate with Trp, due to hydrogen bonding and S-aromatic interactions [61]. When M 10 was substituted to Ala, AChE 586-599 lost the ability to switch to a b-sheet conformation and to fibrilize. However, the M 10 /A mutant was able to form oligomers with a size distribution identical to AChE 586-599 but less abundant, which reinforces that it is not merely the hydrophobicity of the side-chain that drives AChE 586-599 oligomerization. It is possible that these oligomers did not bind ThT and were not able to further assemble into larger species, resulting in an absence of signal during the ThT assays. A role of M 35 has been described in the dimerization of the Ab protofibril [62]. Therefore, it is possible that M 10 interacted with W 6 , through hydrophobic or S-aromatic interactions, to stabilize the AChE 586-599 assembly. The similar effects observed for the substitutions of both W 6 and M 10 on fibrilization and oligomerization reinforce this hypothesis.
Another important factor able to stabilize proteins is metal chelation [63]. Studies on the binding of metal ions on amyloid proteins or peptides demonstrated that Cu(II) ions induce b-sheet formation of the unstructured amyloidogenic region of the prion protein, and Cu(II) and Zn(II) ions strongly induce Ab fibrilization [64,65,66,67,68]. In the case of AChE 586-599 , the substitution of H 12 to Ala affected both the fibrilization rate and the oligomerization, with a slower lag phase than AChE 586-599 , a decrease in plateau height and an unbalanced distribution of oligomers. Moreover, the substitution of H 4 to Ala affected the morphology of the fibrils, thinner than AChE 586-599 and tangled. Thus, it appeared that H 12 was more ''important'' than H 4 , which was in agreement with the significant loss of propensity for conversion to b-strand within the sequence YMVH (the strongest propensity within AChE 586-599 ), when H 12 was mutated to Ala. A putative role for H 4 and H 12 in metal chelation will be the subject of further studies.
AChE 586-599 is amphiphilic due to an alternating pattern of polar and non-polar residues, which would trigger the burial of the non-polar faces by aggregating into b-sheet structure. Although nature and evolution have disfavored such alternating pattern, sequences containing it were shown to self-assemble [8]. Polar side-chains have the advantage of forming hydrogen bonds, in addition to the van der Waals interactions. In general polar residues interact with the solvent or other polar residues. The effects of substitutions of S 7 and S 8 on all the AChE 586-599 properties tested were similar and consistent. Indeed, the decrease oligomer amount for the S 7 /A and S 8 /A mutants were in agreement with the absence of fibrilization or their longer lag phases of fibrilization. The similarity and consistency upon substitution suggests that the two Ser residues might interact together rather than with other residues, creating a polar-polar interaction through hydrogen bonding, which would be in agreement with the strong correlation found between Ser-Ser pairing in b-sheets [39].
It is thought that extended parallel b-sheets are less stable than antiparallel ones [69,34]. This is based on the fact that in antiparallel b-sheets, most contacts along the fibril axis are between non-identical and complementary residues, which allow more variability in the hydrogen bonds and side-chain interactions, and also in the geometry of the interactions. This variability would allow a greater number of possible conformations and alignments for the strands. By contrast, in a parallel arrangement, the contacts are in between identical residues and the optimal geometry of the hydrogen bonding would therefore be linear. The presence of uncompensated opposite charges on each peptide plays a fundamental role in favoring an arrangement in which the distance between identical charges is maximized. In the case of AChE 586-599 , this would favor an antiparallel organization to avoid high electrostatic repulsion due to E 2 and K 14 . This is a conclusion already tentatively suggested by ELISA on these mutants, using the MAb 105A [23]. A good agreement and correlation were found between all the assays and properties for AChE 586-599 aggregation, and are summarized in Figure 10A. In accord with the experimental observations, and taking all the previous arguments and interactions into consideration, we attempted to model the assembly of AChE 586-599 , in which we have considered only an antiparallel arrangement for the b-sheets ( Figure 10B and C). Our model fits with the position dependence of assembly (restrictive and permissive positions for substitutions) and the putative interactions described above. Indeed, figure 10B shows an electrostatic interaction between E 2 and K 14 (blue and red shaded box); two hydrophobic patches, the first one at the edges of the assembly (A 1 -W 13 and F 3 -V 11 ) and the second within the core of the assembly (W 6 -M 10 , which could also include an S-aromatic interaction) (green shaded boxes); cation-p interactions between R 5 and Y 9 (pink boxes); putative site for metal chelation between H 4 and H 12 (brown boxes); and polar-polar interaction between S 7 and S 8 (non boxed). All these interactions fit with the recent proposal of a steric-zipper forming the basic surface from which the b-sheet stacking occurs and the fibril elongates [70]. Figure 10C represents the formation of the b-sheets along the fiber axis and the putative quaternary interactions stabilizing and reinforcing such assembly. Such interactions during stacking of the strands within the b-sheet would be p-p between aromatic rings (Phe, Tyr and Trp residues) and metal chelation (His and Tyr residues). Our experiments were not able to ascertain the orientation of the strand edges (both edges 'up', or one 'up' and one 'down',) or of the strand faces (face-to-face or face-to-back, with the same or different faces adjacent to one another) during the stacking. The differences between the types of orientation would see the assignment to different classes of steric-zipper, according to the nomenclature of Sawaya et al., and the identity of the aromatic residues involved in the p-p interactions [70]. Class 2, 3, 6, 7 and 8 of steric zippers were ruled out due to the parallel arrangement of their b-sheets. We found that optimal p-p stacking, as represented and highlighted on Figure 10C, could be achieved only when AChE 586-599 b-sheets have the same sides facing each other ('face-to-face') and the orientation of the sheet edges facing up ('up-up'). According to the nomenclature of Sawaya et al., this type of arrangement and orientation corresponds to a class 1 steric-zipper [70]. The strands within a b-sheet are stacking in a parallel arrangement, whereas the bsheets are antiparallel. We noted that the only other class with similar side-chain interaction within the steric-zipper, class 5 (the strands within a b-sheet are stacking in an antiparallel arrangement; the b-sheets are antiparallel, and have the same sides facing each other, 'face-to-face'), led to a model with poor p-p stacking ( Figure 11). However, the orientation of the stacking remains to be determined.
In summary, the analysis of stabilizing or destabilizing effects of residue substitutions on the amyloid assembly of AChE 586-599 has provided evidence for the critical role of specific side-chain interactions in the stabilization of nascent aggregates and for the position dependence of these side-chains upon polymerization and fibril formation. Systematic dissections of the critical residues and interactions driving amyloid assembly, and of the chemical details underlying the molecular recognition process could provide invaluable information on such a poorly understood and complicated process. The benefits of such an understanding could be applied to the wider field of protein folding since an increasing number of non-pathogenic polypeptides have been shown to form amyloid fibrils under certain conditions [71,72,73,74]. Another important application would be in the biological and medical fields by helping in the design of synthetic molecules to prevent the critical interactions occurring during amyloidogenesis (e.g. capping peptides abolishing Ab fibrilization and blocking of p-stacking interactions) [26,27].
Synthetic peptides and antibodies
AChE 586-599 and AChE 586-599 mutants were prepared as described [23]. Specific mouse Mab (105A) anti-AChE 586-599 in a b-sheet conformation was previously described [23]. Preparation of amyloid oligomers AChE 586-599 and AChE 586-599 mutants oligomers (12 mM) were covalently cross-linked by photo-activation using the photoinduced cross-linking of unlabelled proteins (PICUP) as previously described [24]. However, the light was filtered through a 450 nm UV filter and the reaction mixture was exposed to 5 flashes of light (xenon lamp).
SDS-PAGE and Western-blot
Amyloid oligomers were resolved on 10% Tris-Tricine SDS-PAGE and electro-blotted onto nitrocellulose. Nitrocellulose membranes were blocked with 5% (w/v) non-fat milk in PBS and incubated with the Mab 105A recognizing AChE 586-599 in bsheet conformation, followed by anti-mouse IgG conjugated to horseradish peroxidase (HRP). Products were visualized by enhanced chemiluminescence.
Circular dichroism
CD-spectra were recorded from 250 to 190 nm (far-UV) and from 320 to 240 nm (near-UV) at 20uC in a quartz cuvette (1 mm path length) using a Jasco J-720 spectropolarimeter. The mean spectra of multiple scans (scan speed of 50 nm min 21 and response time 4 sec) were collected. The spectra were blank subtracted and normalized to molar ellipticity. At least three independent assays were performed and analyzed with the twosample Student's t-test.
Fibrilization experiments
The assays were performed in a 96-well plate (black wall, clear bottom; Greiner, UK) with 165 mM ThT in PBS. ThT fluorescence (excitation 450 nm, emission 480 nm) was measured at 37uC every 6 min, with 5 min shaking after every measurement, on a BMG Polarstar plate reader. The values of buffer-ThT were subtracted from the values of peptide-ThT. At least three independent assays were performed and analyzed with the twosample Student's t-test.
To investigate the effect of ionic strength, the fibrilization experiment were carried as described above except that the peptides were incubated with 165 mM ThT in 1.8 mM KH 2 PO 4 and 10.1 mM NaH 2 PO 4 with varying concentration of NaCl (from 0 to 1.4 M) and KCl (from 0 to 27 mM).
Surface tension measurement
Analyses were performed in a 96-well plate format, as described [29,24]. Briefly, AChE 586-599 and AChE 586-599 mutants were re-suspended in 80 mL 200 mM sodium acetate pH 3 and surface tension measured at 450 nm (BMG Polarstar plate reader) before and at various time points after neutralization (20 mL 1M NaH 2 PO 4 , pH7.2). DOD = (OD offset position -OD central position ) neutral pH 2min -(OD offset position -OD central position ) acidic pH . At least three independent assays were performed and analyzed with the two-sample Student's t-test.
Electron microscopy
200 mM AChE 586-599 and AChE 586-599 mutants in 50 mM NaH 2 PO 4 pH 7.2 were incubated for 36 hours. Then the samples were adsorbed onto Formvar-coated 400 mesh copper grids, air dried, washed with distilled water, negatively stained with 2% aqueous uranyl acetate and viewed with a Zeiss Omega 912 microscope.
Structural Model Building
The fibrillar model was built with the DeepView program [75]. Starting with one b-strand aligned along the x-axis, a second bstrand, which is a copy of the first strand rotated by 180u along the z-axis, was placed next to the first strand along the y-axis separated by 10 Å . A third strand, an exact copy of the first strand is placed next to the second strand along the y-axis, again separated by 10 Å . Adjustment was made to the second and third strands to ensure that the side-chains on separate strands are intercalating with each other. These three strands constitute a layer or stericzipper. For Class 1 fibril, the second and third layers were added by translating the original layer by 4.8 Å and 9.6 Å along the zaxis. For the Class 5 fibrils, the second layer was obtained by rotating the original layer by 180u along the y-axis and then by translation so that it laid on top of the original layer along the zaxis with a separation of 4.8 Å . The third layer was a translation of the first layer by 9.6 Å along the z-axis. Adjustments were made to the second and third layers to allow for the correct configuration for hydrogen bonding. Computations for energy minimization were done in vacuo using the GROMOS96 43B1 parameter set without reaction field, as implemented within Swiss-PdbViewer. | v3-fos-license |
2018-04-03T04:53:24.656Z | 2014-06-12T00:00:00.000 | 14234448 | {
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} | pes2o/s2orc | Immobilized Lentivirus Vector on Chondroitin Sulfate-Hyaluronate Acid-Silk Fibroin Hybrid Scaffold for Tissue-Engineered Ligament-Bone Junction
The lack of a fibrocartilage layer between graft and bone remains the leading cause of graft failure after anterior cruciate ligament (ACL) reconstruction. The objective of this study was to develop a gene-modified silk cable-reinforced chondroitin sulfate-hyaluronate acid-silk fibroin (CHS) hybrid scaffold for reconstructing the fibrocartilage layer. The scaffold was fabricated by lyophilizing the CHS mixture with braided silk cables. The scanning electronic microscopy (SEM) showed that microporous CHS sponges were formed around silk cables. Each end of scaffold was modified with lentiviral-mediated transforming growth factor-β3 (TGF-β3) gene. The cells on scaffold were transfected by bonded lentivirus. In vitro culture demonstrated that mesenchymal stem cells (MSCs) on scaffolds proliferated vigorously and produced abundant collagen. The transcription levels of cartilage-specific genes also increased with culture time. After 2 weeks, the MSCs were distributed uniformly throughout scaffold. Deposited collagen was also found to increase. The chondral differentiation of MSCs was verified by expressions of collagen II and TGF-β3 genes in mRNA and protein level. Histology also confirmed the production of cartilage extracellular matrix (ECM) components. The results demonstrated that gene-modified silk cable-reinforced CHS scaffold was capable of supporting cell proliferation and differentiation to reconstruct the cartilage layer of interface.
Introduction
The rupture of the anterior cruciate ligament (ACL) is one of the most common injuries of the knee with an incidence of 1 in 3000 [1], which can result in severe limitations in mobility, pain, and inability to participate in sports and exercise. Due to the limited capacity to regenerate, ACL heals poorly in response to the repair by suturing the injured tissue back together. So the grafts are required for ACL reconstruction [2]. Currently, there are approximately 125,000 ACL reconstruction surgeries performed worldwide each year, most using biological grafts (autografts and allografts). However, there are still drawbacks including the risk of disease transmission, lack of appropriate donors, immune response, and high costs [3,4]. Recently tissue engineering has emerged as a promising strategy for the regeneration of injured ligament with similar biomechanical and biochemical properties [4,5]. The scaffold plays an important role in constructing the tissue-engineered ligament by providing appropriate mechanical integrity and biochemical stimulation.
The integration between soft graft and hard bone-tunnel is particularly critical for biological fixation. The native structure of ACL-bone insertion has four distinct yet continuous regions of ligament, noncalcified fibrocartilage, calcified fibrocartilage, and bone. Interface tissue engineering is a new strategy aiming at regeneration of interface and ultimately enabling the biological fixation of soft grafts in bone-tunnel [6]. The intricate multitissue organization of insertion indicates that interface scaffold design must consider the need to regenerate more than one type of tissue, as well as exercising spatial control over the respective cell populations indigenous to different interface regions. Initial attempts to improve ligament graft to bone fixation focused on augmenting the surgical graft with a material that could encourage bone tissue ingrowth. In previous studies, the scaffold modified by calcium phosphate or tricalcium phosphate cement was found to enhance healing and promote integration [7][8][9]. Additional approaches to improve osteointegration included the addition of periosteum grafts to the region of graft that interacted with the bone and growth factors such as rhBMP-2 [10][11][12][13][14]. Although these methods have improved osteointegration between graft and bone-tunnel, the efforts do not result in regeneration of fibrocartilage layer of interface. Moreover, single-phase scaffold does not fully mimic the complexity of natural interface. The ideal biomimetic scaffold should be designed to recapitulate the inherent complexity of multilayered ligament-to-bone interface. It might direct the growth of the multitissue and overcome shortcomings mentioned above. This multilayer interface scaffold was reported to be loaded with different cells (fibroblast, chondrocyte, and osteoblast) for reconstructing insertion. But it is difficult to conduct due to complicated process.
In recent years, silk has been increasingly studied as the scaffold for ligament tissue engineering due to its biocompatibility, slow degradability, and remarkable mechanical capability [15,16]. In our previous work, the knitted scaffold was demonstrated to possess good mechanical strength and internal connections. It could facilitate nutrients' transmission, tissue infiltration, and matrix deposition. After implantation the scaffold could successfully regenerate ACL in small and large animal models. However, the typical four layers of insertion were not observed, which might decrease the stability of graft [17]. Growth factors have long been delivered to cells to promote cell growth, proliferation, and differentiation. Transforming growth factor-3 (TGF-3) was substantiated to help to maintain a round or polygonal shape of chondrocytes and stimulate total collagen synthesis, which is crucial in maintaining the cartilage functions [18,19]. Recombinant TGF-3 protein has also been delivered to mesenchymal stem cells (MSCs) for in vitro chondrogenesis. In our previous work, the immobilized TGF-3 on scaffold efficiently induced the chondrogenic differentiation of MSCs [20]. In this study, the bone-tunnel part of scaffold was modified with lentivirus carrying TGF-3 gene. We hypothesize that MSCs transfected by lentivirus can regenerate cartilagelike tissue between ligament and bone-tunnel to resemble cartilage layer of native insertion.
In order to prove this hypothesis, the study was designed (1) to prepare a silk cable-reinforced chondroitin sulfatehyaluronate acid-silk fibroin (CHS) scaffold modified with lentiviral-mediated TGF-3 gene, (2) to observe cell proliferation and collagen production in modified area of scaffold, and (3) to examine expressions of cartilage related genes in mRNA and protein level with real-time quantitative RT-PCR, western bolt, and histological stains. The purpose of current study is to find whether the cartilage layer of ligament-bone junction can be reconstructed in vitro using gene-modified scaffold and MSCs.
Scaffold Fabrication.
Four sericin-free silk fibers were braided into one bundle and then two bundles were braided into one cable. Twelve cables placed in parallel were fixed across a customized polypropylene cylinder mold (diameter: 6 millimeters; length: 60 millimeters). The raw Bombyx Mori silk fibers were immersed in 0.02 M NaHCO 3 solution at 90 ∘ C for 1 hour to remove sericin. The fibers were rinsed for 20 minutes 3 times, squeezed out excess water, and allowed to dry overnight. To prepare silk fibroin (SF) solution, the sericin-free silk fibers were added to 9.3 M lithium bromide (BrLi) solution on top of silk fibers and incubated at 60 ∘ C for 4 hours [21]. After dialysis with SnakeSkin (Thermo Scientific Co., 3500 MWCO, USA), the final concentration of SF was 2.0 wt%. The 0.1 g of chondroitin sulfate sodium (Sigma Co., St. Louis) was mixed with 5 mg of hyaluronate (C. P. Freda Pharmacy Co., Shandong, China) and 12 mL of 2.0 wt% SF solution and then poured into the cylinder mold containing the 12 silk cables. Before lyophilization the cylinder mold was kept at −80 ∘ C for 1 hour and then put into lyophilizer (Christ Alpha 1-2 LD, Germany) for 24 hours. The freezedried scaffold was immersed in 90% methanol solution for 30 minutes to induce -sheet structural transition and then washed several times to remove the residual chemicals. Thereafter, the end of scaffold (length 20 millimeters) was immersed in phosphatidylserine (PS) chloroform solutions for 5 minutes. Finally, the scaffold was dried overnight in hood. The process was schematically depicted in Figure 1.
Scaffold Characterization.
The structures and mechanical properties of scaffold were characterized. Mercury porosimetry (Pascal 140, GA) was used to assess the pore size, porosity, and total surface area of scaffold ( = 4). Scanning electronic microscopy (SEM) (HITACHI S-4800, Japan) was used to observe the morphology of micropores. To measure hydrophilicity, the dried scaffolds ( = 6) were hydrated in phosphate buffered saline (PBS) after being weighed. Then the swollen scaffolds were weighed again and the degree of swelling was calculated as the ratio of the weight of PBS absorbed by scaffolds normalized to the initial dry weight [22]. Mechanical test was performed using Shimadzu mechanical testing system (AGS-10kNG, Shimadzu Inc., Japan) with a maximum loading capacity of 500 N. After being hydrated in PBS, the 12 cable-reinforced CHS hybrid scaffolds ( = 6) were performed with a gauge length of 20 millimeters at a loading speed of 2 mm/min. The maximum tensile load (max-load), maximum tensile distance (max-disp), break tensile load (break-load), and break tensile distance (break-disp) were determined.
Isolation and Expansion of MSCs.
MSCs were generated from bone marrow aspirates of rabbits (12 weeks old, 2.5-3.0 kg). According to previous methods [20], mononuclear cells were separated by centrifugation in a Ficoll-Paque gradient (Sigma Co., St. Louis) and suspended in 20 mL of Dulbecco's modified eagle medium (DMEM) supplemented with 15% fetal bovine serum (FBS) (HyClone Logan, Utah). Cultures were incubated at 37 ∘ C and 5% carbon dioxide for 72 hours; then the nonadherent cells were removed by changing medium. When reaching 70-80% confluence, adherent cells were detached from the flask using 0.25% trypsin and subcultured. A homogenous MSCs' population was obtained after 2 weeks of culture and cells of passage 3 were harvested for further use. The adipogenic, osteogenic, and chondrogenic differentiations of cells were tested to ensure the multilineage potential.
Cell Adhesion, Metabolism, Viability, and Transfection.
Cell adhesion to scaffold was examined as reported [23]. The hybrid scaffolds were sliced into circular disc (diameter: 6 mm; length: 4 mm), sterilized by brief treatment with 75% ethanol. The lentivirus vector (1 × 10 8 copies/mL) with a promoter encoding both TGF-3 and enhanced green fluorescent protein (eGFP) was prepared by GeneCopoeia Co.
Then the scaffold was incubated with transfection medium (40 × 10 6 of virus in 1 mL of DMEM) at 37 ∘ C and 5% CO 2 4 hours for immobilization of lentivirus vector. The scaffold was placed into a polypropylene tube (length: 25 millimeters; inner diameter: 6.0 millimeters), loaded with 1 × 10 6 MSCs (passage 3), and incubated. At different time points (i.e., 0, 0.5, 1, 2, 4, and 8 hours) the MSCs/scaffolds ( = 6 for each time point) were gently rinsed with PBS. The detached cells collected from rinsing solution and medium were counted using light microscopy.
The metabolism of MSCs on scaffold was evaluated at 1, 3, 5, 7, 9, 11, 13, and 15 days by alamar blue assay following the vendor's instructions (ABD Serotec, Oxford, USA). The alamar blue assay is a useful method for cell monitoring on 3D porous scaffold [17]. A brief description was as follows: the MSCs/scaffold ( = 6 for each culture period) was incubated in culture medium supplemented with 10% (v/v) alamar blue fluorescent dye for 2 hours. Then 100 L of medium was extracted from each sample and measured at 570/600 nm in a microplate reader (Sunnyvale, CA). The culture medium supplemented with 10% alamar blue was used as a negative control.
The viability of MSCs seeded on scaffolds ( = 4) was examined at culture periods of 1 and 2 weeks using live/dead cells assay. The assay was based on the combination of the fluorescein diacetate (FDA), which stains living cells green, and propidium iodide (PI), which stains dead cells red. Briefly, the MSCs/scaffolds were thoroughly rinsed with PBS in a six-well plate. Then 2 mL of PBS supplemented with 2 L of PI (2 mg/mL) and 6 L of FDA (5 mg/mL) was added into each well. The system was allowed to incubate at room temperature for 10 minutes. Then, each sample was washed with PBS twice and observed with confocal microscopy (Olympus Fluo View FV-1000, Japan).
The transfection of MSCs was examined after 1 and 2 weeks using fluorescence microscope (Leica DMI6000B Inverted Microscope, Germany). The MSCs/scaffolds ( = 4 for each time point) were gently rinsed with 1 mL of PBS, added to 3 mL of trypsin-EDTA solution (Beyotime Co., China), and shaken for 20 minutes. The detached cells were collected, resuspended in DMEM, and transferred into flask for cells adhesion. After 4 hours of culture, the transfected cells which expressed the enhanced green fluorescent protein (eGFP) were observed with fluorescence microscope.
Collagen Production.
One million MSCs were loaded onto scaffold and cultured in vitro for periods of 1 and 2 weeks. The collagen deposited on scaffold was then quantified using sircol collagen dye binding assay kit (Biocolor Ltd., Newtownabbey, Ireland). The dye reagent specifically binds to the [Gly-X-Y]n helical structure of collagen but not to unwound triple helix or random chains of gelatin. Briefly, the samples ( = 6) were incubated with 500 L of pepsin solution (0.25 mg/mL) and shaken for 2 hours. Then 1 mL of dye reagent was added to 300 L of soluble collagen and mixed for 30 minutes at room temperature. The pellet of dyed collagen was precipitated by centrifugation for 5 minutes and then dissolved with 1 mL of releasing reagent. The absorbance was measured at 540 nm. The standard curve was set up on the basis of collagen standard provided by the vendor. The collagen produced was presented as amount of collagen per scaffold.
Histological Assessment.
MSCs seeded on the scaffolds ( = 6) were cultured and harvested at the end of 1 and 2 weeks. The samples were washed with PBS and fixed in 10% neutral buffered formalin. Thereafter, they were dehydrated through a series of graded alcohols and embedded in paraffin. Sections of 5 m thickness were cut and collected on slides. For immunohistochemistry stains, the slides were incubated with collagen II (Sigma Co.) and TGF-3 (Sigma Co.) antibodies. Then detection was applied using streptavidin-biotin immunoenzymatic antigen detection system (UltraVision Detection System, LabVision, USA). The results were assessed by three individuals who were blinded to the treatment. The total reaction volume was 20 L. Real-time PCR reactions were performed at 95 ∘ C for 15 minutes, followed by 40 cycles of amplification consisting of denaturation step at 95 ∘ C for 15 seconds and extension step at 60 ∘ C for 1 minute. The transcription level normalized to GAPDH was then calculated using the 2ΔCt formula with reference to the undifferentiated MSCs.
Western Blot Analysis.
Proteins were extracted from MSCs/scaffold ( = 4) at the end of 1 and 2 weeks with pepsin (200 g/mL in 0.08 M acetic acid, Sigma) for 72 hours at 4 ∘ C. The pepsin was subsequently inactivated with 1 M NaOH. The extract was concentrated using a Nanosep 30 centrifugal filter (30,000 Mw cutoff, Pall Life Sciences, USA). Samples were then separated by electrophoresis in NuPAGE Novex Trisacetate mini gels (Invitrogen, USA) and electrophoretically transferred to a supported nitrocellulose membrane (Biorad Laboratories). The membranes were tested using western blot kit (Invitrogen, USA) according to the manufacturer's instructions. A brief description was as follows: the membranes were blocked with buffer for 1 hour and incubated overnight at 4 ∘ C with monoclonal antibodies against collagen II and TGF-3 diluted to 1 : 500 in blocking buffer. The membranes were then washed five times with washing buffer and incubated for 30 minutes with secondary antibodies diluted to 1 : 200 in blocking buffer. The membranes were rinsed with washing buffer again and incubated with ECL working solution for 5 minutes. The signal was detected using VersaDoc Imaging System (Biorad Laboratories) and relative intensities of positive bands were compared between groups.
Statistical Analysis.
The mean and standard deviation were used to describe the data. The data analysis was performed using SPSS Statistics 20.0 statistical software package. A statistical analysis of quantitative results was carried out with the unpaired Student's t-test and one-way analysis of variance (ANOVA). The statistical significance level was set at 0.05.
Characterization of Scaffold.
Twelve cables placed in parallel were fixed across a customized polypropylene cylinder mold (Figure 2(a)). The cable-reinforced CHS hybrid scaffold exhibited an elastic texture and porous morphology. Each end of scaffold (length 20 millimeters) in the E areas (Figure 2(b)) was immersed in PS chloroform solutions. The porosity of the microporous sponge was found to be 63.4 ± 4.2% and the average pore diameter was 172.3 ± 52.6 m. The surface area of the hybrid scaffold was 2.1 ± 0.3 m 2 /g. The SEM images also indicated that the pore size ranged from 80 to 230 m (Figures 2(c) and 2(d)). The swelling ratio of the scaffold was measured to be 635.7 ± 48.3%. Figure 3 showed the summary of mechanical properties of the scaffold. The max-load was 151.9 ± 11.7 N and the max-disp was 7.1 ± 0.6 mm. The breakload was 51.3 ± 1.0 N and the break-disp was 8.0 ± 0.6 mm.
Cell Adhesion, Metabolism, Viability, and Transfection.
The results indicated that the number of nonadherent cells decreased proportionately with increasing culture time. The number of nonadherent cells at the 4 h group was 7.7 ± 1.4 × 10 4 . This was significantly lower than cell number of 0 h group ( < 0.05). Although the cell number continued to decrease after 4 hours, there was no significant difference between 4 h and 8 h groups. Therefore, 4 h incubation was deemed sufficient for MSCs to attach onto hybrid scaffold (Figure 4(e)). The metabolism of MSCs seeded on the hybrid scaffold increased rapidly with culture time at early stage. The value of alamar blue test increased from 9.9 ± 1.6% to 70.8 ± 5.1% after 5 days of culture. Subsequently it increased gradually and reached a plateau. The value after 15 days was 85.7 ± 3.4% (Figure 4(f)). Viability of MSCs seeded on scaffold was evaluated by confocal microscope. Based on live/dead assay, no significant cell death was observed (Figures 4(a) and 4(b)). The transfected cells expressing eGFP were observed in the interconnective pores with fluorescence microscope. The number of transfected cells at 2-week time points significantly increased compared to that of 1-week ones (Figures 4(c) and 4(d)).
Collagen Production.
Collagen deposition on hybrid scaffold was found to increase proportionately with culture time. The MSCs produced an average of 79.2 ± 4.5 and 112.1 ± 6.3 g collagen after culture periods of 1 and 2 weeks, respectively. The difference between these two groups was found to be significant ( < 0.05) ( Figure 5).
Histological Analysis.
Histological examination revealed that microporous structure of the hybrid scaffold was well preserved and few micropore walls collapsed. Thus, the exchange of nutrients between scaffold and environment was ensured. The MSCs proliferated robustly along the wall of micropores and exhibited fibroblast morphology with elongated nucleus. The 2-week group showed higher cell density and more ECM production when compared with 1week group. Immunohistochemistry staining for collagen II and TGF-3 was found to be positive after 1 week. ECM formation was observed to increase with culture time. The staining of collagen II and TGF-3 was more intense at 2 weeks when compared with that at 1 week ( Figure 6).
Transcription Level of Cartilage-Specific Genes.
MSCs cultured on hybrid scaffold were harvested to assess their gene transcription level using real-time quantitative RT-PCR. The transcription levels of collagen II gene were 0.290 ± 0.046-fold and 0.462 ± 0.053-fold at 1 and 2 weeks ( < 0.05), respectively. The results showed that TGF-3 gene transcription was 0.048 ± 0.005-fold and 0.086 ± 0.006-fold at at 1-week and 2-week time points. The difference at these two time points was found to be significant ( < 0.05) (Figures 7(a) and 7(b)). The results suggested that the hybrid scaffold could support the differentiation of MSCs towards chondrocytes.
3.6. Western Blot Analysis. The protein expressions of TGF-3 and collagen II were compared between groups after 1 week and 2 weeks of culture. Collagen II and TGF-3 were expressed more prominently in 2-week groups in comparison with 1-week groups. The relative intensities of positive staining band for collagen II were 54.2 ± 4.4% and 76.2 ± 8.6% after 1 week and 2 weeks, respectively. There was a significant difference ( < 0.05). The relative intensities of positive staining band for TGF-3 were 46.5 ± 7.4% and 75.9 ± 6.8% after 1 week and 2 weeks, respectively. There was a significant difference ( < 0.05) (Figures 7(c) and 7(d)).
Discussion
This study demonstrated that the lentivirus vector could be immobilized by PS on scaffold, which transfected MSCs continuously. The transfected cells could differentiate into chondrocyte-like cell. This tissue-engineered cartilage layer mimicked the chondral part of natural ligament-bone junction. It might reduce the stress concentration between hard bone and elastic ligament. The results provide potential application of gene-modified scaffold for constructing the cartilage-zone of ligament-bone junction.
The adherence between vector and material was achieved by the binding between lentivirus and PS coating, a component of the plasma membrane. Shin et al. reported that PS could be incorporated into PLG microspheres, which was subsequently combined with scaffold. Large numbers of transduced cells and increased gene expression were observed on the gene-modified scaffold [24]. In this study, lentiviral vector was immobilized on scaffold by incorporation with PS.
The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. The gene delivery methods include viral and nonviral methods. The usually used physical, nonviral methods are microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration. However, the clinical application of nonviral methods is still restricted by some limitations including low transfection efficiency and poor transgene expression. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations. The commonly used vectors mainly include adenovirus, adeno-associated virus (AAV), and retroviruses. Adenovirus and AAV are hardly integrated into target cell genome. Lentivirus belongs to a genus of retrovirus; it can effectively integrate exogenous gene into the host chromosome, so as to achieve the persistence [25]. It is very important that the lentivirus transfection system does not produce cell damage and immune response [26].
A significant role of PS is to increase the amount of virus that associates with the material. Thus a higher dosage of vector can be delivered locally. For scaffold modification, we once demonstrated an increase in lentivirus activity when compared with virus alone (unpublished data). In this study, we found very high transduction efficiency of MSCs with an MOI (multiplicity of infection) of 40 on the scaffold immobilized by PS. At this MOI, over 90% of the cells were identified to be transfected by fluorescence microscope. Despite these high transfection efficiencies, there was no evidence of cell death at this MOI. Lentiviral vectors can transfect both replicating and nonreplicating cells. It can be incorporated into the host genome, thereby theoretically offering prolonged protein production. Although long-term protein production may enhance cartilage repair, there are also concerns about the formation of potential oncogenic effects on surrounding cells.
Several methods have been used to immobilize virus on scaffold. Although these approaches have been effective, vector biotinylation can influence its activity [27,28]. PS has a specific interaction with the VSV-G protein, which is influenced in part by electrostatic interactions. PS is a relatively low molecular weight lipid that is soluble in organic solvents, which is relatively easy compared with the immobilization of antibodies or avidin for virus binding. In general, a scaffold with a minimal pore size of 150 m is suggested for bone tissue engineering. For soft tissue engineering, the pore size of the scaffold usually ranges from 150 to 250 m. The newly formed ECM on the surface would further prevent the cells from infiltrating into the scaffold [29]. It has been estimated that the ligament typically bears peak loads of about 169 N during normal ambulation, with a threefold increase from 400 N to 500 N during strenuous athletic activity [30]. In this study the average pore size of the hybrid scaffold was 172.3 ± 52.6 m, which was suitable for ligament tissue engineering. The enlarged pores and high porosity of scaffold facilitated tissue ingrowth, as demonstrated by histology examination. The hybrid scaffold has a microporous structure with interconnecting pores, consequentially enlarging the surface area for better cell adhesion. However, microporous structure can result in poor mechanical properties. To overcome this drawback, braided silk cables were introduced to increase tensile strength. In this study, the hybrid scaffold was designed to have an average max-load of 151.9 ± 11.7 N. This would meet the mechanical requirement of ACL for activities of daily living. Because the max-load transmittable through the hybrid scaffold is proportional to the number of silk cables, the tensile strength of scaffold can be easily adjusted by changing the number of supporting silk cables.
An important aspect of ECM is its ability to store water, which is necessary to support various cell activities and nutrients exchange. The weight swelling ratio of silk cablereinforced CHS scaffold was 635.7 ± 48.3%. This is about four times higher than that of sericin-free silk fiber (137 ± 12%) [31]. The discrepancy may be due to different composition ratio and fabricating method used [32]. The excellent swelling property of scaffold facilitated cell seeding and cell adhesion. The number of detached cells in the rinsing solution and medium dropped steeply from 96.0 ± 1.9 × 10 4 to 49.5 ± 2.4 × 10 3 within 2 hours. Then it continued to decrease with culture time. Therefore, the scaffold had both excellent biomechanical properties and enlarged surface area for cells adhesion, tissue ingrowths, and nutrients supply [33].
The ligament-bone junction plays a key role in ACL reconstruction. The lack of biological fixation remains the primary cause of graft failure. Due to the complicated structure and various cells, the ligament-bone junction is difficult to successfully reconstruct. In our previous work, the structure of interface was not successfully reconstructed with knitted silk scaffold and MSCs in bone-tunnel. In this study the gene-modified scaffold was used to regenerate the ligament-bone junction.
MSC is known to possess the ability of self-renewal and differentiation into various lineages [34]. In this study MSCs seeded on the hybrid scaffold proliferated robustly and showed good viability. The expression of cartilage-specific markers (collagen II) was upregulated at high levels with the increase of TGF-b3. This is correlated with other reports [35]. The collagen production was also found to increase with culture time.
Conclusions
This study has successfully developed a gene-modified silk cable-reinforced CHS hybrid scaffold with potential application in reconstructing the cartilage layer of ligament-bone junction. Lentivirus vector was imported by PS coating on scaffold. TGF-3 released by transfected MSCs could induce the chondral differentiation of MSCs. The reinforced silk cables significantly increased the tensile strength of scaffold to meet the mechanical requirements. Future study will focus on introducing multivectors into the hybrid scaffold in order to reconstruct the structure of ligament-bone junction. | v3-fos-license |
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} | pes2o/s2orc | The Characteristics of Oil Migration due to Water Imbibition in Tight Oil Reservoirs
In tight oil reservoirs, water imbibition is the key mechanism to improve oil production during shut-in operations. However, the complex microstructure and composition of minerals complicate the interpretation of oil migration during water imbibition. In this study, nuclear magnetic resonance (NMR) T2 spectra was used to monitor the oil migration dynamics in tight oil reservoirs. The factors influencing pore size distribution, micro-fractures, and clay minerals were systematically investigated. The results show that the small pores corresponded to a larger capillary pressure and a stronger imbibition capacity, expelling the oil into the large pores. The small pores had a more effective oil recovery than the large pores. As the soaking time increases, the water preferentially entered the natural micro-fractures, expelling the oil in the micro-fractures. Subsequently, the oil in the small pores was slowly expelled. Compared with the matrix pores, natural micro-fractures had a smaller flow resistance and were more conducive to water and oil flow. Clay minerals may have induced micro-fracture propagation, which can act as the oil migration channels during water imbibition. In contrary to the inhibitory effect of natural micro-fractures, the new micro-fractures could contribute to the oil migration from small pores into large pores. This study characterized the oil migration characteristics and provides new insight into tight oil production.
Introduction
As a key unconventional energy, tight oil has received increased attention by many scholars and has gradually become another research topic following shale gas [1]. The commercial exploitation of tight oil has greatly affected the energy structure of the world, especially North America [2,3]. A tight oil reservoir is characterized by low permeability (<0.1 mD), low porosity (<10%), and micro/nano pores. Currently, multistage hydraulic fracturing is the key technology for commercial exploitation. This technology can induce complex fracture networks by injecting large-scale fracturing fluid into the formation, thus increasing the reservoir's exposed area and improving oil production [4]. After hydraulic fracturing stimulation, shut in operations for a period are beneficial to the production increase of tight oil. Extended shut in time can promote fracturing fluid imbibition into reservoirs to displace crude oil and has been proven to be effective for enhanced oil recovery [1].
In tight oil reservoirs, complex imbibition characteristics result in the uncertainties of appropriate shut-in operations. Lan et al. (2014) [5] conducted a series of spontaneous imbibition experiments to establish dimensionless time model and calculate shut-in time. However, a dimensionless time model cannot involve the effects of pore structure and clay minerals and is not suitable for tight oil reservoirs. Jiang et al. (2018) [6] considered that the water imbibition is generated under confining pressure and carries out a large number of forced imbibition experiments by means of nuclear magnetic resonance (NMR). A modified model of dimensionless time was established by considering the relationship between pore radius and confining pressure [7,8]. Field studies found that not all tight oil wells are suitable for shut-in operation and extending the shut-in time may intensify the damage of the fracturing fluid to the reservoirs, which was not conducive to oil production [9]. Understanding the imbibition characteristics and relevant influencing factors (e.g., complex microstructure and minerals) contributes to conduct-appropriate shut-in operations.
Understanding water imbibition into matrix pores is the key for clarifying microscopic physical mechanism during the shut-in periods. Many published studies have focused on shale, tight sandstone and volcanic rock reservoirs [10,11]. The capillary pressure caused by interfacial tension and the osmotic pressure induced by water activity difference are the main forces that imbibe water into matrix pores. Capillary pressure is inversely proportional to pore radius. Micro/nano pores may result in an ultra-high capillary pressure to imbibe the fracturing fluid [12,13]. The capillary pressure also increases pore pressure within the matrix pores, triggering the propagation of tensile fractures [14,15]. In addition, tight reservoirs have a more complex pore structure than conventional reservoirs, leading to complex imbibition behaviors. Hu et al. (2012) [16] performed imbibition experiments on Barnett shale and found the imbibition behavior to present low pore connectivity. The existence of micro-fractures and bedding planes can enhance the imbibition rate of fracturing fluid. For clay-rich rock, the water imbibition of osmotic pressure is much stronger than that of the capillary pressure, which may result in micro-fracture propagation to increase the imbibition rate [17]. Xu et al. (2018) [1] found that the current imbibition tests were carried out under the atmospheric pressure without considering the effect of forced pressure. Based on low-field nuclear magnetic resonance, a large number of forced imbibition experiments have been conducted on tight sandstone to clarify the imbibition characteristics and influencing factors.
At present, the fracturing fluid imbibition involves physical and chemical process that are not well understood in the tight reservoirs. Oil migration among the multiscale pore structure and the effects of clay minerals on oil migration still need further study. In this study, a large number of imbibition experiments were conducted and the NMR T 2 spectra was used to monitor oil migration dynamics. The imbibition oil recovery of different diameters pores was studied and the effects of pore size distribution, micro-fractures, and clay minerals were analyzed.
Rock Samples and Fluids
The tight reservoirs samples were taken from Ordos Basin, Songliao Basin and Junggar Basin, which are the greatest potential reservoirs for tight oil production in China. Considering that these formations are characterized by a different pore structure and mineral composition, this study can present the oil migration characteristics influenced by microstructure and minerals. The core plugs of 2-5 cm in length and 2.5 cm in diameter were drilled from larger core materials. The end faces of the plugs were cut by a line cutting machine. The X-ray diffraction test for mineral composition was performed on the rock fragments collected from the drilling process. After removing residual oil from the core plugs, the porosity and permeability measurement was conducted on the core plugs. Table 1 presents the basic properties of tight reservoirs samples that involve source, size, porosity and permeability. The porosity of the samples was measured by a helium porosimeter and ranged from 2.7% to 9.2%. It showed that tight oil reservoirs have the low porosity properties. According to the measurement method of Brace et al. (1968) [18], the pulse-decay permeability was determined under the condition of confining pressure (8 MPa), pore pressure (5 MPa) and room temperature (25 • C). The permeability of samples is not corrected by Klinbenberg effects. In contrary to the porosity, the permeability of samples was significantly different, ranging from 0.0053 to 0.24 mD. The different permeability characteristics may result in the existence of micro-fractures that can connect the matrix pores and increase the permeability. The results of the mineral composition are presented in Table 2. The formation of UC7 and LC7 was mainly composed of quartz and feldspar. The content of clay minerals was lower than 16%. Therefore, clay expansion was not considered during water imbibition. The UC7 and LC7 formation samples were used to study the effects of pore size distribution and micro-fractures on oil migration. The content of the clay minerals was larger than 20% in the formation of QT and WEH. The water absorption of clay minerals may change the pore structure. A moderate content of clay minerals (20.5-30.2%) may induce micro-fracture propagation to relieve the reservoir damage and the high content of clay minerals (36.8%) may destroy matrix pores to aggravate the reservoir damage. The QT and WEH formations were used to study for the pore structure change due to clay minerals and their effects on oil migration. In addition, the samples are not organic-rich and the TOC measurement are not conducted. The experimental fluids included deionized water, MnCl 2 solution and kerosene. The basic properties of density, viscosity and surface tension are shown in Table 3. The deionized water was prepared for chemical solutions of MnCl 2 . The NMR signal of hydrogen atom decreased significantly with the MnCl 2 solution concentration, and the high concentration of MnCl 2 solution did not have the NMR signal [11]. During the imbibition experiments, an MnCl 2 solution of 15 wt.% was used as the wetting fluid to displace the non-wetting fluid. The non-wetting fluid was kerosene instead of crude oil.
Experimental Apparatus
The experimental device for sample mass determination was a Mettler Toledo balance (ME204E, Mettler Toledo, Shanghai, China) with an accuracy of 0.0001 g, as shown in Figure 1a. The volume of the imbibition water and the expelled oil was estimated by sample mass change and the density difference between oil and water. The NMR device (MinNMR, Niumag Analytical Instrument Corporation, Suzhou, China) is shown in Figure 1b. NMR is a non-destructive test method, and the T 2 spectra can well reflect the pore structure and fluid distribution characteristics. The higher the T 2 value, the larger the pore size of the fluid concentration. The T 2 spectra amplitude is positively related to fluid volume in the certain aperture pores. By measuring the changes of T 2 spectra amplitude during the imbibition process, it is possible to understand the oil and water migration characteristics due to capillary pressure imbibition. density difference between oil and water. The NMR device (MinNMR, Niumag Analytical Instrument Corporation, Suzhou, China) is shown in Figure 1b. NMR is a non-destructive test method, and the T2 spectra can well reflect the pore structure and fluid distribution characteristics. The higher the T2 value, the larger the pore size of the fluid concentration. The T2 spectra amplitude is positively related to fluid volume in the certain aperture pores. By measuring the changes of T2 spectra amplitude during the imbibition process, it is possible to understand the oil and water migration characteristics due to capillary pressure imbibition.
The setting parameters during the NMR test had a large influence on the test results. For the different reservoir rocks, the test coefficients needed to be determined. In general, the NMR test had four test coefficients: waiting time (RD), echo time interval (TE), signal superposition times (SCANS), and echo numbers (NECH). If the RD is too short, the large aperture signals would be lost. On the other hand, if the RD is too long, the measurement time significantly increases. In conventional sandstone, RD > 3000 ms is appropriate. As for tight sandstones, RD > 8000 ms is suitable. Similarly, the large values of NECH and SCANS can improve the test accuracy, but they also can increase the test time. In this study, the values of NECH and SCANS were set to 2048 and 64 respectively. In addition, the TE was set to 0.3 ms, which is the minimum value of low-field NMR equipment, which contributes to the recognition of micro-pores. The intensity of the magnetic field was about 0.3 ± 0.05 T.
Experimental Procedure
During the imbibition experiments, the NMR T2 spectra as a function of time was measured and oil recovery of different-scale pores was comparatively analyzed. The experimental process is presented as follows: The setting parameters during the NMR test had a large influence on the test results. For the different reservoir rocks, the test coefficients needed to be determined. In general, the NMR test had four test coefficients: waiting time (RD), echo time interval (T E ), signal superposition times (SCANS), and echo numbers (NECH). If the RD is too short, the large aperture signals would be lost. On the other hand, if the RD is too long, the measurement time significantly increases. In conventional sandstone, RD > 3000 ms is appropriate. As for tight sandstones, RD > 8000 ms is suitable. Similarly, the large values of NECH and SCANS can improve the test accuracy, but they also can increase the test time. In this study, the values of NECH and SCANS were set to 2048 and 64 respectively. In addition, the T E was set to 0.3 ms, which is the minimum value of low-field NMR equipment, which contributes to the recognition of micro-pores. The intensity of the magnetic field was about 0.3 ± 0.05 T.
Experimental Procedure
During the imbibition experiments, the NMR T 2 spectra as a function of time was measured and oil recovery of different-scale pores was comparatively analyzed. The experimental process is presented as follows: (1) Before the experiments, the tight reservoirs samples should be cleaned to remove the residual oil. The cleaning solvent are mixture of toluene and ethyl ether, and the process lasts about one month. (2) The samples were placed in a saturating device and evacuated for 2 to 3 h to remove air.
Then, the kerosene was injected under a 20 MPa pressure. The saturation process lasted for 72 h to fill the samples fully with kerosene. (3) The samples were taken out and the mass and size of the samples were measured. They were immersed in the MnCl 2 solution. After a period of time, the masses of the samples were measured and the T 2 spectra was tested using NMR apparatus. (4) Step (3) was repeated, and the T 2 spectra variation was drawn over soaking time.
The schematic diagram of the experimental procedure is shown in Figure 2. (1) Before the experiments, the tight reservoirs samples should be cleaned to remove the residual oil. The cleaning solvent are mixture of toluene and ethyl ether, and the process lasts about one month.
(2) The samples were placed in a saturating device and evacuated for 2 to 3 h to remove air. Then, the kerosene was injected under a 20 MPa pressure. The saturation process lasted for 72 h to fill the samples fully with kerosene.
(3) The samples were taken out and the mass and size of the samples were measured. They were immersed in the MnCl2 solution. After a period of time, the masses of the samples were measured and the T2 spectra was tested using NMR apparatus. (4) Step (3) was repeated, and the T2 spectra variation was drawn over soaking time.
The schematic diagram of the experimental procedure is shown in Figure 2.
Effects of Pore Size Distribution on Oil Migration
The water entered into matrix pores due to the capillary pressure and the oil droplet was gradually expelled to adhere to the surface of the sample. As the volume of the oil droplets was relatively small, the droplets tended to be more evenly distributed on the sample surface, as shown in Figure 3. As for the tight reservoirs, the capillary imbibition can spontaneously displace the oil in the pores, which contributes to the tight oil production. Figure 4 shows the T2 spectra changes of UC7-1 and LC7-1 over time. As Figure 4 shows, the UC7-1 and LC7-1 had the same T2 spectra characteristics. In the initial stage of imbibition, the water preferentially enters into the small pores to displace the oil, causing a rapid decline of oil saturation.
When the soaking time exceeded 520 h, the T2 spectra area did not change substantially, indicating that the residual oil in the pores could not be expelled only by capillary pressure ( Figure 4). The oil saturation was no longer reduced and the imbibition process ended to some extent. In addition, the samples UC7-1 and LC7-1 have different pore size distribution characteristics. The sample of UC7-1 has a dual-peak distribution with a range of 0.06 to 5 ms for the left peak and 5 to 600 ms for the right peak. The sample LC7-1 has a single peak distribution with a range of 0.06-10 ms. Thus, the permeability (0.0062 mD) of LC7-1 is much smaller than that of UC7-1 (0.24 mD). At a soaking time of 520 h, the imbibition oil recovery of UC7-1 was about 34.2%, and that of LC7-1 was about 35.7%. The sample LC7-1 had a slightly larger imbibition oil recovery than UC7-1.
Effects of Pore Size Distribution on Oil Migration
The water entered into matrix pores due to the capillary pressure and the oil droplet was gradually expelled to adhere to the surface of the sample. As the volume of the oil droplets was relatively small, the droplets tended to be more evenly distributed on the sample surface, as shown in Figure 3. As for the tight reservoirs, the capillary imbibition can spontaneously displace the oil in the pores, which contributes to the tight oil production. Figure 4 shows the T 2 spectra changes of UC7-1 and LC7-1 over time. As Figure 4 shows, the UC7-1 and LC7-1 had the same T 2 spectra characteristics. In the initial stage of imbibition, the water preferentially enters into the small pores to displace the oil, causing a rapid decline of oil saturation. [19], the tight rock pores were divided into micro-pores (<1 ms), small pores (1-10 ms), largish pores (10-100 ms) and large pores (>100 ms). The largish pores and large pores included micro-fractures and matrix pores. Figure 5 presents oil recovery in different diameter pores. The oil recovery of the micro-pores was larger than 70%, that of small pores and largish pores was much lower than 30% and that of largish pores was about 40%. Unexpectedly, the smaller pores did not have a much larger imbibition oil recovery.
In Figure 5a, the oil in micro-pores began to decrease at 0 h, but the oil in large pores began to decrease at 100 h. The water preferentially enters the micro-pores under the capillary pressure and the oil drop displaced by water enters the large pores. Figure 6 presents the schematic diagram of oil When the soaking time exceeded 520 h, the T 2 spectra area did not change substantially, indicating that the residual oil in the pores could not be expelled only by capillary pressure (Figure 4). The oil saturation was no longer reduced and the imbibition process ended to some extent. In addition, the samples UC7-1 and LC7-1 have different pore size distribution characteristics. The sample of UC7-1 has a dual-peak distribution with a range of 0.06 to 5 ms for the left peak and 5 to 600 ms for the right peak. The sample LC7-1 has a single peak distribution with a range of 0.06-10 ms. Thus, the permeability (0.0062 mD) of LC7-1 is much smaller than that of UC7-1 (0.24 mD). At a soaking time of 520 h, the imbibition oil recovery of UC7-1 was about 34.2%, and that of LC7-1 was about 35.7%. The sample LC7-1 had a slightly larger imbibition oil recovery than UC7-1.
According to the T 2 relaxation time of Meng et al. (2016) [19], the tight rock pores were divided into micro-pores (<1 ms), small pores (1-10 ms), largish pores (10-100 ms) and large pores (>100 ms). The largish pores and large pores included micro-fractures and matrix pores. Figure 5 presents oil recovery in different diameter pores. The oil recovery of the micro-pores was larger than 70%, that of small pores and largish pores was much lower than 30% and that of largish pores was about 40%. Unexpectedly, the smaller pores did not have a much larger imbibition oil recovery. [19], the tight rock pores were divided into micro-pores (<1 ms), small pores (1-10 ms), largish pores (10-100 ms) and large pores (>100 ms). The largish pores and large pores included micro-fractures and matrix pores. Figure 5 presents oil recovery in different diameter pores. The oil recovery of the micro-pores was larger than 70%, that of small pores and largish pores was much lower than 30% and that of largish pores was about 40%. Unexpectedly, the smaller pores did not have a much larger imbibition oil recovery.
In Figure 5a, the oil in micro-pores began to decrease at 0 h, but the oil in large pores began to decrease at 100 h. The water preferentially enters the micro-pores under the capillary pressure and the oil drop displaced by water enters the large pores. Figure 6 presents the schematic diagram of oil In Figure 5a, the oil in micro-pores began to decrease at 0 h, but the oil in large pores began to decrease at 100 h. The water preferentially enters the micro-pores under the capillary pressure and the oil drop displaced by water enters the large pores. Figure 6 presents the schematic diagram of oil migration among the different pores. The smaller pores correspond to a larger capillary pressure and a stronger imbibition capacity. Therefore, the oil of micro-pores tended to migrate into small and largish pores and that of small and largish pores tends to migrate into large pores. The small and largish pores act as bridges to connect the micro-pores and large pores. migration among the different pores. The smaller pores correspond to a larger capillary pressure and a stronger imbibition capacity. Therefore, the oil of micro-pores tended to migrate into small and largish pores and that of small and largish pores tends to migrate into large pores. The small and largish pores act as bridges to connect the micro-pores and large pores.
Effects of Natural Fractures on Oil Migration
The UC7-2 samples contained many micro-fractures that were visible to the naked eye. They were in the direction of bedding planes. Microscopic observation shows that the crack width was about 100-350 μm (Figure 7b).
Effects of Natural Fractures on Oil Migration
The UC7-2 samples contained many micro-fractures that were visible to the naked eye. They were in the direction of bedding planes. Microscopic observation shows that the crack width was about 100-350 µm (Figure 7b). Figure 8 shows the NMR imaging at a soaking time of 0 h and 59 h. The water imbibition experiments on UC7-2 can help understand the effects of natural fractures on oil migration. migration among the different pores. The smaller pores correspond to a larger capillary pressure and a stronger imbibition capacity. Therefore, the oil of micro-pores tended to migrate into small and largish pores and that of small and largish pores tends to migrate into large pores. The small and largish pores act as bridges to connect the micro-pores and large pores.
Effects of Natural Fractures on Oil Migration
The UC7-2 samples contained many micro-fractures that were visible to the naked eye. They were in the direction of bedding planes. Microscopic observation shows that the crack width was about 100-350 μm (Figure 7b). Figure Figure 9 shows the relationship between T 2 spectra and soaking time. The distribution range of fractures corresponds to >10 ms, which involves largish pores and large pores. The sample LC7-2 contained a small amount of micro-fractures that were speculated by T 2 spectra. The samples of UC7-2 and LC7-2 were embedded by micro-fractures and characterized by different oil migration features. The amplitude decline velocity of largish pores and large pores was larger than that of micro-pores. Compared with the micro-pores, the micro-fractures were the dominant channels for oil migration. According to the NMR imaging shown in Figure 8, a large amount of oil exited in the micro-fractures. As the soaking time increased, the water preferentially entered the natural micro-fractures, expelling the oil in the micro-fractures. Subsequently, the oil in the small pores was slowly expelled. When the soaking time was 500 h, the imbibition oil recovery of sample UC7-2 and LC7-2 were about 36.6% and 34.1% respectively. Sample UC7-2 had more micro-fractures that were beneficial to imbibition oil recovery to some extent. micro-pores oil recovery of UC7-2 and LC7-2 was much smaller than that of UC7-1 and LC7-1.
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Therefore, the existence of micro-fractures may hinder oil migration from micro-pores into large 228 pores. This results in higher residual oil saturation in the micro-pores. In the UC7-2, the oil recovery of micro-pores, small pores, largish pores and large pores was 32.1, 23.5, 55.2 and 82.7% respectively. In the LC7-2, the oil recovery of micro-pores, small pores, largish pores and large pores was 45.6, 1.5, 30.5 and 66.8% respectively. The oil recovery of large pores was much larger than that of micro-pores. Water preferentially enters the micro-fractures to expel the oil. The micro-pores oil recovery of UC7-2 and LC7-2 was much smaller than that of UC7-1 and LC7-1. Therefore, the existence of micro-fractures may hinder oil migration from micro-pores into large pores. This results in higher residual oil saturation in the micro-pores.
Effects of Fracture Propagation on Oil Migration
The Figure 11 shows the observations of QT-1 before and after experiments. It can be seen that a large amount of oil droplets was precipitated, and the volume of the oil droplets significantly exceeded that of the sample UC7-1. Figure 12 presents the NMR images at different soaking times. Evidently, there was no fractures in the sample at the beginning of the experiment. When the soaking time exceeded 43 h, a large number of micro-fractures were generated on the surface of
Effects of Fracture Propagation on Oil Migration
The Figure 11 shows the observations of QT-1 before and after experiments. It can be seen that a large amount of oil droplets was precipitated, and the volume of the oil droplets significantly exceeded that of the sample UC7-1. Figure 12 presents the NMR images at different soaking times. Evidently, there was no fractures in the sample at the beginning of the experiment. When the soaking time exceeded 43 h, a large number of micro-fractures were generated on the surface of sample. The micro-fractures were not natural fractures and they were induced during the experiments. It should be noted that nuclear magnetic signals can only be detected when oil is contained in the micro-fractures. This suggests that a large amount of oil migrates into the micro-fractures during the process of micro-fracture propagation. The new fractures were in the direction of the formation bedding planes, which may result from the opening of bedding planes. The QT reservoir had a clay mineral content of about 23.7%, which formed a strong expansion stress to induce the fracture propagation after encountering water [11].
Effects of Fracture Propagation on Oil Migration
The Figure 11 shows the observations of QT-1 before and after experiments. It can be seen that a large amount of oil droplets was precipitated, and the volume of the oil droplets significantly exceeded that of the sample UC7-1. Figure 12 presents the NMR images at different soaking times. Evidently, there was no fractures in the sample at the beginning of the experiment. When the soaking time exceeded 43 h, a large number of micro-fractures were generated on the surface of sample. The micro-fractures were not natural fractures and they were induced during the experiments. It should be noted that nuclear magnetic signals can only be detected when oil is contained in the micro-fractures. This suggests that a large amount of oil migrates into the micro-fractures during the process of micro-fracture propagation. The new fractures were in the direction of the formation bedding planes, which may result from the opening of bedding planes. The QT reservoir had a clay mineral content of about 23.7%, which formed a strong expansion stress to induce the fracture propagation after encountering water [11]. Figure 13 presents the curves of T 2 spectra in the QT-1 and QT-2 samples. The T 2 spectra of QT formation had single peak features and the T 2 value ranged from 0.1 ms to 800 ms. It contained micro-pores, small pores, largish pores and large pores. As the soaking time increased, the total area of T 2 spectra shows a downward trend, suggesting that the water was imbibed into matrix pores to expel the oil. The amplitude of pores corresponding to 5-50 ms decreased at the beginning, rose in the medium term, and fell in the later period. At the beginning, the oil in the pores was gradually displaced, resulting in a T 2 amplitude drop. In the medium term, the clay mineral expansion induced a lot of micro-fractures and the oil gradually migrated into new micro-fractures, leading to a T 2 amplitude rise. In the later period, the oil in the micro-fractures was expelled and the velocity of oil entry was larger than that of oil departure, causing the T 2 amplitude to drop. The imbibition oil recovery of QT-1 and QT-2 was 44.3 and 32%, respectively. Figure 13 presents the curves of T2 spectra in the QT-1 and QT-2 samples. The T2 spectra of QT formation had single peak features and the T2 value ranged from 0.1 ms to 800 ms. It contained micro-pores, small pores, largish pores and large pores. As the soaking time increased, the total area of T2 spectra shows a downward trend, suggesting that the water was imbibed into matrix pores to expel the oil. The amplitude of pores corresponding to 5-50 ms decreased at the beginning, rose in the medium term, and fell in the later period. At the beginning, the oil in the pores was gradually displaced, resulting in a T2 amplitude drop. In the medium term, the clay mineral expansion induced a lot of micro-fractures and the oil gradually migrated into new micro-fractures, leading to a T2 amplitude rise. In the later period, the oil in the micro-fractures was expelled and the velocity of oil entry was larger than that of oil departure, causing the T2 amplitude to drop. The imbibition oil recovery of QT-1 and QT-2 was 44.3 and 32%, respectively. Figure 14 presents the oil recovery in different diameter pores of the QT-1 and QT-2 samples. In the QT-1, the oil recovery of micro-pores, small pores, largish pores and large pores was 81.5, 35.4, 21.9 and 63.3% respectively. In the QT-2, the oil recovery of micro-pores, small pores, largish pores and large pores was 57, 27.3, 10.2 and 37.0% respectively. The oil recovery order of different diameters pores was micro-pores > large pores > small pores > largish pores. In addition, the oil recovery of largish pores and large pores descended below the zero at first and then increased above zero. The negative value of oil recovery suggests that the fractures propagated to form new space during water imbibition and oil gradually entered the new fractures to decrease the oil recovery factor. The new micro-fractures correspond to largish pores and large pores. Therefore, part of the oil in micro-pores and small pores may also have migrated into the largish pores and large pores. Figure 14 presents the oil recovery in different diameter pores of the QT-1 and QT-2 samples. In the QT-1, the oil recovery of micro-pores, small pores, largish pores and large pores was 81.5, 35.4, 21.9 and 63.3% respectively. In the QT-2, the oil recovery of micro-pores, small pores, largish pores and large pores was 57, 27.3, 10.2 and 37.0% respectively. The oil recovery order of different diameters pores was micro-pores > large pores > small pores > largish pores. In addition, the oil recovery of largish pores and large pores descended below the zero at first and then increased above zero. The negative value of oil recovery suggests that the fractures propagated to form new space during water imbibition and oil gradually entered the new fractures to decrease the oil recovery factor. The new micro-fractures correspond to largish pores and large pores. Therefore, part of the oil in micro-pores and small pores may also have migrated into the largish pores and large pores. Figure 15 presents pictures of the samples before and after the imbibition experiments. At 1 h, the samples began to expand and the consolidated strength of samples decreased. At 60 h, the samples had broken into grains, resulting in the interruption of the imbibition experiments. This can be explained by an abundance of clay minerals in WEH formation. Figure 16 presents the change of T 2 spectra during water imbibition. The T 2 spectra of WEH-1 had single peak features and that of WEH-2 had dual-peak features. The T 2 spectra of WEH-1 and WEH-2 decreased gradually over the soaking time. When the soaking time exceeded 43 h, the samples turned into grains and imbibition experiments ends. The imbibition oil recovery of WEH-1 and WEH-2 was 37.5 and 47% respectively. The WEH-2 had larger oil recovery because of more micro-pores and small pores. To an extent, the imbibition oil recovery of WEH-1 and WEH-2 may be close to 100%. The pore structure of the samples completely disintegrated and all the oil trapped by matrix pores was released. Figure 17 shows the oil recovery in different diameter pores of the WEH-1 and WEH-2 samples. In the WEH-1, the oil recovery of micro-pores, small pores, largish pores and large pores was 33.3, 44.4, 31.4 and 54.0% respectively. The two peak features of WEH-1 suggest that it contained a large amount of micro-fractures. The WEH formation are tight conglomerate formation containing a large amount of gravels. These micro-fractures are well developed along the edge of gravels, which are gravel-edge fractures. The water was imbibed into gravel-edge fractures to displace the oil, resulting in larger oil recovery. In the WEH-2, the oil recovery of micro-pores, small pores, largish pores and large pores was 52.7, 53.1, 26.1, and −0.89% respectively. It did not contain the gravel-edge fractures, and the pores were the main channels for water imbibition. Therefore, the smaller pores had a larger oil recovery. The negative value of oil recovery in >100 ms pores suggests that the micro-fracture propagation and oil migration into new micro-fractures decreased the oil recovery during water imbibition.
Effects of Clay Mineral on Oil Migration
in larger oil recovery. In the WEH-2, the oil recovery of micro-pores, small pores, largish pores and large pores was 52.7, 53.1, 26.1, and −0.89% respectively. It did not contain the gravel-edge fractures, and the pores were the main channels for water imbibition. Therefore, the smaller pores had a larger oil recovery. The negative value of oil recovery in >100 ms pores suggests that the micro-fracture propagation and oil migration into new micro-fractures decreased the oil recovery during water imbibition.
Scaling the Imbibition Results of Different Reservoirs
The ratio of final spectra area to original spectra area is regarded as imbibition oil recovery. In order to study the influencing factors of imbibition oil recovery, the authors used the dimensionless time tD to scale the experimental results. According to Ma et al. (1997) [20], the dimensionless time tD is given by where k is the rock permeability, φ is the fractional porosity of rock, t is the soaking time, σ is the interfacial tension,
Scaling the Imbibition Results of Different Reservoirs
The ratio of final spectra area to original spectra area is regarded as imbibition oil recovery. In order to study the influencing factors of imbibition oil recovery, the authors used the dimensionless time t D to scale the experimental results. According to Ma et al. (1997) [20], the dimensionless time t D is given by where k is the rock permeability, φ is the fractional porosity of rock, t is the soaking time, σ is the interfacial tension, µ o is the oil viscosity, µ w is the water viscosity, and L s is the characteristic length defined by Ma et al. (1997) [20] that involves the effects of sample shapes and boundary conditions. During the imbibition experiments, the experimental fluids were MnCl 2 solution and kerosene. Considering that all the experiments used the same fluids, the surface tension could be set to 45 mN/m, which is the surface tension of kerosene-water. This may not have had significant effects on the analytical results. Figure 18 shows the results of scaling the imbibtion results using the Ma's model. The Ma's dimensionless time t D did not function satisfactorily for all the different samples, indicating that it was not suitable for tight oil reservoirs. The mineral composition and pore structure were very different, and they were not scaled by this method. According to Akin et al. (2000) [21], the effects of these influncing factors on the imbibition rate can be studied based on the plots of imbibition oil recovery vs dimensionless time t D 0.5 (Figure 18b). In Figure 18b, the different lines represent the different experimens of tight reservoirs samples. Despite of the marked differences in physical property, pore structure and mineral composition, similar trends are found in these curves. The relationship between oil recovery and dimensionless t D 0.5 is close to a straight line. The slope of curves represents the imbibition rate that was affected by mineral composition and pore structure. According to Yang et al. (2016) [22], the slope of curves could be defined as a dimensionless imbibition rate, which suggests a stong imbibition potential for water. The imbibition process of tight reservoirs was very slow and need a long time for water front to arrive at the end or center of sample. Therefore, it was difficult to obtain the final oil recovery with the imbibition experiments. However, it can be speculated that the final imbibiton oil recovery was about 35-45% in tight oil reservoirs.
structure. According to Yang et al. (2016) [22], the slope of curves could be defined as a dimensionless imbibition rate, which suggests a stong imbibition potential for water. The imbibition process of tight reservoirs was very slow and need a long time for water front to arrive at the end or center of sample. Therefore, it was difficult to obtain the final oil recovery with the imbibition experiments. However, it can be speculated that the final imbibiton oil recovery was about 35-45% in tight oil reservoirs. The dimensionless imbibition rate of different reservoirs samples is presented in Figure 19. The pore (P) type rock represents the tight reservoirs that only developed matrix pores for the oil migration channel. The natural fracture (NF) type corresponds to the tight reservoirs that developed both matrix pores and micro-fractures. The fracture propagation (FP) type means that the clay expansion could induce fracture propagation during water imbibition. The effects of in situ stress on fracture propagation were not taken into account. The clay (C) type refers to the tight reservoirs that are characterized by a high content of clay minerals (>40%). These different types of tight reservoirs were comparatively studied ( Figure 19).
For the same type reservoirs, the pore size distribution of single peak tended to have a larger capillary pressure, resulting in a stronger imbibition potential than that of dual-peak. As for the tight reservoirs with the pore size distribution of a single peak, the dimensionless imbibition rates of the P type, NF type, FP type and C type were 0.16, 0.73, 0.33-0.96, and 1.02, respectively. Evidently, the dimensionless imbibition rates of the NF type, FP type and C type rival surpassed those of P type due to clay minerals and micro-fractures. The imbibition rate of the NF type was 4.5 times that of the tight rock P type. Compared with the matrix pores, the micro-fractures had smaller flow resistance and were more conducive to water and oil flow. The imbibition rates of the FP type and C type were six times those of the P type. The more the clay mineral, the larger the imbibition rate. This can be explained by driving force. The imbibition driving force was only capillary pressure in low clay content sample, but the imbibition driving forces were both capillary pressure and osmotic pressure in high clay content sample. However, when the clay mineral content exceeded 23%, the increase in The dimensionless imbibition rate of different reservoirs samples is presented in Figure 19. The pore (P) type rock represents the tight reservoirs that only developed matrix pores for the oil migration channel. The natural fracture (NF) type corresponds to the tight reservoirs that developed both matrix pores and micro-fractures. The fracture propagation (FP) type means that the clay expansion could induce fracture propagation during water imbibition. The effects of in situ stress on fracture propagation were not taken into account. The clay (C) type refers to the tight reservoirs that are characterized by a high content of clay minerals (>40%). These different types of tight reservoirs were comparatively studied ( Figure 19). clay mineral content had little effect on the imbibition rate [11]. Excessive clay minerals can also give reservoirs a strong water sensitivity and seriously disperse in water ( Figure 15). Under the reservoir condition, solid particle may plug the pores and decrease the in-place permeability, which is not conducive to the production of tight oil. In this studies, the spontaneous imbibition experiments were mainly carried out at atmospheric pressure, which does not reflect the actual situation. In the future, it will be necessary to perform the imbibition experiments under the reservoir conditions.
Conclusions
In this study, a series of imbibition experiments were conducted on tight reservoirs samples and the NMR T2 spectra was used to monitor oil migration dynamics. The oil recovery in different diameters pores was comparatively analyzed. The effects of pore size distribution, micro-fractures, and clay minerals were studied by scaling the imbibition results. The conclusions are as follows: (1) Concerning the tight reservoirs without clay minerals and micro-fractures, the oil migration due to water imbibition was mainly determined by the pore size. The smaller pores corresponded to a larger capillary pressure and a stronger imbibition capacity. Therefore, the oil of micro-pores tended to migrate into small and largish pores and that of the small and largish pores tended to migrate into large pores. The small and largish pores acted as bridges to connect the micro-pores and large pores. Compared with small and largish pores and large pores, the micro-pores had the largest oil recovery.
(2) As the soaking time increased, the water preferentially entered the natural micro-fractures, expelling the oil in the micro-fractures. Subsequently, the oil in the small pores was slowly expelled. Compared with the matrix pores, micro-fractures have smaller flow resistance and are more conducive to water and oil flow.
(3) The clay minerals of middle content may have induced the fracture propagation. A large Figure 19. The dimensionless imbibition rate of different reservoirs samples.
For the same type reservoirs, the pore size distribution of single peak tended to have a larger capillary pressure, resulting in a stronger imbibition potential than that of dual-peak. As for the tight reservoirs with the pore size distribution of a single peak, the dimensionless imbibition rates of the P type, NF type, FP type and C type were 0.16, 0.73, 0.33-0.96, and 1.02, respectively. Evidently, the dimensionless imbibition rates of the NF type, FP type and C type rival surpassed those of P type due to clay minerals and micro-fractures. The imbibition rate of the NF type was 4.5 times that of the tight rock P type. Compared with the matrix pores, the micro-fractures had smaller flow resistance and were more conducive to water and oil flow. The imbibition rates of the FP type and C type were six times those of the P type. The more the clay mineral, the larger the imbibition rate. This can be explained by driving force. The imbibition driving force was only capillary pressure in low clay content sample, but the imbibition driving forces were both capillary pressure and osmotic pressure in high clay content sample. However, when the clay mineral content exceeded 23%, the increase in clay mineral content had little effect on the imbibition rate [11]. Excessive clay minerals can also give reservoirs a strong water sensitivity and seriously disperse in water ( Figure 15). Under the reservoir condition, solid particle may plug the pores and decrease the in-place permeability, which is not conducive to the production of tight oil. In this studies, the spontaneous imbibition experiments were mainly carried out at atmospheric pressure, which does not reflect the actual situation. In the future, it will be necessary to perform the imbibition experiments under the reservoir conditions.
Conclusions
In this study, a series of imbibition experiments were conducted on tight reservoirs samples and the NMR T 2 spectra was used to monitor oil migration dynamics. The oil recovery in different diameters pores was comparatively analyzed. The effects of pore size distribution, micro-fractures, and clay minerals were studied by scaling the imbibition results. The conclusions are as follows: (1) Concerning the tight reservoirs without clay minerals and micro-fractures, the oil migration due to water imbibition was mainly determined by the pore size. The smaller pores corresponded to a larger capillary pressure and a stronger imbibition capacity. Therefore, the oil of micro-pores tended to migrate into small and largish pores and that of the small and largish pores tended to migrate into large pores. The small and largish pores acted as bridges to connect the micro-pores and large pores. Compared with small and largish pores and large pores, the micro-pores had the largest oil recovery. (2) As the soaking time increased, the water preferentially entered the natural micro-fractures, expelling the oil in the micro-fractures. Subsequently, the oil in the small pores was slowly expelled. Compared with the matrix pores, micro-fractures have smaller flow resistance and are more conducive to water and oil flow. (3) The clay minerals of middle content may have induced the fracture propagation. A large amount of oil migrated into the new micro-fractures during the process of micro-fracture propagation. In contrary to the inhibitory effect of natural micro-fractures, the new micro-fractures could have contributed to the oil migration from micro-pores into large pores. The clay minerals of high content can completely decentralize pore structure and significantly increase the imbibition oil recovery at atmospheric pressure. Under the reservoir condition, the effects of excessive clay minerals need to be studied in the future. More clay minerals may result in water sensitivity damage and do not contribute to oil production. | v3-fos-license |
2020-11-04T14:08:27.086Z | 2020-11-02T00:00:00.000 | 226243498 | {
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} | pes2o/s2orc | Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout
Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.
Recently, there has been a major push to use smartphone technology for infectious disease detection instead of expensive equipment, enabling smartphone-based point-of-care diagnostic applications 22 . For example, Song et al. 23 combined the bioluminescent real time reporter (BART) of LAMP with a smartphone to rapidly quantify Zika virus and spatiotemporally map the disease. Damhorst et al. 24 developed a smartphone-based LAMP platform for HIV virus detection from whole blood samples. Yin et al. 25 used the smart cup for synergistically enhanced colorimetric LAMP detection of HPV virus. Rodriguez-Manzano et al. 26 combined the LAMP assay with smartphone-based detection to develop a rapid, portable and affordable lab-on-a-chip platform for the detection of mobilized colistin resistance. However, their detection signals were not specifically generated by the sequence-specific probes, which potentially leads to false positive results. Due to the low fluorescence signal of the fluorescence probes, it still remains a challenge to adopt a smartphone to directly detect the fluorescence signals generated by the energy-transfer-tagged oligonucleotide probes.
In this study, we developed a novel energy-transfer-tagged oligonucleotide probe, termed cleavable hairpin beacon (CHB), to create a highly sensitive and specific nucleic acid isothermal amplification assay with an improved fluorescence change, enabling smartphone-based point of care detection. The CHB is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonucleases to simultaneously initiate rapid nucleic acid amplification and generate sequence-specific fluorescence. As an application demonstration, the CHB probe was coupled with LAMP assay, termed CHB-LAMP, to detect the DNA of Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. In addition, the CHB probe was used for the IMSA assay to detect a specific gene sequence (VP1 gene) of Enterovirus (EV) 71 virus, the leading causative pathogen of hand, foot, and mouth disease (HFMD).
Results and discussion
CHB-LAMP assay. As shown in Fig. 1A, the CHB probe typically containing loop and stem parts is a single fluorophore-tagged (fluorophore 6-FAM at the 5′-end and quencher DABCYL at the 3′-end) hairpin oligonucleotide with five continuous ribonucleotides close to its stem in the 3′ direction. The loop size of the CHB probe is 16-18 nucleotides (nt). To ensure the stable hairpin structure at elevated temperature (e.g., 60 °C), the stem size of the CHB probe was increased to 8 nt, which is longer than that of conventional molecular beacons (e.g., 6 nt) 27 . The loop sequence of the CHB probe is completely target-specific. To study the formation of the hairpin structure, the minimum free energy (MFE) of the CHB probe was analyzed by using an online NUPACK software. Figure 1B shows the MFE analysis result for the CHB probe designed to detect B. burgdorferi recA gene of tick DNA by the CHB-LAMP assay. Due to the increased stem length, the CHB probe can maintain the stable hairpin structure at both elevated temperature (e.g., 60 °C) (MFM = − 1.97 kcal/mol) and room temperature (e.g., 25 °C) (MFM = − -8.54 kcal/mol), which significantly reduces background fluorescence and enables realtime quantitative isothermal amplification detection.
Once the CHB probe anneals to the target DNA sequence, its hairpin structure is destroyed due to the formation of the hybrid DNA-RNA pairing in its ribonucleotide sites (Fig. 1C). The ribonuclease of RNase H2 can specifically recognize the DNA-RNA pairing and hydrolyse the phosphodiester bonds of RNA to create a free 3′-OH end. The introduction of five continuous ribonucleotides improves the cleavage efficiency of the CHB probe by the RNase H2 28 . The cleaved CHB probe can serve as a new primer to further accelerate the amplification process. Figure 1D showed the principle of the CHB-LAMP assay. Since previous study showed that loop primers plays a crucial role in accelerating LAMP amplification reaction 29 , the loop backward (LB) primer was targeted to design the CHB probe in our CHB-LAMP assay. During the CHB-LAMP reaction, strong fluorescence signal was produced due to the specific cleavage of the CHB probe (Fig. 1D).
Optimization of the CHB-LAMP assay. As proof-of-concept assay, the B. burgdorferi recA gene sequence was targeted to develop real-time CHB-LAMP. Firstly, we used conventional real-time fluorescence LAMP with EvaGreen dye to optimize the LAMP reaction conditions, such as reaction temperature, sequences of primers, and concentration of different compositions (e.g., primers, MgSO 4 , dNTPs). As shown in Supplementary Figs. S1 and S2, the optimal reaction temperature was 60 °C and the optimal reaction mixture contained 4 mM MgSO 4 , and 1.6 mM dNTPs. Next, we designed the CHB probe to develop the CHB-LAMP assay. As shown in Supplementary Fig. S3A, the performance of the real-time CHB-LAMP was influenced by the concentration of the ribonuclease RNase H2. In our experiment, the concentration of 2.0 U/mL was optimal because of its strong fluorescence signal. The success of developing real-time CHB-LAMP may be attributed to the extended stem (e.g., up to 8 nt) of the CHB probe that stabilizes the hairpin structure at elevated temperature (e.g., 60 °C), thereby dramatically decreasing the background fluorescence.
To further confirm the specificity of the fluorescence signal generated in the CHB-LAMP assay, a denaturing 15% polyacrylamide gel electrophoresis (PAGE) with 8 M urea was carried out to analyse the amplified products. As shown in Supplementary Fig. S3B, self-probed fluorescence signal was readily observed from the amplicons of the CHB-LAMP with the RNase H2 and template, whereas not in the CHB-LAMP without either RNase H2 or template. These results demonstrated that the fluorescence produced in the CHB-LAMP was highly specific to target DNA. In addition, it further confirmed that the cleaved CHB indeed served as new LB primer to participate in the enzymatic amplification.
Sensitivity of the CHB-LAMP assay.
To determine the sensitivity of the CHB-LAMP assay, we tested a tenfold serial dilution of plasmids containing B. burgdorferi recA gene by the real-time fluorescence CHB-LAMP assay. For comparison, the MB-LAMP and real-time PCR assays were run in parallel. As depicted in Fig. 2A, the CHB-LAMP was able to detect as few as 100 copies of target DNA with an excellent linearity (R 2 = 0.984), within about 25 min, which was almost two times faster than that of the conventional MB-LAMP assay (Fig. 2B).
Scientific Reports
| (2020) 10:18819 | https://doi.org/10.1038/s41598-020-75795-y www.nature.com/scientificreports/ Figure 2A also indicated that the CHB-LAMP assay was more reproducible, with relative standard deviation (RSD; n = 3) ranging from 0.44 to 2.66%, compared to the MB-LAMP assay with RSD ranging from 0.91 to 9.72% (Fig. 2B). In addition, the CHB-LAMP produced an almost two times greater fluorescence signal than the MB-LAMP assay, enabling us to directly adapt a smartphone for fluorescence signal readout and agent detection at the point of care. Further, the CHB-LAMP assay achieved a comparable sensitivity (e.g., 100 copies of target DNA) with that of the real-time PCR method (Supplementary Figs. S4 and S5). Thus, our CHB-LAMP showed high sensitivity with better quantitative ability compared to the conventional MB-LAMP.
CHB-enhanced fluorescence detection and smartphone readout. To achieve sensitive, cost-effective point of care detection in the context of mobile health, it is critical to generate a strong enough fluorescence signal that can be detected with a smartphone. We compared the endpoint fluorescence detection of the CHB-LAMP with other methods: (i) conventional EvaGreen-based LAMP, (ii) CHB-LAMP without RNase H2, and (iii) MB-LAMP. As shown in Fig. 3A, the CHB-LAMP with the RNase H2 significantly enhanced the endpoint fluorescence change between positive and NTC. Particularly on detecting low copy number of targets (e.g., 100 copies targets), the enhanced fluorescence change (delta FI in Fig. 3B) by the CHB-LAMP with RNase H2 was www.nature.com/scientificreports/ 187.0 ± 1.8 (n = 6), which was more than 48, 1.8, and 2 times higher than those of the EvaGreen-based LAMP, CHB-LAMP without RNase H2, and MB-LAMP, respectively. Further, the amplified products were subjected to denaturing PAGE ( Supplementary Fig. S6), confirming the highly specific amplification detection.
We also compared the CHB-LAMP with the previous CBP-LAMP assay 20 in which the CBP probe only contained one ribonucleotide. As shown in Fig. 3C,D, the fluorescence change [Delta fluorescence intensity (FI) = 195.6] in the CHB-LAMP was about 1.3 times higher than that (Delta FI = 154.1) of the CBP-LAMP assay. This is likely attributed to the introduction of more ribonucleotides in the CHB probe, which increases the cleavage efficiency of the RNase H2 during isothermal amplification.
To further adapt the smartphone for fluorescence detection at the point of care, we developed a smartphone app which we entitled "Fluorescence Reader" to quantitatively detect the fluorescence intensity of the CHB-LAMP products. After 60-min amplification at 60 °C, the tubes were firstly placed on a portable LED blue light illuminator. Next, the programmed smartphone was used to record the fluorescence image, analyse the fluorescence signals and quantitatively report the fluorescence intensity (Fig. 4). As shown in Fig. 4A, the CHB-LAMP products with RNase H2 showed the most remarkable fluorescence change between positive and negative compared with the EvaGreen-based LAMP, CHB-LAMP without RNase H2, and MB-LAMP. To quantify the fluorescence, the average green value of each tube in the photo was calculated automatically by the smartphone app (Fig. 4B,C). As shown in Fig. 4C, the green value of each CHB-LAMP reaction tube could be quantitatively reported by the app without need for complex optical equipment. Therefore, the enhanced fluorescence detection of the CHB-LAMP assay was beneficial for smartphone readout, enabling point-of-care, cost-efficient pathogen diagnostics in resource-limited settings.
Performance of the CHB-LAMP assay on real sample test. Lyme disease is one of the most common tick-borne illnesses that is caused by B. burgdorferi sensu lato transmitted by infected Ixodes scapularis ticks, commonly known as deer ticks. Rapid and accurate detection of B. burgdorferi in tick species plays an important role in assessing the epidemiology and risk of tick-borne diseases, and predicting the disease outcome for tickbitten patients 30,31 . Conventional methods (e.g., PCR, culture) for B. burgdorferi detection are labour-intensive www.nature.com/scientificreports/ and time-consuming, which is not suitable for rapid, sensitive, field-deployable detection. To further verify the application potential in testing real samples, we have adapted the CHB-LAMP to detect B. burgdorferi gene in the DNA samples extracted from ten different tick species. As shown in Fig. 5A,B, four positive samples and six negative samples were accurately detected by both real-time CHB-LAMP and endpoint CHB-LAMP assays, which were well in agreement with the real-time PCR results (Fig. 5C,D).
Versatility of the CHB probe in the IMSA assay.
To evaluate the versatility of the CHB probe in other isothermal amplifications, we coupled the CHB probe with the IMSA assay to detect a specific gene sequence (VP1 gene) of Enterovirus (EV) 71 virus, which is the leading causative pathogen of hand, foot, and mouth disease (HFMD) [32][33][34] . The principle of the CHB-IMSA assay is given in Supplementary Fig. S7. Since the stem primers of the IMSA assay play a critical role in initiating a rapid and highly efficient amplification detection 7 , we designed the CHB probe to target the stem primer of SteR. To optimize the structure design of the CHB probe, its minimum free energy was analysed at different temperature. The simulation results of the CHB probe showed that it maintained a hairpin structure at both 63 °C (MFM = − 2.04 kcal/mol) and 25 °C (MFM = − 9.17 kcal/ mol) (Supplementary Fig. S7A). To further optimize the CHB-IMSA assay, we tested different concentrations of the CHB probe ranging from 0.4 to 1.6 μM and obtained an optimal concentration of 0.4 μM ( Supplementary Fig. S8). To determine the sensitivity, we detected tenfold serial dilution of plasmids bearing VP1 gene sequence by the CHB-IMSA assay. Our results showed that the CHB-IMSA can detect as low as 100 copies of the EV71 VP1 gene (Fig. 6) with a good linearity (R 2 = 0.981) between the threshold time and the log 10 of template's copy number (from 10 7 to 10 3 copies). These results demonstrate that the CHB probe is compatible with the IMSA assay and has great potential to be utilized in different nucleic acid amplification detection.
Conclusions
In this study, we developed a novel, cleavable, energy-transfer-tagged probe for rapid, sensitive and specific nucleic acid detection with enhanced fluorescence signals by coupling with isothermal amplifications. The CHB-LAMP was successfully demonstrated to detect B. burgdorferi DNA in samples extracted from ticks. Compared with conventional MB 17 and the previously reported CBP probe 20 , our CHB probe provides several advantages www.nature.com/scientificreports/ for nucleic acid amplification detection: (i) Enhanced fluorescence signal readout. The introduction of multiple ribonucleotides in the CHB probe increases the cleavage efficiency and produces higher fluorescence signal, and furthermore, the longer stem of the CHB probe ensures it to maintain a stable hairpin structure at elevated temperature (e.g., 60 °C), significantly reducing background fluorescence and enabling highly sensitive, realtime fluorescence quantitative detection. By combing microfluidics-or droplet-based detection technology, the CHB-LAMP can be adapted for absolute quantification of nucleic acids. (ii) Shorter detection time. The CHB probe facilitated nucleic acid isothermal amplification due to its unique structure design and excellent cleaving ability. (iii) Higher reliability for quantitative detection of nucleic acids. Our real-time CHB-LAMP has high sensitivity with better quantitative ability than the conventional MB-LAMP. (iv) Easy coupling with smartphonebased point of care detection. The enhanced fluorescence caused by the CHB probe can readily be read by smartphone, enabling smartphone-based point of care diagnostics. (v) Applicability to different nucleic acid detection methods. Apart from the LAMP assay, the CHB probe has been used for other isothermal amplification, such as IMSA assay. Regarding these advantages, we have shown that CHB-enhanced fluorescence detection is beneficial for the development of simple, affordable smartphone-based point-of-care pathogen diagnostics in resource-limited settings. For endpoint detection, the reaction solutions in the tubes were imaged using the Bio-Rad Gel Imaging System at room temperature after 60-min incubation. The images were analyzed by ImageJ software or a customized smartphone app entitled "Fluorescence Reader". Statistical analysis was conducted using the unpaired t-test in Prism 8 software. MB-LAMP and PCR assays. Molecular beacon-based LAMP (MB-LAMP) assay and a real-time PCR assay were run as references. The molecular beacon (Supplementary Table S1) specific to B. burgdorferi recA gene sequence was used as described previously 17 . The other primers used for the MB-LAMP were the same as those in the CHB-LAMP assay above. The MB-LAMP reaction system consisted of 1 × Isothermal Amplification Buffer, 4 mM MgSO 4 , 1.6 mM each of dNTPs, 0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.8 μM MB, 0.8 μM LF, 0.32 U/μL Bst 2.0 WarmStart DNA polymerase, and 1 μL of the plasmid template solutions or the DNA samples extracted from tick species. For the PCR assay, the SsoAdvanced Universal SYBR Green Supermix kit from the Bio-Rad Laboratories was used and the primers were shown in Supplementary Table S1. Based on the manufacturer's instructions, the reaction contained 1 × Supermix, 400 nM each of primers, and 1 μL of DNA sample. The thermal cycling protocol was 2.5 min at 98 °C for initial denaturation, 35 cycles of 15 s at 95 °C for denaturation and 30 s at 60 °C for annealing and extension, followed by a melt-curve analysis (from 65 to 95 °C with 0.5 °C increment). | v3-fos-license |
2018-10-21T23:03:03.131Z | 2018-09-27T00:00:00.000 | 52909756 | {
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"Chemistry",
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} | pes2o/s2orc | Drug Resistance Mutation L76V Alters Nonpolar Interactions at the Flap–Core Interface of HIV-1 Protease
Four HIV-1 protease (PR) inhibitors, clinical inhibitors lopinavir and tipranavir, and two investigational compounds 4 and 5, were studied for their effect on the structure and activity of PR with drug-resistant mutation L76V (PRL76V). Compound 5 exhibited the best Ki value of 1.9 nM for PRL76V, whereas the other three inhibitors had Ki values of 4.5–7.6 nM, 2–3 orders of magnitude worse than for wild-type enzymes. Crystal structures showed only minor differences in interactions of inhibitors with PRL76V compared to wild-type complexes. The shorter side chain of Val76 in the mutant lost hydrophobic interactions with Lys45 and Ile47 in the flap, and with Asp30 and Thr74 in the protein core, consistent with decreased stability. Inhibitors forming additional polar interactions with the flaps or dimer interface of PRL76V were unable to compensate for the decrease in internal hydrophobic contacts. These structures provide insights for inhibitor design.
■ INTRODUCTION
The protease (PR) encoded by the human immunodeficiency virus (HIV) is an important drug target for treatment of the pandemic disease HIV/AIDS. A decrease in AIDS-associated deaths was observed in the mid-90s because of the inclusion of both PR inhibitors (PIs) with reverse transcriptase inhibitors in therapy. 1 Despite this notable success, the rapid evolution of drug-resistant viral strains poses a critical challenge, and drugresistant mutations have been observed for all classes of antiviral drugs. 2 HIV PR performs an essential role in viral replication by processing the viral precursor polyproteins into mature viral proteins. Inhibitors bind in the active-site cavity of dimeric HIV PR and block its catalytic activity. More than 100 mutations in the PR gene have been associated with drug resistance. 3 Second-generation inhibitors, such as darunavir (1), lopinavir (2), and tipranavir (3) (Figure 1), were designed to target resistant variants of HIV-1 PR. The peptidomimetic inhibitor 2 resembles the natural PR substrate with P2−P3′ groups. 4,5 Inhibitor 3 is a highly potent, nonpeptidic PI with a unique oxygen that displaces conserved active-site water and forms direct hydrogen bonds with the main chain amides of Ile50/Ile50′. 6,7 The most-recently approved inhibitor, 1, exhibits low toxicity and is equipped with P2/P2′ groups that form strong hydrogen bonds with conserved, main chain atoms, resulting in high binding affinity for PR. 8−10 Because of these favorable factors, resistance mutations rarely develop during treatment with 1. 11 The evolution of resistance toward second-generation inhibitors has fueled the design of novel investigational inhibitors. Compounds GRL-0519 (4) and GRL-5010 (5), derived from the scaffold of 1, are highly potent against several drug-resistant variants ( Figure 1). Inhibitor 4 has an enlarged tristetrahydrofuran P2 group, which fits better in the S2 pocket of PR and reinforces a water-mediated network at the dimer interface. 12−14 Inhibitor 5 differs from 1 by addition of a gemdifluoro moiety on the P2 bis-THF group, which improves lipophilicity and forms halogen bond interactions with the carbonyl oxygen of flap residue Gly48. 15−17 Mutation L76V is associated with clinical resistance to 1, fosamprenavir, 2, and indinavir; 2,18 however, it is also linked with increased susceptibility to first-generation inhibitor saquinavir (6), atazanavir, and 3. 19,20 This mutation occurs with a frequency of around 3% in PI-experienced patients 20, 21 and can be transmitted to treatment-naive patients. 22,23 Inclusion of L76V in mutants bearing three other resistance mutations is associated with two-and eightfold increase in resistance to 1 and 2, respectively, and an eightfold increase in susceptibility to 6. 20 Previous studies of PR with the single mutation of L76V (PR L76V ) showed about 100-fold worse inhibition by 1 compared to wild-type PR (PR WT ) as assessed by isothermal titration calorimetry, 24 although another group using an enzyme inhibition assay reported only 1.5-fold loss in potency. 25 Investigational inhibitor GRL-02031 showed a twofold increase in inhibition constant (K i ) for PR L76V in comparison to PR WT . 26 Crystal structures of PR L76V in complexes with 1 and 6 showed loss of interactions with 1 and gain of a water-mediated interaction with 6 relative to PR WT , 24,25 consistent with the effects on resistance. 20 These effects on inhibitors must be indirect as the side chain of Leu76 lies in the interior of the PR dimer and has no van der Waals contacts with these antiviral inhibitors. In the mutant structure, the smaller Val76 side chain has lost hydrophobic interactions with neighboring side chains of Asp30, Lys45, Ile47, and Thr74 compared with those of the wild-type Leu76, consistent with decreased stability and slower autoprocessing observed for the mutant and its precursor. 24,26 Here, we have assessed the effect of four antiviral inhibitors, clinical inhibitors 2 and 3 and investigational inhibitors 4 and 5, on the structure and activity of the PR L76V mutant. Clinical inhibitor 2 was selected because L76V is associated with an eightfold increase in resistance to this PI as inferred from genotype−phenotype data. 20 Fluorine-containing inhibitors, 3 and 5, form direct interactions with flap residues of the PR, 7,16 potentially stabilizing the PR dimer. The larger P2 group and reinforced dimer interface interactions of compound 4 also might overcome the decreased dimer stability observed for PR L76V . 12,24 The results show that these chemically diverse inhibitors lose potency against PR L76V , and suggest that local rearrangements in the hydrophobic core because of mutation L76V act to decrease the effectiveness of the inhibitors.
■ RESULTS
Tested Inhibitors Have Higher K i Values for PR L76V Relative to PR WT . Inhibition constants (K i ) of the compounds for PR L76V were determined using a fluorescent substrate analog of the HIV-1 p2/NC cleavage site. Table 1 lists K i values for the mutant in comparison with values reported previously for wild-type enzymes. 7,12,16 As reported previously, inhibitor 1 has the best inhibition of this mutant, 24 whereas inhibitor 6 retains a similar inhibition of mutant relative to wild-type enzymes. 27,28 Compound 5 is the most potent of the new inhibitors for PR L76V with a K i of 1.9 ± 0.7 nM, whereas 3 and 4 are the worst of the tested inhibitors with K i of 7.6 ± 0.3 and 7.2 ± 1.4 nM, respectively. Therefore, the tested inhibitors are within a 10-fold difference in potency from each other for PR L76V inhibition. With the exception of 6, all K i values measured for inhibition of PR L76V were significantly higher than the picomolar K i values reported for wild-type PR with changes of 1200-fold for 4, 400-fold for 3, 300-fold for 5, 150fold for 2, and 80-fold for 1. 7,12,16 These higher K i values imply that L76V confers resistance toward the four tested inhibitors.
Structures of the PR L76V Dimer with Inhibitor Resemble Each Other as Well as Their PR WT Counterparts. Crystal structures of PR WT complexed with PIs 2 and 3, and of PR L76V complexed with each of the four inhibitors, were determined at high resolution to identify any structural changes because of the single mutation ( Table 2). The dimer of PR L76V with 2 and the location of residue 76 are illustrated in Figure 2A. All PR L76V structures were solved in the P2 1 2 1 2 space group with one dimer in the asymmetric unit, as were PR WT complexes with inhibitors 2 and 3 in the present study and in previous studies [PR WT complexes with 4 and 5 at 1.27 Å (PDB ID 3OK9) and 1.30 Å (PDB ID 4U8W), respectively]. 12,16 The unit cell dimensions were almost identical for all structures, although 2 complexes had ∼1 Å longer a axis and ∼1 Å shorter b axis compared to the other structures. The six structures were refined to R-factors of 15.1−19.8% with diffraction data at a 1.20−1.75 Å resolution. Atoms were unambiguously modeled, as shown by the electron density map in Figure 2B for the single conformation of 2 in complex with PR L76V . Coordinate errors estimated from Luzzati plots ranged from 0.14 to 0.18 Å for the highest to lowest resolution structures.
The new PR WT complexes with 2 and 3 were refined at nearatomic resolutions of 1.26 and 1.20 Å, respectively, which is a significant improvement compared to previously reported structures determined at 1.54 and 1.80 Å resolutions, respectively. 7 The previous PR WT structures in complex with 2 (PDB ID 2O4S) or 3 (PDB ID 2O4P) were solved in the same space group, and the Cα atoms superimposed with the new higher resolution structures with root-mean-square deviation (rmsd) values of 0.19 Å for complexes with 3 and 0.20 Å for complexes with 2. Therefore, the overall folds are very similar.
The four new PR L76V inhibitor structures were superimposed with their corresponding PR WT inhibitor complexes. The overall backbone structures were essentially identical, with low rmsd values ranging from 0.12 to 0.17 Å for all Cα atoms. Therefore, mutation L76V does not produce major alterations in the overall structure of the PR dimer. Furthermore, all PR L76V structures were also very similar to each other, regardless of the inhibitor, with pairwise rmsd values ranging from 0. 25
ACS Omega
Article and inhibitor, both direct and water-mediated, were compared for wild-type and mutant PR complexes. Halogen bonds were also observed for two inhibitors, 3 and 5, containing fluorine atoms. The interactions are described for the major conformations of inhibitors in the complexes. Similar interactions were observed for the minor conformations except where noted.
Hydrogen bond interactions in X-ray crystal structures of proteins must be interpreted with caution as hydrogen atoms are poorly scattered by X-rays. Neutron crystallography, however, provides direct evidence for the position of protons. The neutron crystal structure of HIV PR with amprenavir showed nonideal hydrogen bond geometry for inhibitor interactions with the carbonyl oxygen of Gly27, the amide of Asp29, and a water-mediated interaction with the amide of Ile50′. 29 Similar effects were observed for the neutron structure of amprenavir complexed with PR mutant V32I/I47V/V82I. 30 No neutron crystal structures have been reported for the inhibitor complexes in this study; hence, hydrogen bonds for L76V complexes are described by the same criteria as for previously published wild-type complexes ( Figure 3).
All clinical inhibitors contain a central hydroxyl group that interacts with the catalytic aspartates, residues 25 and 25′, of PR. The protonation state and hydrogen bond interactions of the catalytic residues, Asp25 and 25′, have been examined in several neutron structures. The side chain carboxylate oxygens of Asp25 and 25′ are almost coplanar with about 2.7 Å between the closest "inner" oxygens of the two side chains. Our neutron structure of wild-type HIV PR with amprenavir showed two protons located on the inner carboxylate oxygen of Asp25 and the outer carboxylate oxygen of Asp25′, which formed hydrogen bonds with the inhibitor hydroxyl. 29 Subsequent neutron studies of 1 and amprenavir complexes with a mutant PR demonstrated that the location of the two protons varies, depending on the pH, inhibitor, and mutated residues. 30,31 In our X-ray structures of inhibitor-bound PR L76V , we cannot distinguish which hydrogen bond interactions occur with the inhibitor hydroxyl group. In the absence of neutron structures corresponding to the PR complexes with the inhibitors described here, we have indicated distances for four possible interactions between the hydroxyl oxygen of the inhibitor and carboxylate oxygens of
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Article Asp25 and 25′ in Figure 3. These interactions are excluded from the description of inhibitor−PR hydrogen bonds in the following sections. Although inhibitor 2 occurs in two alternative conformations with relative occupancy of 75:25 in the wild-type complex, only a single conformation was observed for 2 bound to PR L76V . Excluding interactions with the catalytic Asp25 and 25′, compound 2 shows only three direct hydrogen bonds with PR L76V and three water-mediated interactions with main chain atoms ( Figure 3A). The interactions are in agreement with those reported previously. 7 Compound 2 has a pseudosymmetric structure for the central P1−P1′ region. The watermediated interactions with the amides of Ile50 and 50′ in the flaps are conserved in the majority of PR−inhibitor complexes. The pyrimidine acetamide group of 2 forms hydrogen bonds with the main chain amide and carboxylate side chain of Asp29 and a water-mediated interaction with the carbonyl oxygen of Gly27. These hydrogen bonds contribute to a network of interactions at the dimer interface connecting Gly27 and Asp29 in one subunit with Arg8′ in the second subunit. The hydrophobic dimethylphenoxy group on the opposite end of 2 has van der Waals interactions with the side chains of Ala28′, Asp29′, Asp30′, Val32′, Ile47′ and Ile84′. Inhibitor 2 shows highly conserved hydrogen bond interactions in the complexes with PR WT and PR L76V with differences in hydrogen bond length of no more than 0.2 Å. Therefore, the decreased potency of 2 toward PR L76V cannot be explained by changes in PR interactions with the inhibitor.
The distinctive features of the interactions of compound 3 with PR are the presence of direct hydrogen bonds of the inhibitor with the amides of flap residues Ile50 and Ile50′, as well as fluoride halogen bonds with the guanidinium side chain group of Arg8′. None of the other clinical inhibitors can form these interactions. Furthermore, 3 adopts a bent conformation at the sulfonamide group, and does not bind in the same pockets as more peptidic inhibitors. As shown in Figure 3B, inhibitor 3 has five direct hydrogen bonds with the PR, two halogen interactions with the guanidinium side chain of Arg8′, and a water-mediated interaction with the amide of Gly48. The carboxylate groups of the catalytic Asp25 and Asp25′ are rotated in the 3 complex relative to their orientation in the complexes with other inhibitors and show altered distances to the hydroxyl of 3. Inhibitor 3 retains wild-type hydrogenbonding interactions with the PR L76V mutant, with 0.1 Å difference in distance observed for most interactions. The largest increases of 0.2 and 0.3 Å in the mutant are seen for the hydrogen bond to the carbonyl oxygen of Gly48 and the watermediated interaction with the amide of Gly48, respectively. Thus, 3 forms direct hydrogen bonds with the main chain atoms of flap residues, Gly48, Ile50, and Ile50′ in both wildtype and mutant PRs. Inhibitor 3 shows two alternative conformations in both complexes with 70:30 relative occupancy; the minor conformation with 30% occupancy loses a hydrogen bond to the amide of Asp29 in the mutant structure. The conservation of 3 interactions with PR L76V and wild-type enzyme implies that the poor K i value for the mutant does not arise from altered binding interactions.
In both the PR WT and mutant complexes, compound 4 binds in two alternative conformations with 50:50 relative occupancy. Inhibitor 4 forms five direct hydrogen bonds with PR and three water-mediated interactions in both mutant and wild-type complexes ( Figure 3C). In comparison to the wild-type complex, both conformations of 4 in the mutant show a slight shift of the water interacting with the amides of Ile50 and Ile50′, yet this change maintains the interactions with the flaps. A second water, which is highly conserved in many PR structures and was mentioned earlier in the description for complexes with 2, is integrated into a dimerstabilizing network of interactions that coordinates the tris-THF rings of 4, the carbonyl oxygen of Gly27, and the side
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Article chains of Asp29 and Arg8′. Compared to the wild-type complex, most hydrogen bond interactions between inhibitors and PR L76V have insignificant changes of less than 0.1 Å. Greater variation is observed for the interactions with the flap water with differences of up to 0.4 Å in length. One alternate conformation in both wild-type and mutant structures shows longer 3.4−3.5 Å hydrogen bonds between the tris-THF group of 4 and the amides of Asp29 and Asp30. Once again, the lack of significant differences in PR active-site interactions with 4 indicates that the resistance mechanism induced by L76V relies on an alternate strategy.
Compound 5 crystallized in two alternate conformations in PR WT as well as in PR L76V with 55:45 relative occupancy. Overall, PR WT shows seven direct hydrogen bond interactions, two halogen bond interactions, and three water-mediated interactions with the major conformation of 5 ( Figure 3D). The majority of the direct hydrogen bond interactions of PR L76V with inhibitors are identical in length or within a 0.1 Å range from those observed in the wild-type complex. Asp30 and Asp30′ occur in two alternate conformations, resulting in differences in the interactions of the P2 aniline group of inhibitors with Asp30′ in the PR L76V structure. In the wild-type complex, the amino group of P2 forms hydrogen bond interactions with the side chain carboxylate of Asp30′ and with the main chain amide and carbonyl oxygen. In the PR L76V structure, the P2 aniline has shifted slightly relative to its position in the wild-type complex. In the major conformation of Asp30′ in the mutant, the carboxylate side chain is positioned to form a shorter hydrogen bond interaction with the aniline amino group relative to the wild-type complex, whereas the hydrogen bond of the main chain amide of Asp30′ with the inhibitor NH 2 is elongated to 3.5 Å, and the interaction of the main chain carbonyl oxygen with inhibitor is lost (interatomic distance of 4.4 Å). Similar shifts in P2 aniline and altered interactions with Asp30′ were reported for the PR L76V complex with 1. 24 This major conformation of Asp30′ is stabilized by an ionic interaction with the side chain of Lys45′. The minor conformation of the Asp30′ side chain has rotated away from the inhibitor; however, the main chain amide and carbonyl oxygen form hydrogen bonds of 3.0 and 3.3 Å, respectively, with NH 2 of inhibitors. Although 5 is the most effective of the four tested inhibitors for PR L76V , it did not retain the picomolar inhibition reported for wild-type enzymes. Overall, the interactions of the major conformation of 5 with Asp30′ are altered in the mutant; however, the rest of the hydrogen-bonding network is maintained. Therefore, the loss in potency against the mutant is not completely explained by interactions between inhibitors and PR.
Polar and hydrophobic interactions of inhibitors 1−6 with mutant PR L76V and wild-type enzyme are summarized in Table 3. Hydrogen bond interactions include direct inhibitor− protein interactions and water-mediated interactions and showed little change for alternate conformations of inhibitors. The count of van der Waals contacts is complicated by the existence of alternate conformations of inhibitors, and frequently for adjacent amino acid side chains or main chains. The exact contacts may be impossible to determine when alternate conformations show 50:50 relative occupancy and, in fact, multiple conformations will contribute to the ensemble present in solution. The binding affinities of clinical inhibitors for wild-type PR have been divided into enthalpic and entropic components using isothermal scanning calorimetry. 7,28 The binding of inhibitors 2, 3, and 6 is dominated by the large entropic component, and only inhibitor 1 showed enthalpically driven binding to wild-type PR. This thermodynamic analysis implies that in most cases inhibition cannot easily be assessed by summing inhibitor−PR interactions. In fact, thermodynamic dissection of inhibitor affinity for mutant PRs showed unfavorable changes in both entropic and enthalpic components. 7 Inhibitor 1 loses hydrogen bond interactions with mutant PR L76V , countered by a small increase in hydrophobic contacts, and worse inhibition of the mutant. Inhibitor 6 showed gains in hydrogen bond and hydrophobic interactions with this mutant in agreement with insignificant differences in inhibition compared to wild-type enzymes. Apart from inhibitor 2, the other inhibitors showed fewer hydrophobic contacts with mutants as well as worse inhibition values.
Hydrophobic Interactions of Leu76 are Decreased in the PR L76V Mutant. Residue 76 occupies a region critical for internal hydrophobic contacts between the flap and the core of the protein. Residue 76 lies in the central strand of a threestranded β-sheet forming one flank of the substrate binding cavity near the base of the flap (Figure 2). The mobility of the flaps is essential for substrate binding and catalysis as the flaps act as lids over the active-site cavity and must open to allow substrate entry and release of products. 32 Consequently, altered flap dynamics has been reported for drug-resistant mutants. 25,27,33,34 In all the structures of PR WT and PR L76V with various inhibitors, the main chain of residue 76 forms conserved hydrogen bond interactions with adjacent strands of the βsheet comprising residues 31−33 and 57−59. In addition, the side chain of residue 76 forms hydrophobic interactions with the side chains of Val32, Val56, and Gln58 ( Figure 4). Furthermore, the wild-type residue, Leu76, forms van der Waals interactions with Asp30, Thr74, and with the side chains of Lys45 and Ile47 in the first β-strand of the two-stranded flap ( Figure 4A). These interactions of Leu76 with residues 30−33 and both strands of the flap are conserved in the open conformation of the PR dimer (PDB ID 2PC0). 35 However, in the PR L76V mutant, the shorter side chain of Val76 loses hydrophobic contacts with the first β-strand of the flaps, and additionally loses interactions with Asp30 and Thr74 ( Figure 4B). These changes agree with those reported previously for PR L76V complexes with 1 and 6. 24 The flap−core interface is composed of residues Lys45, Ile47, Ile54, Val56, and Gln58 in the flap, residues Asp30 and Val32 in the inhibitor binding site, and Thr74 and Leu76 in the protein core. The side chains of these residues form hydrophobic interactions and shield the interface from the solvent. Leu76 is a central component of the flap−core
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Article interface and provides extensive hydrophobic interactions connecting the flaps to the active site. The loss of interactions across the flap−core interface in the PR L76V mutant is expected to influence flap mobility and promote inhibitor dissociation from the dimer. This effect is consistent with the observations of increased dimer dissociation and decreased dimer stability. 24 Increased conformational flexibility of the flaps in the PR L76V mutant compared to the wild-type enzyme was reported in 1 ns molecular dynamics simulations of the complexes with inhibitor 2 consistent with increased calculated interaction free energy and clinical resistance. 36
■ DISCUSSION
Drug-resistant mutation L76V is rare in clinical isolates of HIV; however, this mutation alone acts to decrease the stability of the PR dimer, alters precursor processing, and reduces viral fitness. 20, 24,37 In this study, we examined the effect of four antiviral inhibitors, two clinical drugs and two investigational inhibitors, on the single mutant PR L76V . These inhibitors exhibit different chemical structures and binding interactions with the PR. The tested inhibitors have K i values of ∼2−8 nM for PR L76V , which are 2−3 orders of magnitude worse compared to values reported for the wild-type enzyme. 7,12,16 None of these inhibitors were more effective than 1 for the mutant PR L76V . Inhibition of PR L76V by 1 has been reported as 0.79 nM 24 or 1.5-fold worse 25 than for wild-type enzymes in different assays with two distinct substrates. GRL-02031, a different antiviral inhibitor based on the 1 scaffold, also gave an inhibition constant of 0.8 nM for PR L76V , consistent with decreased interactions of the P1′ pyrrolidinone group with PR atoms. 26 In contrast to other tested inhibitors, 6 exhibited similar inhibition of the PR L76V mutant compared to wild-type enzymes, consistent with retaining antiviral effectiveness. 19,20,24 In the current study, 5 was the better of the two investigational inhibitors with K i of 1.9 nM for PR L76V , possibly due to the two halide interactions with the carbonyl oxygen of Gly48 in the flap. Compounds 3 and 4 showed the worst inhibition, which suggests neither the larger tris-THF group at P2 in 4 nor the direct interactions of 3 with the flaps are beneficial for binding to this mutant.
The structures of PR L76V −inhibitor complexes are very similar to the corresponding wild-type enzyme structures. Only inhibitor 5, which was the best inhibitor of the four for PR L76V , showed distinct changes in the interactions of the aniline amine with alternate conformations of Asp30′. Elongations of water-mediated bonds by up to 0.3 and 0.4 Å, respectively, were observed in complexes 3 and 4. The active-site interactions with 2 were essentially identical in both the wild-type and the mutant. Fewer van der Waals contacts occurred with the mutant compared to wild-type enzymes for inhibitors 3, 4, and 5, whereas inhibitors 1, 2, and 6 showed the opposite effect. Therefore, the mechanism of resistance does not seem to rely solely on the loss of active-site interactions with inhibitors. Mutation L76V is not unusual in this respect; mutations L90M and N88D/S also have no direct interactions with inhibitors, yet are strongly associated with resistance to one or more clinical inhibitors. 2, 38 The rarity of L76V as a single mutation in clinical samples, at 0.4%, 20 may be due to its debilitating effects on precursor processing. Furthermore, the slower turnover of substrate and poor inhibition may arise from alterations in the dynamics of flap opening and closing rather than altered interactions with inhibitors. The mutation is likely to confer resistance by a mechanism that is independent of the inhibitor interactions in the active site. Instead, the loss of interactions of Val76 with residues at the base of the flap could increase flap mobility. If the virus accumulates additional mutations, as observed in 3.2% of clinical samples, 20 these mutations might compensate for the loss of stability because of L76V, while retaining resistance to a specific drug.
Interestingly, five of the seven residues adjacent to Leu76 in the PR structure are sites of major resistance mutations, D30N, V32I, I47V, Q58E, and T74P 2 . Of these, only the resistance mutation Q58E is strongly associated with L76V in resistant mutants. 2,39 Experimental studies corroborate findings from statistical analyses. Mutation L76V, which is associated with resistance to 1, is selected during viral passage with increasing concentrations of 1. 40 In contrast, L76V is associated with increased susceptibility to 6 and atazanavir, and experiments suggest that L76V re-sensitizes multi-drug resistant viruses to therapy with those two inhibitors. 19 Flap mutation M46I is strongly associated with L76V as shown by a large proportion of coprevalence in L76V-containing sequences. 20,39,41 Impaired autoprocessing of precursor PR-containing L76V is partly rescued by addition of a second mutation of M46I 24 and, although L76V reduces viral replication, viral fitness is partly rescued by combination with this mutation. 37 The effects of combining L76V with other mutations on the structure and dynamics of the PR dimer have not yet been explored.
An inhibitor capable of forming strong interactions with residues 45−47 of the flaps might assist in retaining a closed conformation dimer, and be effective against resistant mutants with defects such as those observed for L76V. Therefore, this hydrophobic region between the flaps and the outer edge of the active site in each monomer is a potential target site that should be considered during the design of next-generation inhibitors for resistant viruses.
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Article ■ EXPERIMENTAL SECTION Enzymes and Inhibitors. PR WT and PR L76V proteins contain optimizing mutations Q7K/L33I/L63I to reduce autoproteolysis and C67A/C95A to prevent thiol bond formation. 42 Recombinant PR was expressed in Escherichia coli BL21 DE(3) and purified by size-exclusion chromatography followed by reverse phase chromatography as described previously. 43 PR was refolded via buffer-exchange dialysis in 25 mM formic acid and 1 mM dithiothreitol. PR was activated via buffer-exchange dialysis in 50 mM sodium acetate, pH 5.0. PR was concentrated to 3.5−5.0 mg/mL for crystallization or further diluted for inhibition assays as needed. Inhibitors 2 and 3 with HPLC purity of 99.3 and 100%, respectively, were obtained from the AIDS Reagent Program, Division of AIDS, NIAID, NIH. Inhibitors 4 and 5 (>95% purity by HPLC) were provided by Dr. Arun Ghosh at Purdue University.
Enzyme Inhibition Assays. A continuous kinetic assay employing a Foster resonance energy transfer substrate analog of the HIV-1 p2/NC cleavage site (Abz−Thr−Ile−Nle−pnitro-Phe−Gln−Arg−NH 2 where Abz is anthranilic acid, Nle is norleucine, and p-nitro-Phe is p-nitrophenylalanine) was performed as previously described. 26 Microtiter plates (96well) were loaded with 10 μL PR stock (final well concentration 12−26 nM determined via active-site titration with tight binding inhibitor), 98 μL reaction buffer (0.1 M 2morpholinoethanesulfonate (MES), pH 5.6, 0.4 M NaCl, 1 mM ethylenediaminetetraacetic acid, and 5% glycerol), and 2 μL inhibitor in dimethyl sulfoxide (DMSO) (final well concentration of 0−100 nM). Samples were equilibrated at 25°C for 5 min and the reactions were measured at the same temperature. The reactions were initiated by addition of 90 μL substrate at a final well concentration of 81 μM. Reactions were measured under steady-state conditions using a PolarStar Optima (BMG Labtech) with emission wavelength at 340 nm and excitation wavelength at 420 nm. Fluorescence resulting from substrate hydrolysis at each inhibitor concentration [I] was measured and plotted against time. Initial velocities (V 0 ), corresponding to the slope per minute of each reaction, were determined using MARS software (BMG Labtech). A dose− response plot of V 0 versus [I] was constructed using SigmaPlot (Systat Software) by nonlinear regression curve fitting to determine IC 50 . Reactions were performed in triplicate, and K i values were calculated from IC 50 using the tight-binding inhibitor equation K i = (IC 50 − 0.5[E])/(1 + [S]/K m ). 44 The K m for PR L76V at 37 μM was determined previously under similar conditions. 24 Protein Crystallization. Each inhibitor suspended in DMSO was complexed with PR on ice at a molar ratio of at least 5:1 and crystallized using vapor diffusion hanging drop method. Each drop contained equal volumes of protein and reservoir solution. Crystals grew in 0.8−1.5 M sodium chloride as precipitant. Crystals for most complexes grew in 0.1 M sodium acetate, pH 4.0−5.4, as buffer. Buffers used for crystallization for the following were exceptions: 0.1 M MES, pH 5.6, for PR WT in complex with 2, and 0.1 M citrate phosphate, pH 6.0, for PR L76V in complex with 2. Moreover, 5% DMSO was used for crystallization conditions of PR L76V complexes with 2 and 3. Crystals were soaked in reservoir solution with 30% glycerol (v/v) as cryoprotectant and then flash-frozen in liquid nitrogen.
X-ray Diffraction, Processing, and Refinement. X-ray diffraction data were collected remotely using Southeastern Regional Collaborative Access Team ID-22 and BM-22 beamlines of the Advanced Photon Source in Argonne National Laboratory (Argonne, IL). Diffraction data were indexed, integrated, and scaled using HKL-2000. 45 Molecular replacement was performed using CCP4 Phaser 46,47 with PR WT in complex with amprenavir (PDB 3NU3) as the initial model. Structures were refined iteratively with Coot 48 and SHELX. 49 The inhibitor and any side chains with incomplete 2F o − F c density were removed during the initial rounds of refinement to avoid bias, and atoms were added according to density in F o − F c omit maps. Alternate conformations were modeled if visible in the electron density maps for the inhibitor and protein residues. Anisotropic B factors were included in the last stages of refinement for all structures, except for the lowest resolution structure of PR L76V in complex with 4. Structures were analyzed using Coot, CCP4 Superpose, CCP4 Baverage, CCP4 Contact, and CCP4 Sfcheck. Illustrations were created using PyMol (Schrodinger, LLC.).
Crystallographic coordinate and structure factors have been deposited in the Protein Data Bank with accession codes 6DJ1 for PR WT −2, 6DJ2 for PR L76V −2, 6DIF for PR WT −3, 6DIL for PR L76V −3, 6DJ5 for PR L76V −4, and 6DJ7 for PR L76V −5. The authors will release the atomic coordinates and experimental data upon article publication. . We thank the staff at the Southeast Regional-Collaborative Access Team (SER-CAT) at the Advanced Photon Source, Argonne National Laboratory, for assistance during X-ray data collection. Supporting institutions may be found at http:// www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. W-31-109-Eng-38.
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Article | v3-fos-license |
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} | pes2o/s2orc | Characterisation of Crevice and Pit Solution Chemistries Using Capillary Electrophoresis with Contactless Conductivity Detector
The ability to predict structural degradation in-service is often limited by a lack of understanding of the evolving chemical species occurring within a range of different microenvironments associated with corrosion sites. Capillary electrophoresis (CE) is capable of analysing nanolitre solution volumes with widely disparate concentrations of ionic species, thereby producing accurate and reliable results for the analysis of the chemical compositions found within microenvironment corrosion solutions, such as those found at crevice and pit corrosion sites. In this study, CE with contactless conductivity detection (CCD) has been used to characterize pitting and crevice corrosion solution chemistries for the first time. By using the capillary electrophoresis with contactless conductivity detection (CE-CCD) system, direct and simultaneous detection of seven metal cations (Cu2+, Ni2+, Fe3+, Fe2+, Cr3+, Mn2+, and Al3+) and chloride anions was achieved with a buffer solution of 10 mM 2,6-pyridinedicarboxylic acid and 0.5 mM cetyltrimethylammonium hydroxide at pH 4 using a pre-column complexation method. The detection limits obtained for the metal cations and chloride anions were 100 and 10 ppb, respectively. The CE-CCD methodology has been demonstrated to be a versatile technique capable of speciation and quantifying the ionic species generated within artificial pit (a pencil electrode) and crevice corrosion geometries for carbon steels and nickel-aluminium bronze, thus allowing the evolution of the solution chemistry to be assessed with time and the identification of the key corrosion analyte targets for structural health monitoring.
Introduction
The failure of a structure, such as an airframe or marine vessel, can cause considerable economic loss, and even loss of life. Using an array of sensors to continuously monitor such structures, structural health monitoring can provide an early indication of problems such as damage to the structure from fatigue, corrosion or impact, and this information can be used to undertake corrective action before the damage develops to a stage where a catastrophic failure occurs [1,2]. However, there are a wide variety of corrosion types that routinely occur in aircraft and marine structures. For example, corrosion can occur in situations where the metal components are generally expected to be corrosion-resistant and can result in the destruction of integrity of metal-metal and metal-nonmetal junctions (crevice corrosion) such as may exist at bolted and lock-tight joints, flanges, and poor quality welds. For ferrous metals and aluminium alloys, the chemical microenvironment in these affected areas becomes acidic and eventually the crevice becomes sufficiently aggressive to depassivate the metallic surface [3]. Likewise, crevice corrosion can also occur for copper-based systems, for example nickel-aluminium bronze (NAB) where there is a selective phase attack within the occluded zone plus an additional accelerated attack at the crevice edge [4].
Although non-destructive evaluation for corrosion detection is becoming available, corrosion is often found using visual inspection methods. This means that corrosion of internal or inaccessible structures could go undetected [5]. To date, the only practical solution to check for corrosion damage has been to strip and inspect. Frequently, no corrosion is found, but the time and cost of performing the inspection has already been lost and damage may even result if the refurbishment is to a lower standard than the original construction. The objective of corrosion surveillance is to predict attack before significant damage is sustained, the condition of components is thus monitored whilst in service and not just intermittently at routine inspections. This minimises the inspection requirements yet ensures that maintenance is carried out as it becomes necessary. Recognition of the advantage gained from predictive management of corrosion is becoming more widespread, following such developments in the nuclear, chemical and offshore oil industry [6,7], with cost savings achieved equivalent to approximately half the cost of using the conventional maintenance approach [8].
However, the ability to predict structural degradation in-service is limited in many cases by a lack of understanding and detailed knowledge of the evolving chemical processes occurring within a range of different microenvironments containing micro-solution volumes. Generally, there is a lack of data with regard to the levels of metal cations within the microenvironments that form in situations such as crevice corrosion due to the small quantities of materials of interests and the inherent analytical difficulty of measuring small amounts of one species in the presence of large amounts of another. CE has been proven to be a powerful and robust analytical technique for corrosion solution analysis with a low level (e.g., parts per million, ppm) detection of some metallic cations and inorganic anions in the presence of high levels (in the order of 1 M) of chloride ions [9][10][11][12][13][14].
Capillary electrophoresis (CE) separates different ionic species within a buffer solution filled capillary under the influence of an external electric field based on differences in their electrophoretic mobilities. Only requiring very small sample volumes of less than 10 µL to detect both anions and cations, it offers high resolution, fast analysis, and simple sample preparation methodology. The most widely used mode of detection in CE is ultraviolet (UV) absorbance detection, which works well for organic species with chromophoric moieties. Unfortunately, due to their lack of significant UV absorbance, most inorganic anions and metal cations can only be detected by indirect UV detection, a method which is relatively insensitive and has a limited linear range. For example, Kelly et al. [11][12][13][14] have quantified local chemistries within the corrosion-induced blisters in organic coatings on aluminium alloys using CE with UV detection, but four different buffer electrolytes were used for the indirect UV detection of inorganic anions and cations existing within the corrosion microenvironments. However, these small inorganic ions can be directly determined with simpler buffer compositions by measuring conductivity changes in the capillary with contactless conductivity detection (CCD). Capillary electrophoresis with contactless conductivity detection (CE-CCD) has been proven to allow direct and simultaneous detection of inorganic anions and cations at extremely low concentration levels down to parts per billion (ppb) and even parts per trillion ranges [15][16][17]. In addition to conventional CE, CCD has also proven to be preferred and well suited for micro-CE, due to its high sensitivity, low cost, simple fabrication and easy implementation [17][18][19].
In this paper, a capillary electrophoresis analysis approach using contactless conductivity detection has been established for the first time to allow direct and simultaneous detection of chloride anions and the most common metal alloy cations, including cupric ions, nickel ions, ferric ions, ferrous ions, chromium ions, manganese ions and aluminium ions. Using this new CE methodology, the solution chemistries sampled from either a pencil electrode (an artificial pit electrode, often called one-dimensional pit geometry) or crevice corrosion assembly for carbon steel and nickel-aluminium bronze have been determined.
Chemicals and Test Solutions
All reagents (analytical grade) were supplied by Sigma-Aldrich (Poole, UK) and dissolved in 18 MΩ cm deionised water as received. All the standard solutions of 2000 ppm copper ions, nickel ions, ferric ions, ferrous ions, chromium ions, manganese ions and aluminium ions were prepared from their chloride salts. The CE buffer solution was prepared using a solution having appropriate amounts of 2,6-pyridinedicarboxylic acid (PDCA) with additional appropriate amounts of cationic surfactant cetyltrimethylammonium hydroxide (CTAH) or tetramethylammonium hydroxide (TMAH), and the pH was adjusted with 2 M sodium hydroxide.
Specimen Materials
A carbon steel, EN3A grade (equivalent to 090M20-BS970 Part 1, AISI 1020 or UNS G10200), was used for accelerated corrosion tests (chemical composition: Fe balance, C 0.17-0.23, Mn 0.60-0.90, S 0.05 (max.) wt %). Cast nickel-aluminium bronze (UNS C95800, British Naval spec. NES 747 Part 2) was also used for accelerated corrosion tests (chemical composition: Cu balance, Al 9.32, Ni 5.38, Fe 5.00, Mn 1.10, Si 0.05 wt %). The specimens for accelerated corrosion tests were machined as pencil electrodes and embedded in epoxy resin, as shown in Figure 1. Prior to testing, the pencil electrodes were wet abraded sequentially with 600 and 1200 grit SiC papers and polished using 6 µm and 1 µm diamond pastes, then degreased in acetone and air-dried. In addition, a high strength low alloy Q1N carbon steel (HY80) was used for the Cortest crevice corrosion tests (chemical composition: Fe balance, Ni 2.25-3.25, Cr 1.00-1.80, Mo 0.3-0.6, Si 015-0.35, C 0.18 (max.) wt %). The Q1N specimens for Cortest experiments were 50 mm × 50 mm × 5 mm with a central 12 mm diameter hole. Prior to testing, the Cortest specimens were wet abraded with 600 SiC grit paper on both sides to provide a uniform surface finish, then degreased in acetone and air-dried.
Instrumentation
The CE experiments were performed using a Prince Technologies PrinCE-560 capillary electrophoresis system (Prince Technologies B.V., Emmen, The Netherlands) with a TraceDec contactless conductivity detector. All CE measurements and data processing were carried out with Data Acquisition and Analysis Software (DAx 8 Analysis, Prince Technologies B.V., Emmen, The Netherlands). A bare fused silica capillary was used for all the CE measurements with an inner diameter of 50 μm, total length of 90 cm and effective length of 60 cm. The CE separation was carried out at a temperature of 20 °C.
Electrochemical tests were performed within a Faraday cage using a Gamry Reference 600 potentiostat with Gamry Framework and Software Package 5.63 (Gamry Instruments, Warminster, PA, USA). A standard reference electrode of Ag/AgCl in 3.5 M KCl was used for all the electrochemical measurements, and a graphite rod was used as the counter electrode. pH values were measured using a Hanna pH meter HI211 (Hanna Instruments, RI, USA) with a MI-4156 combination microelectrode, with an outer diameter of 1.3 mm (Microelectrodes, Inc., Bedford, NH, USA). All electrochemical testing was carried out in 3.5% NaCl solution at room temperature.
Pencil Electrode Assembly (Artificial One-Dimensional Pit)
Accelerated corrosion processes were investigated for the EN3A carbon steel and NAB to identify those ionic species that are indicative of corrosion occurring. All the accelerated corrosion tests were performed with pencil electrode assemblies as shown in Figure 1. When encased in epoxy resin, immersed in a 3.5% NaCl solution and appropriately biased using a potentiostat, the small surface area of the exposed pencil electrode is progressively corroded leaving a cavity within the epoxy resin containing a small volume of the corrosion products, which were collected with a micropipette at predefined time intervals and diluted to 4 to 200 folds with the running buffer solution for pre-column complexation treatment and CE analysis.
Cortest Crevice Corrosion Assemblies
As described in [9], crevice formers were created using the Cortest assembly (large single crevice area of 4.6 cm 2 ) manufactured from polymethylmethacrylate (PMMA) and fastened using commercially pure titanium nuts, bolts and washers, as shown in Figure 2. Crevice assembly tightness was controlled using a torque wrench set to 0.6 N.m. To prevent electrical connection between the Q1N specimen and the titanium parts, ploytetrafluoroethylene (PTFE) tape was used to electrically insulate the bolt. In total eight crevice corrosion assemblies were immersed into a tank containing 4.6 L of a continuously aerated 3.5 wt % NaCl test solution (bulk solution). The bulk solution dissolved oxygen content was measured with a Hanna Instruments HI9143 probe and found to be approximately 7.0 ppm. At set time intervals, a single crevice assembly was retrieved from the bulk solution and placed in a freezer for about half an hour. This step was taken to ensure that crevice solution samples remained on the specimen surface during the crevice former disassembly. The melted solution was collected from the crevice surface in the region immediately under the crevice former using a micropipette. Prior to CE analysis, the collected samples were mixed with the CE running buffer solution for pre-column complexation and 4 to 200-folds dilution.
Capillary Electrophoresis Method for Corrosion Solution Chemistries Analysis
The most common alloying components are copper, nickel, iron, chromium, manganese and aluminium. Most of the widely used marine and aerospace alloys normally contain four to six of these metal alloying elements. In order to identify the possible metal cations produced during corrosion processes, it is important to conduct an analysis such that it is capable of detecting all of the corrosion products in a single CE run, especially when considering the very small corrosion solution volumes that can occur. To date, the separation of all aforementioned seven metal cations as well as chloride ions in a single analysis run has previously not been reported for CE or ion chromatography.
Anions and cations migrate in opposite directions in CE, and it is difficult to simultaneously determine metal cations and chloride anions in a single CE run. Thus, all the metal ions need to be transformed at first into negatively charged complexes to achieve simultaneous separation of all the target metal cations with negatively charged chloride ion in a single CE run. In addition, because these metal ions possess similar mobilities, it is also necessary to use a chelating agent to enhance separation efficiency and selectivity of these metal ions in order to realize the simultaneous and direct determination of these target metal ions in a single CE run. It has been reported that PDCA can form highly stable, negatively charged complexes with some heavy metal ions [9,[20][21][22][23]. PDCA was therefore used as a complexation agent and running buffer additive to establish CE methodology in our experiments. A buffer solution pH range of 3.5 to 4.0 was also reported as having achieved best separation selectivity and sensitivity for metal ions [9,[20][21][22][23]. In this study, a number of parameters, including PDCA concentrations in the buffer solution, surfactant types and their concentrations in the buffer solution, pH, sample pre-treatment methods, separation temperature and applied potentials, were investigated to optimise the separation conditions. The results from our investigations into the effect of buffer solution composition on metal ion separation is summarised in Table 1, along with the effects of employing on-column or pre-column complexation sample pre-treatment methodologies.
For these optimisation experiments, all the measurements were performed with a bare fused silica capillary at a temperature of 20 °C with a running time of 1 h by using the standard metal ion solutions prepared from chloride salts of metal ions with a concentration of 10 ppm. For all the measurements, the peak for chloride ion was always detected and completely separated from those for the metal ions. Table 1 shows that regardless of the buffer solution composition, better separation was achieved with pre-column complexation than on-column complexation. The results also show that when no surfactant was included in the buffer solution, the baseline signal at the detector exhibited significant levels of noise, making it difficult to discriminate peaks in the conductivity signal attributable to the different ions (i.e., selectivity and sensitivity were poor). It was also observed that when CTAH was employed as the surfactant, a lower concentration of PDCA produced better separation efficiency irrespective of the complexation method used. Further experiments with the buffer solution containing 5 mM PDCA and 0.25 mM CTAH confirmed that the optimum buffer solution for reliable separation of all of the ions of interest is 10 mM PDCA and 0.5 mM CTAH at pH 4.0. The electric field strength within the capillary is another important parameter in CE optimisation since it directly governs electrophoretic migration. The electric field strength is changed when either the applied voltage is varied or the capillary length is altered. In CE, increasing the voltage could improve separation resolution and shorten the analysis time, but the production of heat possibly limits the resolution improvement. Therefore, separation of all the target ions was investigated under different field strengths with applied voltages of −10 kV, −15 kV and −30 kV. The obtained results indicate that reasonable separation resolution and analysis time was achieved under an applied voltage of −15 kV with the used fused silica capillary under the optimized buffer solution compositions.
As a result, the best separation for all the seven metal cations and chloride anion can be achieved with the buffer solution of 10 mM PDCA and 0.5 mM CTAH at pH 4 using a pre-column complexation method and with the analysis performed at a temperature of 20 °C with an applied voltage of −15 kV. Electropherograms demonstrating the ion separation efficiency under these conditions are displayed in Figure 3. Under the optimized conditions, chloride ion appeared with a very sharp peak before the seven metal ions due to its high mobility, see Figure 3a. At the concentration of 10 ppm, all seven metal ions were separated into individual peaks. Moreover, as demonstrated in Figure 3b, the peaks for Cu 2+ and Ni 2+ ions are positive while those for Fe 2+ , Fe 3+ , Mn 2+ , Cr 3+ , and Al 3+ ions are negative. This indicated that compared to the conductivity of the running buffer solution, the conductivities within the zones formed by the complexation of Cu 2+ ions with PDCA and the complexation of Ni 2+ ions with PDCA during the CE separation are greater, while those for PDCA and the other five metal ions are lower.
Calibration Curves for CE-CCD Analysis of Chloride and Metallic Ions
For the CE analysis, sample component identification is achieved by comparing the migration times with those of standard solutions obtained under the same experimental conditions. In order to quantify the composition of the corrosion sample components, calibration curves for each metal ion were first established by plotting the sample concentration versus peak area. Under the optimised conditions, CE measurements were carried out for chloride, ferrous, ferric, manganese, chromium, aluminium, cupric and nickel ions in the concentration range 10 ppb to 500 ppm. For all ions investigated, linear relationships were observed with all the coefficients of determination R 2 above 0.99 between the logarithm of the peak area and the logarithm of the concentration over the range 2.5 ppm to 250 ppm. For chloride ions, the linear relationship extended down to the 50 ppb level and the limit of detection for chloride ion was determined as 10 ppb, as shown in Figure 4.
Solution Chemistries Analysis of Accelerated Pitting Corrosion of EN3A Carbon Steel
Potentiodynamic polarisations were performed for the carbon steel pencil electrode assembly in a 3.5% NaCl solution to determine an appropriate potential for the accelerated corrosion study. Figure 5 shows the EN3A carbon steel exhibited typical polarisation behaviour for an actively corroding metal, with the corrosion potential (E corr ) at −0.618 V. An applied potential of −0.400 V, where carbon steel is within an active corrosion region, was selected for the accelerated corrosion tests in order to rapidly generate corrosion products within a microenvironment for CE identification.
The pH change was also monitored at the corrosion site of the pencil working electrode and around the counter electrode at pre-determined time intervals. As shown in Figure 5, the pH of the solution around the counter electrode initially increased and reached a value of pH 11.5 after 5 h. By comparison, the solution pH initially decreased at the corrosion site and then stabilised at around pH 5.5 after 5 h. The predominant cathodic process in an aerated 3.5% NaCl solution is oxygen reduction reaction (ORR): In these circumstances, the products of the ORR are hydroxide ions, thus the solution around the counter electrode (cathode) becomes alkaline during the accelerated corrosion test.
The dissolution of the EN3A pencil electrode resulted in the formation of a pit cavity within the epoxy resin. Ferrous ions are initially considered to form as a primary corrosion product: Current density / A cm As corrosion continues this will lead to an increase in the concentration of dissolved metal ions within the pit cavity, and oxides or hydroxides are precipitated [24]. These hydrolysis and precipitation reactions cause acidification within the pit cavity, in accordance with the following reactions.
In the chloride background media, the increase in the concentrations of Fe 2+ and H + cations in the cavity will be counterbalanced by an increase in the concentration of Cl − ions, driven by diffusion from the bulk solution towards the cavity interior to maintain electroneutrality. This is confirmed by the chemical analysis results of corrosion solutions collected from the pit cavity during the accelerated corrosion process, see Figure 6. From the CE analysis, the concentration of chloride and ferrous ions reach about 42,000 ppm (1.2 M in terms of NaCl) and 21,000 ppm (0.4 M in terms of FeCl 2 ) after 24 h, respectively.
Solution Chemistries Analysis of Cortest Crevice Corrosion of Q1N Carbon Steel
Q1N is a low alloy carbon steel and it exhibited a similar polarisation behaviour as that observed for the EN3A carbon steel. However, due to nickel and chromium contents, the corrosion potential at −0.570 V was slightly more positive than −0.618 V of EN3A steel in 3.5% NaCl solution. The CE analysis revealed the presence of ferric ions and nickel ions, as well as chloride within the Q1N crevice solution. The concentration profiles of the chloride and metal ions with time were established for the Q1N crevice corrosion for a period of about 8 weeks, see Figure 7. As expected, in order to maintain charge neutrality, the chloride ions were drawn into the crevice site, and the concentration of chloride ions in the crevice increased significantly with corrosion time. The chloride ion concentration in the crevice solution reached a maximum of 106,000 ppm (ca. 3.0 M in terms of NaCl) after 2 weeks, before decreasing to 40,000 ppm after 8 weeks. The Fe 3+ and Ni 2+ ions exhibited a similar concentration profile with time, rapidly increasing during the initial corrosion phase and then gradually decreasing. The observed trends in concentration profiles with time indicated that crevice corrosion of Q1N activated immediately once immersed in NaCl solution, followed by a resilient period, possibly due to precipitation of metal hydroxides and oxides on the metallic surface (thus hindering corrosion progress). The ferric ions detected within the crevice corrosion solution reached a concentration maximum of 6,700 ppm (ca. 0.12 M in term of FeCl 3 ) at day 5, while the highest level of nickel ions (1,500 ppm, 0.026 M in terms of NiCl 2 ) was detected at the initial stage of crevice corrosion, just after 2 day in the 3.5% NaCl test solution.
Solution Chemistries Analysis of Accelerated Pitting Corrosion of NAB
NAB is a complex copper-based alloy and it is utilised in numerous marine applications due to its good corrosion performance. As previously reported [25], the microstructure of NAB is predominantly constituted of copper-rich α-phase and retained martensitic β-phase, surrounded by a series of intermetallic κ-phases all different in shape and composition. In comparison with the two carbon steels, NAB exhibits a more localised corrosion mechanism. NAB corrosion usually initiates at selective sites, associated with the α-phase where dissolution of copper components occurs to form a cuprous dichloride anion complex (CuCl 2 − ) in the presence of concentrated chloride, or they precipitate on the metal surface in the form of cuprous chloride (CuCl, a white solid). In neutral solutions, the presence of high concentrations of CuCl 2 − at the metal surface may result in a hydrolysis reaction and the formation of Cu 2 O according to: Alternatively, CuCl precipitates in a chloride media may lead to further Cu 2 O growth: 2CuCl + H 2 O → Cu 2 O + 2H + + 2Cl − (8) As shown in Figure 8, NAB exhibits active-passive corrosion behaviour in the 3.5% NaCl test solution. In the active region between the corrosion potential (E corr = −0.208 V) and the pseudo-passivation potential (E pp = −0.010 V), the predominant process is the dissolution of copper to (7) and (8)] [9,25]. Due to the porous microstructure and the electrical conductivity of the Cu 2 O passive layer, the current density is maintained at a relatively high and steady level in the passive region, where the corrosion rates are about two orders of magnitude higher than the corrosion rate at the corrosion potential. Once breakdown of the oxide protective film starts at E pit = +0.630 V, the current increased significantly with applied potential. Therefore, for these accelerated corrosion tests, an applied potential of +0.730 V was chosen, which is 0.1 V greater than the passive film breakdown potential, so that corrosion products could be quickly generated.
During accelerated corrosion testing, a white solid was observed to cover the pit cavity with evidence of some red/brown coloured Cu 2 O deposits. This is in agreement with previous reports on copper pitting mechanisms [25][26][27], where the pit interior is covered with solid CuCl thus hindering the formation of a protective Cu 2 O layer at the metal interface and also the low solubility of CuCl maintains a low activity of copper ions, thereby facilitating anodic dissolution. The formation of CuCl is often attributed to local conditions that restrict diffusion, for example a Cu 2 O layer. Lucey reported that pitting required the formation of a Cu 2 O layer that is sufficiently porous to allow the restricted diffusion of CuCl 2 − through it and showed that the Cu 2 O behaves as an electrically conductive layer, with the upper surface acting as a cathode and the lower surface as an anode [27]. Thus, the CuCl trapped under the Cu 2 O layer may be anodically oxidised to cupric on the lower surface of the Cu 2 O layer and the resultant cupric ions can further attack the metallic copper alloy to reform cuprous ions. However, due to the restricted diffusion through the porous Cu 2 O layer and the block of accumulated CuCl solid in the cavity, the corrosion rate will decrease to a very low level in the long run as observed from current response with corrosion times, as shown in Figure 8. The solution pH around counter the electrode became alkaline due to the cathodic oxygen reduction reaction, while the solution at the accelerated corrosion cavity became acidic due to hydrolysis of corrosion products, see Figure 9. It is noteworthy that the pH measured at the NAB corrosion site decreased to pH 2 during the initial stage of artificial pitting corrosion, and then rose slightly to pH 5. The lower pH levels in the NAB pitting cavity in comparison with the carbon steels may have resulted from the hydrolysis of other metal cations, such as the aluminium species.
Conclusions
A capillary electrophoresis methodology has been established with contactless conductivity detection for direct simultaneous determination of chloride ions and seven metal ions of the most commonly used alloy elements in a single run. The direct and simultaneous detection of all seven metal ions and chloride ions was achieved with a buffer solution of 10 mM PDCA and 0.5 mM CTAH at pH 4 using a pre-column complexation method and with the analysis performed at a temperature of 20 °C with an applied voltage of −15 kV. The detection limits obtained in this study for the metal cations and chloride ions were 100 ppb and 10 ppb, respectively. The established methodology has been demonstrated to be a versatile technique capable of speciation and quantifying the corrosive species generated within microenvironment chemistries found in artificial pit (a pencil electrode) and crevice corrosion geometries for carbon steels and nickel-aluminium bronze. Ultimately, knowledge of the metal ions, as well as chloride ion, concentration vs. time profiles during the corrosion processes will assist in the identification of the key corrosion analyte targets for critical aerospace, marine and microenvironments for structural health monitoring. In light of our results demonstrating contactless conductivity as a preferred detection methodology for capillary electrophoresis systems, we envisage a time when micro-CE systems with CCD could be feasibly used in autonomous, in-situ structural health monitoring. | v3-fos-license |
2017-11-10T18:26:05.545Z | 2003-01-01T00:00:00.000 | 36225466 | {
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} | pes2o/s2orc | EXCHANGEABLE ALUMINUM EVALUATION IN ACID SOILS
One of the main factors limiting agricultural production in tropical climate regions is mainly related to the presence of exchangeable aluminum (Al) in highly weathered acid soils. Four methods of Al determination extracted with neutral 1 mol L KCl solution were evaluated: three colorimetric methods (aluminon plus ascorbic acid, and eriochrome cyanine R by FIA) and the usual titrimetric method with backtitration. Surface samples from 20 soils of different Brazilian regions, with active acidity (0.01 mol L CaCl2 pH) ranging from very high to medium (3.82 to 5.52), were used. The variance analysis revealed significant interaction among Al determination methods and soil. Mean methods comparisons within each soil (Tukey, P < 0.05) indicated that, for most of the soils, the methods differed among each other, although there were high correlations between the obtained values. Al values determined for soil samples by titration varied between 0.15 and 14.71 mmolc dm . The colorimetric methods showed higher values than the titration method, mainly for those with aluminon (up to 18.75 mmolc dm ). The Al contents of colorimetric methods correlated quadraticaly with the titration values, for the soil samples with Al > 10 mmolc dm . Among colorimetric methods, in operational terms, the eriochrome with FIA method presented analytical performance up to 50 samples per hour, easiness and sensibility for routine Al analysis in soil samples. However, due to the specificity, the titration/back-titration method should be used, despite the moroseness, when the Al ions are the objective.
INTRODUCTION
In acid mineral soils of tropical climate regions, the high aluminum (Al) content, associated to high acidity and low fertility, is one of the main constraints for agricultural production (McLean, 1965;Pavan, 1983;Coscione et al., 1998), since toxic concentrations of Al are often concerned as having detrimental effects on plants.
At soil pH 4 or bellow, the predominant aluminum form is Al 3+ .As soil pH increases mononuclear hydrolysis species such as Al(OH) 2+ and Al(OH) 2 + are formed.If pH increases high enough, these species are precipitated as Al(OH) 3 0 , and with further increases in pH, the amphoteric Al(OH) 4 -species appears (Barnhisel & Bertsch, 1982;Thomas & Hargrove, 1984).There are contradicting results on the relative phytotoxicity of Scientia Agricola, v.60, n.3, p.543-548, Jul./Sept.2003 mononuclear Al species, except for the nonphytotoxicity of Al(OH) 4 - (Marschner, 1997).The extraction of Al 3+ (usually called as exchangeable Al) from soil samples using neutral solutions of nonbuffered salts has been employed since the 1960's (Brauner, 1966).The 1 mol L -1 KCl solution has been considered the most appropriate extractant (McLean, 1965;Raij et al., 1987;Hiradate et al., 1998), besides the fact of this solution being less susceptible to ion interferences (Coscione et al., 1998).The Al 3+ determined in these extracts is subject to overestimation due to the dissolution of non-exchangeable aluminum species (hydroxi-Al).The dissolution of hydroxi-Al species is affected by pH, concentration, and chemical feature of the extractant, as well as the extraction time (Kissel et al., 1971;Oates & Kamprath, 1983).Although the extraction with KCl removes both exchangeable and some nonexchangeable Al (Oates & Kamprath, 1983), the contribution of non-exchangeable Al may be considered insignificant in Brazilian acid soils (Pavan, 1983).
The Al 3+ is always displaced from exchange sites by non-buffered salt solutions.Since the soil pH raises and monomers are formed, there gradually occur increments on the OH/Al relation and of the polymerization of these monomers.The formed polymers, of variable size and charges, neutralize negative charges but are not displaced (Thomas & Hargrove, 1984).Despite of this, expressive amounts of low stability hydroxi-Al forms are supposed to occur in KCl extracts.The hydrogen ions (H + ), obtained by titration in KCl extracts of an acid mineral soil containing 1:1 clay minerals and Al, Fe oxides, are indeed a result of low stability hydroxi-Al hydrolysis.According to Kissel et al. (1971), the lower the hydroxi-Al form contents, the higher is the proportion of their hydrolysable forms, due to lower polymerization (stability) of these Al forms, which produce H + in KCl extract.Consequently, as the back-titration step is essential when Al 3+ determination in KCl extract is the main target, the titrimetric method should be used.
The titrimetric method may present inferior sensitivity in relation to the colorimetric methods for the determination of Al (Reis, 1978;Logan et al., 1985), but it is less subject to interferences from ions present in the extract (McLean, 1965;Coscione et al., 1998).Both titrimetric and colorimetric methods present low determination rates in face of the intrinsic slowness of these methods.Therefore, the determination of Al by colorimetry through flow injection analysis (FIA) using eriochrome cyanine R has been suggested (Zagatto et al., 1981;Kronka, 1996).
The objective of this work is to compare titrimetric (standard) and colorimetric methods, with aluminon, aluminon plus ascorbic acid (conventional) and eriochrome cyanine R using FIA system, for the determination of Al 3+ in different extracts by 1 mol L -1 KCl of Brazilian soil samples, to improve routine analysis.
MATERIAL AND METHODS
The experiment was carried out using samples of the surface layer (0 -0.2 m depth) of 20 soils from different Brazilian regions (Table 1).
Air dried samples were homogenized, divided into three subsamples, ground in a porcelain crucible, passed through a 0.5 mm mesh sieve, and conditioned in plastic bags.For the chemical characterization of soil samples (Table 2), the methods described in Raij et al. (1987;2001) were used, except for the exchangeable sodium which was extracted with 0.05 mol L -1 HCl + 0.0125 mol L -1 H 2 SO 4 solution in 1:5 (v/v) soil/extractant ratio and determined by flame photometry.
For the evaluation of the Al 3+ in the extracts of 1 mol L -1 KCl solution, 1:10 (v/v) soil/solution ratio (McLean, 1965), the following procedures were used: a) Titrimetric method (standard method), according to the routine methodology adapted from McLean, (1965).Primarily, the exchangeable acidity (Al 3+ + H + tit ) is determined by titration of 25 mL KCl extract with 0.025 mol L - 1 NaOH, using 1 g L -1 phenolphthalein as an indicator (titration from colorless to pink).Then, the concentration of Al 3+ is obtained by back-titration of the same KCl extract, previously used, after the acidification with a drop of HCl and addition of 40 g L -1 NaF, with 0.025 mol L -1 HCl (titration from pink to colorless); b) Colorimetric methods with aluminon (ammoniacal salt of aurintricarboxilic acid) according to Wolf (1982), adapted for the KCl solution (e.g., 1 mL of KCl extract, 0.4 mL of sodium phosphate, 1 mL hydroxylamine hydrochloride, and 3 mL of aluminon solution).The intensity of developed color is read in a spectrophotometer at 555 nm, zeroing the equipment with the blank (1 mol L -1 KCl); c) Modified aluminon colorimetric methods by addition of ascorbic acid to verify the effect of adding 1 mL of 20 g L -1 ascorbic acid (prepared immediately before the Scientia Agricola, v.60, n.3, p.543-548, Jul./Sept.2003 analysis) on Al 3+ determination in the extract of KCl; and d) Colorimetric methods with eriochrome cyanine R by flow injection analysis (FIA) according to Zagatto et al. (1981), adapted for the KCl solution with modifications in the entrances of the FIA system and in eluent volume, following the Al determination by spectrophotometry of the formed colored complex at pH 6.4, and 546 nm (Figure 1).
Data were submitted to the analyses of descriptive statistics, of correlation and of regression.For the comparison of methods of Al 3+ determination the variance analyses and of test of means (Tukey, P < 0.05), were used after the data transformation (X+0.5).
RESULTS AND DISCUSSION
There were differences among quantities of Al 3+ evaluated by the different methods in the extracts of soil samples in neutral KCl solution (Table 3).The values of Figure 1 -Flow diagram of the system for determining exchangeable aluminum (Al 3+ ) in soil samples, extracted with neutral 1 mol L -1 KCl solution.A is the flow, in mL/min, of 1 mol L -1 KCl extractant containing Al 3+ extracted from the soil sample; C is the flow of carrier KCl solution; R 1 is the flow of 20 g L -1 ascorbic acid solution (prepared just before the analysis); R 2 is the flow of 0.2 g L -1 eriochrome cyanine R solution (prepared just before the analysis by dilution of 2 g L -1 stock solution, pH 2.8, with deionizated water); R 3 is the flow of 4 mol L -1 ammonium acetate buffer solution, pH 6.4; Bp, peristaltic pump; W, waste .Al 3+ , in mmol c dm -3 , in the acid soil samples with pH ranging from 3.82 to 5.52 (Table 2), varied from 0.15 to 14.71 (median of 4.22) when determined by titration; from 0 to 18.75 (median of 3.94) by aluminon colorimetric method; from 0 to 18.27 (median of 4.00) by aluminon colorimetry with addition of ascorbic acid; and from 0 to 15.24 (mean of 4.17 and median of 2.66) by eriochrome cyanine R colorimetry in FIA system.
Quadratic relationships were verified between the Al 3+ concentrations obtained by titration and those of the colorimetric methods (Figure 2), because in the soil samples with Al 3+ > 10 mmol c dm -3 the colorimetric methods gave higher values than by the titration method, mainly in those with aluminon (up to 18.75 mmol c dm -3 , e.g., soil samples LA-1 and PVA-3), owing to the strong acidity of these samples.
For the soil samples with Al 3+ concentration between 1 to 10 mmol c dm -3 , generally, the modified aluminon colorimetric method with the addition of ascorbic acid and the titrimetric method did not differ from each other.However, the aluminon colorimetric method provided the highest Al 3+ values, and the eriochrome cyanine R colorimetric methods by FIA gave the lowest values.The addition of ascorbic acid minimized the interference of Fe 3+ , in the KCl extracts, on the Al 3+ analysis by the aluminon color reagent.The Al 3+ analysis by the eriochrome cyanine R with FIA showed ) low efficiency due to the low concentration of the colorimetric reagent, as this method was adapted from that for analysis of total aluminum in vegetable material (Zagatto et al., 1981), for which the relative Al concentration in the extract is much higher than in KCl soil extract.
Table 2 -Chemical properties of soil samples of the 0 -0.2 m layer.
# Means followed by same letters within a soil sample are not significantly different according to the mean separation Tukey test (P < 0.05) (Variance analysis with X+0.5 data transformation).Brauner (1966) observed, unlike the results of the present work, that in samples with low Al 3+ concentration the difference between the values obtained by titrimetric and aluminon colorimetric was higher than for samples with high concentration and this difference was attributed to the interference of Ca and Fe in the aluminon method.This Ca derives from the calcium chloride solution used as extractant and the Fe from the tioglycolic acid employed in the reagent solution.To remove the interference of these two elements during the Al 3+ determination by the aluninon colorimetric method, Frink & Peech (1962) had proposed the exclusion of the calcium chloride as extractant and the substitution of tioglycolic acid, in the reagent solution, with hydroxylamine hydrochloride.Despite of this, the difference between results observed in the present study and those of Brauner (1966) is also due to the fact that the values of Al 3+ obtained by titration by the last author, are actually equal to the titratable acidity, since the concentration was obtained without back-titration, after addition of NaF.The aluminon colorimetric method described by Wolf (1982) and used in the present work was similar to that proposed by Frink & Peech (1962).
The Al +3 determination by the titrimetric method presented standard deviations (SD, mean of three replicates for each soil sample) between 0.06 to 0.28 mmol c dm -3 (except for soil sample LA-2, with a SD of 0.55 mmol c dm -3 ), and coefficients of variation (CV%, mean of three replicates for each soil sample) between 1.3 to 20.8% (except for sample LV-3, CV% = 43.3).For the colorimetric methods using the aluminon and aluminon + ascorbic acid reagent, the SD varied between 0.04 to 0.68 mmol c dm -3 , 0.07 to 0.23 mmol c dm -3 , and the CV% between 0.7 to 33.2, 0.8 to 7.4 (excluding samples LA-4 and LV-3, CV > 80% due to the inverse relation between Al concentration and CV%), respectively.The lower and narrow range values of SD and CV% for the method with aluminon + ascorbic acid is also associated to the fact that ion interferences, mainly the Fe 3+ ion, which is quite suppressed with ascorbic acid.Using the FIA system with the eriochrome cyanine R reagent, the values of SD and CV% varied between 0.03 and 0.46 mmol c dm -3 and 0.7 and 19.7, respectively.Positive correlations (P < 0.05) were observed between SD and Al 3+ concentration in KCl extracts for all colorimetric methods as there was intrinsic relationship between intensity of developed color and Al content in the extract, and conversely, the titrimetric method presented negative correlations between CV% and Al 3+ concentration, due to the difficulty in the observation of the end-point.
Despite of this, for most of the acid soil samples of pH ranging from 3.8 to 4.7, the relative low values of SD and CV% indicate that all these tested methods presented good reproductibility for the analytical determination of the Al 3+ .The titrimetric method, phenolphthalein being used as indicator, presented a limitation due to the difficulty in observing the end-point and the analytic moroseness, due to the need of two step analysis (back-titration), which limits the number of samples to be analyzed by each batch.The Al 3+ values given by titration may be imprecise and untruthful in relation to solutions of known Al concentration (Logan et al., 1985).Raij et al. (2001) have proposed the Al 3+ determination, when necessary, by some specific methods, such as the spectrophotometry with orange of xylenol in extracts of NH 4 Cl or even by plasma spectrometry (ICP-AES) in KCl or NH 4 Cl extracts.
Both aluminon colorimetric methods presented difficulties for cleaning the glassware and tubes in contact with the aluminon reagent.All glassware was stained with a rose-red coloration, mainly in the quartz vessel of the spectrophotometry, producing accumulative effect and provoking systematic errors for the Al 3+ determination in the soil extracts.The FIA system, employed for Al determination by eriochrome cyanine R method, should also be carefully washed after the analysis to avoid the discoloration, although slow, of the polyethylene tubes by the colorimetric reagent (Zagatto et al., 1981).
Among colorimetric methods, in relation to operationality, the eriochrome cyanine R colorimetric method, with FIA system presented the best analytical quickness (allowing analysis up to 50 samples per hour), easiness and sensibility.In addition, the chemical analysis using FIA system can also turn simultaneous or sequential analysis of several elements easier (Kronka, 1996;Kachurina et al., 2000).The eriochrome-FIA procedure, with some modifications (improvements are needed for soil samples), should be preferred over the titration method when operational gains are the main aim in routine analysis of soil samples; nevertheless when the target is the research, titration should be used, because, despite this moroseness, with back-titration the procedure is specific for Al 3+ ions.
Table 1 -
Legend, classification, and origin of the soil samples used in the experiment. | v3-fos-license |
2019-03-11T17:17:31.045Z | 2019-02-15T00:00:00.000 | 73456999 | {
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} | pes2o/s2orc | Developability Assessment of Physicochemical Properties and Stability Profiles of HIV-1 BG505 SOSIP.664 and BG505 SOSIP.v4.1-GT1.1 gp140 Envelope Glycoprotein Trimers as Candidate Vaccine Antigens
The induction of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an effective vaccine against HIV-1. A soluble, trimeric, germline (gI) bNAb-targeting variant of the HIV-1 envelope glycoprotein (termed BG505 SOSIP.v4.1-GT1.1 gp140, abbreviated to GT1.1) has recently been developed. Here, we have compared this new immunogen with the parental trimer from which it was derived, BG505 SOSIP.664 gp140. We used a comprehensive suite of biochemical and biophysical methods to determine physicochemical similarities and differences between the 2 trimers, and thereby assessed whether additional formulation development efforts were needed for the GT1.1 vaccine candidate. The overall higher order structure and oligomeric states of the 2 vaccine antigens were quite similar, as were their thermal, chemical, and colloidal stability profiles, as evaluated during accelerated stability studies. Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. This developability assessment of the BG505 GT1.1 vaccine antigen supports using the formulation and storage conditions previously identified for the parental SOSIP.664 trimer and enables the development of GT1.1 for phase I clinical studies.
Introduction
Envelope glycoproteins (Env) have been a focus of HIV-1 vaccine research for over 30 years. 1 Because Env spikes are the only virusencoded antigen exposed on the viral surface, they are the only relevant targets for broadly neutralizing antibodies (bNAbs). The Env spike is composed of a trimer of noncovalently linked gp120 and gp41 glycoprotein heterodimers. The soluble BG505 SOSIP.664 gp140 trimer adopts a native-like conformation and presents most of the known bNAb epitope clusters. 2,3 When tested as an immunogen in several animal models, this trimer elicits neutralizing antibodies (NAbs) to the neutralization-resistant (Tier-2) autologous BG505.T332N virus. 4 A soluble, stable, variant trimer termed BG505 SOSIP.664 gp140 has been manufactured under cGMP conditions for evaluation in phase I clinical trials. 5 To support the cGMP manufacture of this trimer, analytical characterization and formulation development experiments were performed to identify conditions that will not only ensure stability of the protein drug product during storage, but also its compatibility with adjuvants that will be used in clinical studies.
An effective HIV-1 Env vaccine will need to induce a more broadly active set of NAbs that could protect against multiple HIV-1 strains, which remains a highly challenging task. One plausible approach involves using an immunogen that targets the human germline (gl) precursors for bNAbs, followed by maturation of the initiated antibody lineage(s) by the use of one or more Env immunogens. [6][7][8][9][10][11] To explore this concept, a variant of the BG505 SOSIP.664 gp140 trimer was developed that, in vitro, can engage the gl-precursors of bNAbs that target the trimer apex and the CD4binding site. 11 The new trimer, BG505 SOSIP.v4.1-GT1.1 gp140 (abbreviated to GT1.1), was created from the parental SOSIP.664 trimer by introducing 18 amino acid substitutions and a 7-residue deletion into the V2 loop, resulting in the removal of 5 glycosylation sites. 11 Despite these changes, the GT1.1 trimer retains the native-like conformation adopted by its parental molecule. 11 As we plan to advance the GT1.1 trimer to early clinical development, we wanted to perform an early developability assessment to understand (1) formulation risks (and whether additional formulation development efforts are needed to produce a stable vaccine dosage form to facilitate rapid initiation of clinical evaluations), and (2) comparative physicochemical properties of GT1.1 trimer, in relation to the parental SOSIP.664 trimer, to evaluate the similarities and difference when formulated in the same (parental trimer) formulation condition. Thus, this work is a case study of evaluating the formulation/stability risk with a new complex protein-based HIV Env vaccine candidate by comparison to a closely related, yet molecularly distinct, HIV Env trimer antigen. Although developability assessments are commonly performed for selection of optimal molecular versions of a new monoclonal antibody drug candidate in terms of improved pharmaceutical properties (with similar biological properties 12 ), this type of evaluation has not been as widely applied to new recombinant protein antigens during early vaccine development.
To this end, the biophysical and biochemical properties of both the SOSIP.664 and GT1.1 recombinant protein trimeric antigens have been systematically evaluated using a set of orthogonal physicochemical techniques that address key aspects of the structural integrity of both molecules. In addition, the conformational, chemical and colloidal stability profiles of both trimers were also evaluated and compared in the currently used formulation buffer for frozen liquid storage of the SOSIP.664 antigen. The prototypic BG505 SOSIP.664 trimer was first analyzed using a larger set of biophysical and biochemical methods. The resulting information allowed for down-selection of the most suitable procedures to apply to the GT1.1 trimer, which was only available in smaller quantities at this stage of the development process. Some of these methods have been used previously for analytical characterization and cGMP quality control analyses for SOSIP.664 gp140 13,14 . Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. Hence, the new GT1.1 vaccine antigen can be formulated and stored as frozen liquid dosage form under the conditions previously defined for the parental BG505 SOSIP.664 trimer, 5 a finding that helps facilitate more rapid advancement of the new GT1.1 vaccine antigen into initial clinical development.
Materials
A frozen stock of BG505 SOSIP.664 gp140 trimers produced in stable CHO cells and purified by a series of column steps and formulated at a concentration of 3 mg/mL in "Formulation Buffer" (20 mM Tris, 100 mM NaCl pH 7.5) 5 was supplied by KBI Biopharma Inc. The BG505 SOSIP.v4.1-GT1.1 trimer (GT1.1) is a modified version of BG505 SOSIP.664, engineered to engage germline antibody precursors, achieved by substitution of 18 amino acids (resulting in the removal of 5 potential N-glycan sites) and deletion of 7 amino acids in the V2 loop. 15 The GT1.1 trimers were produced in stable CHO cells at the Weill Cornell Medical College, purified by the same method and supplied frozen at a concentration of 0.5 mg/mL in the same buffer. To use in method development (sedimentation velocity analytical ultracentrifugation [SV-AUC], size-exclusion chromatography [SEC], etc.), a 250-mg aliquot of CHO celleexpressed BG505 SOSIP.664 gp140 monomers (i.e., one protomer of the trimer) was collected from a 2G12-immunoaffinity and SEC column purification at the Weill Cornell Medical College. Sodium chloride was purchased from Fisher Scientific (Hampton, NH). Sucrose, trehalose from Pfanstiehl Laboratories (Waukegan, Illinois), and sulfobutyl-b-cyclodextrin from Captisol (San Diego, CA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Sample Preparation
Before analysis, BG505 SOSIP.664 and GT1.1 gp140 trimer samples were thawed at room temperature and diluted in "Formulation Buffer" (see the aforementioned) to the desired concentration. Because the pH of Tris-buffered solutions is temperaturedependent, for some techniques described in the following (specifically, those that include a thermal melt), the trimers were dialyzed into PBS buffer (20 mM NaH 2 PO 4 , 100 mM NaCl pH 7.5) overnight at 4 C before use.
UV-Visible Spectroscopy
The UV-visible absorption spectra of the SOSIP.664 and GT1.1 trimers were recorded with an Agilent 8453 UV-visible spectrophotometer (Palo Alto, CA). The concentration of each protein was calculated based on the calculated 16 extinction coefficients 1.590 g/ L at 280 nm for SOSIP.664 and 1.559 g/L at 280 nm for GT1.1. Samples were assayed before and after centrifugation (5000 rpm for 5 min). Spectra were corrected for light scattering using a technique included in the manufacturer's data analysis software (ChemStation UV-Vis analysis software, Agilent Technologies). Second derivative analysis was performed using Origin (OriginLab, Northampton, MA).
SDS-PAGE and BN-PAGE
BG505 SOSIP.664 and GT1.1 samples were mixed with 4X NuPAGE LDS sample buffer (Thermo Fisher Scientific) with and without 5 mM dithiothreitol (Thermo Fisher Scientific) and incubated at 95 C for 5 min. Samples were then treated with 10 mM iodoacetamide (Thermo Fisher Scientific) at 25 C in the dark for 30 min. Approximately, 10 mg of each sample were separated on 4%-12% Bis-Tris gels using NuPAGE MOPS SDS Running Buffer (Thermo Fisher Scientific). SeeBlue Plus2 Pre-Stained Protein Standard (Thermo Fisher Scientific) was used as a molecular weight (MW) ladder. BN-PAGE was performed with the same system used in SDS-PAGE. For Blue-Native gels, samples were mixed with NativePAGE Sample Buffer (Thermo Fisher Scientific) and were separated on NativePAGE Bis-Tris 3%-12% Mini Gels using NativeMark Unstained Protein Standard (Thermo Fisher Scientific) as a MW marker. Protein bands on all gels were visualized by staining with Bio-safe Coomassie Blue G250 stain (Bio-Rad Laboratories, Hercules, CA) or by silver staining.
LC-MS Peptide Mapping
Samples were prepared by drying 80 mg of protein using a SpeedVac (Eppendorf, Hamburg, Germany), followed by resuspension with 250 mM Tris, 6M guanidine HCl, pH 7.5, and reduction with 50 mg/mL DTT (55 C for 20 min). The samples were then alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark, before addition of 180 mL of 50 mM Tris pH 7.5 and 2 mL of PNGase F (Promega, Madison, WI) and incubation of the mixture overnight at 37 C. The following morning, either a trypsin/ LysC mixture (Promega) or chymotrypsin (Promega) was added, and the samples were incubated for 4 h at 37 C. Trifluoroacetic acid (TFA, 0.5%) was added to quench the proteolysis reaction, and 50 mL of the digested protein solution was subjected to liquid chromatographyemass spectrometry (LC-MS), as follows.
The peptides from the digested protein solution were separated by a liquid chromatography system (Thermo Fisher Scientific) before analysis. Peptides were injected onto a C18 column (1.7 mm, TFA; 200 mL/min flow rate) was used for separation. Mass spectrometry (MS) was performed using an LTQ-XL ion trap (Thermo Fisher Scientific) and the Xcalibur 2.0 software (Thermo Fisher Scientific). The instrument was also tuned using a standard calibration peptide (Angiotensin II, Sigma-Aldrich, St. Louis, MO) for maximal sensitivity before running any analyses. The mass spectra were acquired in the LTQ over a mass range of m/z 400-2000, the ion selection threshold was 10,000 counts and the dynamic exclusion duration was 8 s.
Raw experimental files were initially evaluated manually to determine if the ion counts and fragmentation of each peptide were sufficient for further analysis. The raw data files were then processed using PepFinder 2.0 software (Thermo Fisher Scientific). The database used for this experiment consisted of the BG505 SOSIP.664 or GT1.1 primary sequence. Potential Cys carbamidomethylation, Asn deamidation, and methionine (Met)/Trp/His/Cys oxidation events were considered during the analysis. Peptide assignments of MS/MS spectra were validated using a confidence score of !95%.
Size Exclusion, Cation Exchange, and Reversed-Phase High-Performance Liquid Chromatography
Before all HPLC experiments, all samples were centrifuged for 5 min at 12,000 Â g to remove any large aggregates/particles that could interfere with the analysis. Size exclusion high-performance liquid chromatography was performed in triplicate with a Shimadzu HPLC system with UV absorbance detection at 220 with a Tosoh TSKgel Ultra SW Aggregate Column and a corresponding guard column. Experiments were performed at 25 C with a mobile phase containing 0.2 M sodium phosphate, 400 mM NaCl, and pH 7.5, and a flow rate of 0.5 mL/min was used to separate species based on size.
Cation exchange chromatography (CEX) was also performed on a Prominence UFLC system. Thirty microgarm of BG505 SOSIP.664 or GT1.1 trimers in formulation buffer were injected onto a TSKgel BioAssist S column (4.6mm ID Â 5cm, PEEK column, Tosoh Biosciences). The flow rate was set at 0.5 mL/min and the column oven and autosampler temperatures were set at 30 C and 4 C, respectively. Elution was monitored using the absorbance at 214 and 280 nm. Mobile phase A consisted of 10 mM NaH 2 PO 4 , pH 7.0, whereas mobile phase B consisted of 10 mM NaH 2 PO 4, 1 M NaCl, pH 7.0. The mobile phase gradient consisted of 0% B (5 min), 0%-100% B (30 min), 100% B (3 min), and 0% B (2 min).
Fourier-Transform Infrared Spectroscopy
Fourier-transform infrared spectroscopy (FTIR) spectroscopy was performed using a Bruker Tensor-27 FTIR spectrometer (Bruker Optics, Billerica, MA) equipped with a KBr beam splitter. The mercuric cadmium telluride detector was cooled with liquid N 2 and the interferometer was constantly purged with N 2 gas. All instrument validation tests were performed and passed before daily measurements. Two hundred and fifty six scans were recorded from 4000 to 600 cm À1 with a 4 cm À1 resolution using a Bio-ATR cell. Background measurements were acquired with formulation buffer alone and subtracted from the sample spectra. Atmospheric corrections, baseline corrections, and second derivative calculations were applied using OPUS V6.5 (Bruker Optics, Billerica, MA) software. Following Fourier self-deconvolution, between 6 and 8 peaks were fitted to the absorbance spectrum in the Amide I region (1700-1600 cm À1 ) using a mixed Gaussian and Lorentzian function. The areas of the peaks were used to determine the relative percentage of secondary structure components in the trimer samples.
Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) was performed on the trimer samples in triplicate using an Auto-VP capillary differential scanning calorimeter (MicroCal/GE Health Sciences, Pittsburgh, PA) equipped with tantalum sample and reference cells. The SOSIP.664 or GT1.1 trimers (at 0.2 mg/mL), or PBS alone, were loaded in a DSC autosampler tray held at 4 C. Scans were completed from 10 C to 100 C using a scanning rate of 60 C/h. Reference subtraction and concentration normalization were performed using the instrument software. T m and T onset values were determined using Origin (Ori-ginLab, Northampton, MA).
Fluorescence Spectroscopy
Intrinsic tryptophan fluorescence data for the SOSIP.664 and GT1.1 gp140 trimers were collected on a Fluorescence Innovations Plate Reader (Fluorescence Innovations Inc., Minneapolis, MN). Ten millimeter aliquots of 0.2 mg/mL samples were analyzed in sextuplicate in black 386-well plates. An excitation wavelength of 295 nm was used with a 315 nm filter. Samples were heated from 10 C to 90 C using a stepsize of 2.5 C. Peak position was calculated using Fluorescence Innovations data analysis software.
Eight-Anilino-1-naphthalene sulfonate (ANS) was used as an extrinsic fluorescence probe in the presence of the SOSIP.664 or GT1.1 trimers. Experiments were performed in PBS using an Agilent Stratagene Mx3005P QPCR System. A solution containing 100 mM ANS (generally used at 20Â the molar concentration of protein) was excited at 372 nm, and the ANS emission spectrum was collected from 400 to 600 nm every 2 nm. The final protein concentration used was 0.2 mg/mL.
Data Visualization Using Three-Index Empirical Phase Diagrams
Three-index empirical phase diagrams (EPDs) were constructed as described previously 17,18 to analyze the large biophysical stability datasets generated as function of pH and temperature. Intrinsic Trp fluorescence peak position, ANS fluorescence peak intensity, and DSC data were used for construction of the 3-index EPDs for both gp140 proteins. All calculations were performed using the in-house software. Each temperature and pH pair was grouped in regions using a k-means clustering algorithm. The final definition of structural regions was confirmed by visual assessment of the EPD regions versus the trends observed in the biophysical data sets.
Dynamic Light Scattering
The dynamic light scattering (DLS) mode on a ZetaPALS zetasizer (Brookhaven Instruments Corporation, Holtsville, NY) was used, with quartz cuvettes that had been cleaned of any dust and air-dried. The hydrodynamic diameters of filtered SOSIP.664 or GT1.1 samples (at 0.5 mg/mL) were analyzed by generating an autocorrelation decay function after centrifugation at 14,000 Â g for 5 min. Ten measurements were recorded and averaged for 30 s each. Number and intensity distributions were fitted using the cumulant analysis algorithm provided with the instrument software. All measurements were performed in triplicate at 25 C, and the viscosity of the solvent was 0.89 cP.
Sedimentation Velocity Analytical Ultracentrifugation
SV-AUC studies were conducted on a ProteomeLab XL-I analytical ultracentrifuge equipped with a scanning ultravioletvisible optical system (Beckman Coulter, Fullerton, CA). All experiments were performed at 20 C with a rotor speed of 40,000 rpm and detection at 280 nm. Samples (0.5 mg/mL of SOSIP.664 or GT1.1) and formulation buffer alone were loaded into Beckman charcoal-epon 2 sector cells with a 12 mm centerpiece and quartz windows. Sednterp (Professor Thomas Laue, University of New Hampshire) was used to calculate the partial specific volume based on the primary sequence of the protein (around 0.73 mL/g) and the density and viscosity of the formulation buffer. Data were analyzed using Sedfit (Peter Schuck, NIH). A continuous c(s) distribution fitting model was applied with 50 scans. Frictional ratio, radial independent noise, and time independent noise were also fit, whereas the meniscus and bottom positions were set manually. A range of 0 to 25 Svedbergs was used, after verifying that there was no signal that sedimented outside of this range. A resolution of 300 points per distribution and a confidence level of 0.95 were used.
Microflow Imaging
Subvisible particle concentrations were assayed by microflow imaging (MFI) using an MFI 5200 instrument (ProteinSimple, Santa Clara, CA) equipped with a Bot1 autosampler. Before analysis, the instrument was calibrated using 10 mm polystyrene particle standards (Thermo Fisher Scientific), and it was cleaned with filtered water and 2% PCC-54 Detergent. BG505 SOSIP.664 samples were measured undiluted (at 3 mg/mL) while GT1.1 trimers were diluted 10-fold into formulation buffer (final concentration of 0.05 mg/mL) to conserve sample.
Negative Staining Electron Microscopy
Trimer samples were diluted to~0.01-0.03 mg/mL in formulation buffer (see Sample Preparation) before negative staining electron microscopy (NS-EM) analysis. Previously described procedures were followed for grid preparation, imaging, and data analysis. 16,17 A 2% (w/v) solution of uranyl formate was used as the stain, and images were collected on an FEI Tecnai T12 electron microscope equipped with a Tietz TemCam-F416 CMOS camera (120 keV, 2.05 Å/pixel,~25 e -/Å 2 total dose, 1500 nm nominal defocus).
Results
Comparative Physicochemical Characterization of BG505 SOSIP.664 and BG505 SOSIP.v4.1-GT1.1 gp140 Trimers The primary structures of the 2 gp140 molecules (Supplemental Fig. S1) were confirmed and compared by performing LC-MS peptide mapping on BG505 SOSIP.664 (Supplemental Fig. S2) and GT1.1 gp140 proteins (Supplemental Fig. S3). The proteins were digested using either trypsin and LysC (Supplemental Figs. S2A and S3A) or chymotrypsin (Supplemental Figs. S2B and S3B). Chymotrypsin digestion resulted in better sequence coverage for SOSIP.664 gp140 (~95%) than trypsin and LysC digestion (~87%). Similarly, sequence coverage for GT1.1 was notably superior after chymotrypsin (94% coverage) digestion, compared to trypsin and LysC (80% coverage). Before analysis by LC-MS, the peptides were deglycosylated with PNGase to remove the N-linked glycans. The oligosaccharide content differences between the 2 trimers were not examined here because the glycan composition is not expected to change during storage. This LC-MS method can be used to assess whether chemical modifications of amino acid residues in the protein antigens (e.g., Asn deamidation, Met oxidation) occur during accelerated stability studies (see in the following).
The overall secondary and tertiary structures of the SOSIP.664 and GT1.1 gp140 trimers in solution were evaluated by FTIR and second-derivative UV-visible spectroscopy (Fig. 1), respectively. Small differences were observed between the second-derivative UV-visible spectra of the 2 gp140 trimers, indicative of subtle influences of the microenvironments of their aromatic amino acids, which likely arises from primary structure variation (Fig. 1a). FTIR analysis revealed that both proteins had similar overall secondary structure contents, comprising a mixture of a-helices and b-sheets, consistent with previous structural studies 19 (Figs. 1b, 1c and Supplemental Fig. S4).
Size-based analysis of the SOSIP.664 and GT1.1 trimers was performed using a combination of denaturing (SDS-PAGE) and nondenaturing conditions (DLS and SV-AUC), as shown in Figure 2. The results of the SDS-PAGE analysis were similar to those reported previously, 2 with nonreduced SOSIP.664 gp140 migrating as a single band and the reduced sample migrating as a single gp120 band and a poorly resolved gp41 ectodomain (gp41 ecto ) triplet (Fig. 2a, left panel). Similar results were observed for GT1.1 gp140 (Fig. 2a, right panel). The hydrodynamic diameters of the SOSIP.664 and GT1.1 gp140 proteins in solution were determined by DLS. Intensity-weighted cumulant analyses revealed diameters of 18.6 ± 1.6 nm and 22.9 ± 2.5 nm for SOSIP.664 and GT1.1 gp140 proteins, respectively (Fig. 2b). One explanation for this minor discrepancy would be the presence of small amounts of higher MW aggregates in the GT1.1 sample. This was confirmed to be the case when the DLS data were evaluated by an intensity-weighted mean square displacement analysis (Supplemental Fig. S5). In addition, a greater number of subvisible particles are present in GT1.1 samples when analyzed by MFI (see in the following). SV-AUC was then used as an orthogonal technique to estimate the MWs (and percent trimer) for SOSIP.664 and GT1.1. The SV-AUC profiles were similar for both trimers (Fig. 2c). The GT1.1 trimer population gave a calculated mass of 358 ± 8 kDa with a sedimentation coefficient value of 11.9 ± 0 S, whereas the corresponding values for SOSIP.664 were 373 ± 15 kDa and 12.3 ± 0 S (Supplemental Table S1). The differences in sedimentation coefficient values, and the estimated mass reduction of~14 kDa for GT1.1, are consistent with the deletion of 7 amino acid residues and 5 potential N-glycan sites when generating the GT1.1 trimer from the SOSIP.664 prototype (assuming an average amino acid MW of 110 Da and 2000 Da per glycan site). Size exclusion chromatography was also performed on SOSIP.664 (Supplemental Fig. S6) but was found to not be as informative as SV-AUC and was not used in analysis of GT1.1.
Reversed-phase and cation exchange high-performance liquid chromatography (RP-HPLC, CEX-HPLC) were used to assess hydrophobic and charge heterogeneity, respectively, for the SOSIP.664 and GT1.1 gp140s (Fig. 3). In RP-HPLC, SOSIP.664 gp140 eluted as a single main peak comprising over 90% of the total area, but with 2 shoulders (Fig. 3a, left panel). Three minor peaks eluted earlier than the main peak but collectively comprised less than 1% of the total area of the chromatogram. The GT1.1 RP-HPLC chromatograms were notably different from that of SOSIP.664, in that the main species eluted as 2 overlapping peaks (Fig. 3a, right panel). Charge heterogeneity was evaluated by CEX-HPLC. Here, SOSIP.664 gp140 had a more heterogeneous profile than GT1.1 (Fig. 3b). Nearly all (>99%) of the loaded SOSIP.664 protein eluted from the column (determined by injecting sample with and without the column in line). One main peak (at~12.5 min) comprised~50% of the total peak area of the chromatograms but at least 10 individual latereluting peaks were also identified (Fig. 3b, left panel). The GT1.1 profile in the CEX-HPLC analysis was markedly different; while the main peak eluted at approximately the same retention time as SOSIP.664, no later eluting peaks were observed (Fig. 3b, right panel). In an attempt to explain the apparent heterogeneity in the SOSIP.664 CEX chromatographic profile, we enzymatically digested the protein with PNGase F to remove the N-glycans from the protein ( Fig. 3b and Supplemental Fig. S7). Although the SDS-PAGE data indicated that approximately~40 kDa of N-glycans were removed from each SOSIP.664 monomer (Supplemental Fig. S7), no observable changes were seen in the CEX chromatograms (Fig. 3b, left panel). Similarly, no change was seen in the SDS-PAGE and CEX analyses of PNGase F treated GT1.1 gp140 protein (Fig. 3b, right panel and Supplemental Fig. S7).
The aforementioned analyses imply that the GT1.1 trimer is more heterogeneous than SOSIP.664 from the perspective of hydrophobicity (RP-HPLC), but the converse applies to surface charge (CEX-HLPC) with SOSIP.664 being the more heterogeneous protein.
The differences between the 2 proteins could be rooted in the amino acid sequence changes used to create the GT1.1 gp140 trimer from SOSIP.664, including variations in post-translational modifications such as how N-glycosylation sites are utilized. In addition, some of these differences may be due to differences in Met The conformational stability of SOSIP.664 gp140 trimers was evaluated across a pH range of 3.0 to 9.0 and varying temperatures by DSC, intrinsic tryptophan fluorescence peak position, and ANS fluorescence peak intensity (Figs. 4a-4c, respectively). All 3 methods indicated that lower pH values destabilized the SOSIP.664 trimers in a temperature-dependent manner; the overall tertiary structure of the protein molecules was affected at low pH, even at low temperatures, consistent with data reported earlier. 5 At higher pH values (!pH 6), thermal transitions were observed (Figs. 4a-4c) with calculated T m values between 55 C and 70 C (Supplemental Fig. S8). These results are consistent with previous DSC studies. 2,5 Using these biophysical stability data sets, a 3-index EPD 17,18 was constructed to better visualize and compare the temperature and pH stability profiles of both molecules and to identify the contributions of each of the 3 individual techniques (Fig. 4d, left panel). Intrinsic tryptophan fluorescence peak position (red), extrinsic ANS fluorescence intensity (blue), and DSC (green) were used for this analysis, in which 5 distinct colored phases (designated as regions I-V) were identified. Specifically, in addition to the SOSIP.664 native state (region I), 4 structurally altered protein states (regions II-V) were observed. Regions II and III correspond to states that are structurally altered, to varying extents, under low pH conditions. Region IV represents an overall structurally altered conformation, reflecting the pH-and temperature-dependent unfolding processes observed by DSC and intrinsic fluorescence spectroscopy. Region V represents a more extensively structurally altered protein.
For comparison, the GT1.1 gp140 protein was evaluated using the same biophysical methods and by EPD analysis. Overall, its thermal and pH stability profiles were highly similar to SOSIP.664, with region I comprising 30% of the total EPD area for both molecules. The conformational stability profiles of the 2 molecules as a function of pH and temperature, again as measured by intrinsic tryptophan fluorescence, ANS fluorescence, and DSC, were also similar, and no notable differences in the thermal melting temperature values were identified (Figs. 4a, 6b, 6c and Supplemental Fig. S8). As with SOSIP.664, 5 differently colored structural states were identified in the EPD for GT1.1 (Fig. 4d, right panel).
Chemical Stability Profiles of BG505 SOSIP.664 and GT1.1: Oxidative Stress Studies
Chemical stability profiles were determined by subjecting the SOSIP.664 and GT1.1 gp140 proteins to forced oxidation, followed by RP-HPLC, CEX-HPLC analyses, and LC-MS peptide mapping. The protein samples were exposed to 0%, 0.05%, and 0.5% H 2 O 2 for 1 h at room temperature (Fig. 5). As described previously in Figure 3, the RP-HPLC and CEX-HPLC elution profiles of the nonstressed SOSIP.664 and GT1.1 samples (i.e., 0% H 2 O 2 ) were notably different, which was also seen in this set of experiments. Thus, in the RP-HPLC chromatograms, GT1.1 eluted as 2 overlapping peaks (at 24.5 and~25.0 min), whereas only 1 peak (~24.5 min) was seen for SOSIP.664 (Fig. 3a). In the presence of 0.05% H 2 0 2 , however, 2 closely eluting peaks were observed with SOSIP.664 (Fig. 5a). It is therefore possible that the single peak in the unstressed SOSIP.664 sample is composed of 2 coeluting species that behave differently on exposure to H 2 O 2 . We also saw that, in the presence of increasing concentrations of H 2 O 2 , the polarity of both gp140 trimer samples increased, resulting in earlier elution times. In both cases, the extent of this change in retention time was similar at the same H 2 O 2 concentrations. The H 2 O 2 -induced changes to the elution profiles of the SOSIP.664 and GT1.1 gp140 trimers were also similar in the CEX-HPLC analysis (Fig. 5b), despite the notable differences between their elution profiles under nonstressed (control) conditions (Fig. 3b). Thus, in the presence of H 2 O 2 , for both samples, the area of the main peak began to decrease and shift to a later elution volume (consistent with a more positive surface charge), and a new peak began to emerge at~14 min. No notable change in total peak area was observed between samples in either chromatographic analysis, implying that there was no loss of protein (i.e., no precipitation) under the test conditions. Residues susceptible to post-translational modifications under oxidative stress were identified by LC-MS peptide mapping. In total, 80% of the SOSIP.664 or GT1.1 primary sequences were covered in the 0% and 0.05% H 2 O 2 -treated samples after trypsin þ LysC proteolysis. This coverage includes 10 of the 12 Met residues present in both proteins (Met 535 and Met 540 were not identified). No peptides were observed after proteolysis after exposure of either protein to 0.5% H 2 O 2 (Fig. 6), which is consistent with the proteins becoming insoluble under these stressed conditions. The relative amounts of post-translational modifications induced by the oxidative stress in each sample were quantified (Fig. 6c). Met oxidation (þ16 Da) was identified at the same 3 residues in the SOSIP.664 (Met 271 , Met 426 , and Met 475 ) and GT1.1 (Met 271 , Met 426 , and Met 475 ) proteins. For SOSIP.664, Met 271 and Met 426 were more prone to oxidation than Met 475 under these conditions, a pattern also seen for GT1.1 (Met 271 and Met 426 were more susceptible than Met 475 ). A dehydration reaction (indicated by a loss of 18 Da) was also detected in the SOSIP.664 Cys 228 -Arg 273 and GT1.1 Cys 228 -Arg 273 peptides. It is likely that this event arose during or after proteolysis, but this supposition requires experimental confirmation. The amino acid numbers, aforementioned, is based on HIV-1 HxB2 numbering system (Supplemental Fig. S1).
Chemical Stability Profiles of BG505 SOSIP.664 and GT1.1: Elevated Temperature and pH Stresses We evaluated the chemical stability of the SOSIP.664 and GT1.1 gp140 trimers by increasing the temperature or pH. Under such conditions, Asn residues can undergo a deamidation reaction through a succinimide intermediate, which results in the formation of iso-aspartate and aspartate residues in the protein. 20 The SOSIP.664 and GT1.1 gp140 trimers were incubated for 1 week at pH values of 7.5 or 9.0 and at temperatures of 4 C or 37 C, before analysis by RP-HPLC, CEX-HPLC, and LC-MS peptide mapping. In both chromatographic methods, no differences in the elution profiles of either protein were observed between the pH 7.5, 4 C (control) samples and the pH 9.0, 37 C samples (Fig. 7). However, for both trimers, the total peak areas of the pH 9.0, 37 C samples were~10% lower compared to the 3 other conditions. This outcome was consistent with protein loss during the 7-day incubation via aggregation and precipitation. We noted that any such precipitated proteins would probably be removed when the samples were centrifuged before RP-HPLC analysis. The peptide chromatograms derived from LC-MS analysis were similar for both trimers under all the test conditions (Fig. 8). Taken together, these results indicated that neither the SOSIP.664 nor GT1.1 gp140 trimer was highly susceptible to Asn deamination under the test conditions of increased pH and temperature. An initial formulation of these vaccine antigen candidates (to support Phase I clinical trials) needs to provide sufficient stability for long-term storage as a frozen liquid drug product, followed by thawing and immediate (same day) administration to patients. To this end, we examined if the 2 molecules had similar physical properties on freeze-thaw, not only by using the stress test methods described previously but also by comparing the results with 2 already established stability-indicating test methods (immunoassay and NS-EM analysis). 2,5 The experiments were performed with the standard formulation buffer (20 mM Tris, 100 mM NaCl, pH 7.5) already used for SOSIP.664 5 , to identify whether any formulation changes were needed for GT1.1 to be stored as a frozen liquid drug product.
First, the 2 gp140 trimers were subjected to agitation stress to compare their colloidal stabilities in the formulation buffer. The formation of subvisible particles (2-100 mm) was measured by MFI.
To conserve the more limited stocks of GT1.1 gp140 trimers during method development, preliminary agitation studies were performed using SOSIP.664, by shaking the samples for up to 72 h at room temperature. The concentration of subvisible particles increased until the 6 h time point, after which a decrease was seen, possibly because larger aggregates formed and settled out. Accordingly, the GT1.1 and SOSIP.664 gp140 trimers were then subjected to agitation stress for up to 6 h (Fig. 9). Although the initial GT1.1 subvisible particle concentration in the formulation buffer was higher than for SOSIP.664, there was no notable increase in the subvisible particle concentration after agitation stress. Thus, the subvisible particle size distributions were similar for both molecules, with most of the particles in the 2-5 mm size range (data not shown).
Next, the 2 gp140 trimers, in formulation buffer, were frozen at À80 C and thawed at room temperature for 0, 1, and 5 cycles, with their physical stability profiles monitored by SV-AUC and UVvisible spectroscopy. No notable changes in hydrodynamic size were observed for either protein by SV-AUC, as measured by the distribution of sedimentation coefficient values (Fig. 10a). There were also no changes in protein concentration after the freezethaw stress, as assayed by UV-visible spectroscopy after centrifugation (data not shown). Methods already established for assaying SOSIP.664 trimer conformational stability included a BLI-based immunoassay using the trimer-specific antibody PGT145 and NS-EM. 2,5 No notable changes in PGT145 binding were observed before and after subjecting the SOSIP.664 or GT1.1 gp140 trimers to freezethaw stress, indicating that the native-like trimer contents remained stable (Fig. 10b). NS-EM imaging before (i.e., no freezethaws) and after 3 cycles of freeze-thaw stress (Supplemental Fig. S9) confirmed the SV-AUC and BLI findings. Thus, no nonnative Env forms were observed for either vaccine antigen.
Discussion
A detailed understanding of the structural integrity, physicochemical properties, and stability profiles (under both accelerated and real-time storage conditions) is necessary for the successful formulation development and production of stable clinical dosage forms of any recombinant protein antigen-based vaccine candidate. In the present study, a series of biochemical and biophysical techniques were used in a developability study to compare the BG505 SOSIP.664 gp140 protein antigen to its re-engineered, gI-targeting variant, BG505 SOSIP.v4.1-GT1.1 gp140. The stable nature of this SOSIP.664 gp140 trimer has been determined previously, 5 but no such studies have been performed on GT1.1. Using SOSIP.664 gp140 trimer as the comparator, we demonstrated that the GT1.1 trimer has similar biophysical properties and pharmaceutical stability profiles under various environmental stresses (e.g., pH, temperature, agitation, and oxidation).
The GT1.1 gp140 trimer differs from SOSIP.664 by 18 amino acid substitutions (resulting in the removal of 5 potential N-glycan sites) and a 7 amino acid deletion in the V2 loop. The 2 trimers had similar tertiary and secondary structures, as judged by secondderivative UV spectroscopy and FTIR spectroscopy (Fig. 1). Small differences in the UV spectroscopy data can possibly be attributed to mutations that result in the addition or removal of aromatic residues (i.e., A316W and Y173H). However, the hydrophobicities and surface charges of the 2 trimers did differ (Figs. 3a and 3b). Particularly striking is the presence of~10 minor peaks only in the SOSIP.664 CEX-HPLC chromatograms (Fig. 3b). Differences in the characteristics of the extensive array of N-linked glycans present on the 2 trimers might underpin this finding. 19 More specifically, the much simpler elution profile of the GT1.1 trimer might reflect the absence of 5 potential N-glycan sites compared to its SOSIP.664 counterpart. Additional analyses would, however, be needed to identity the minor peaks, such as by fraction collection followed by LC-MS. These striking chromatographic differences were not apparent in the RP-HPLC data, which only show minor differences in the elution profiles. An H 2 O 2 stress test analysis suggests that the latter differences are oxidation-dependent (Fig. 5a).
Similar physical stability profiles of the SOSIP.664 and GT1.1 gp140 trimers were noted by comparisons of their temperature and pH behavior as measured by 3 different biophysical techniques and assessed by data visualization tools. The 3-index EPD stability profiles generated from the biophysical stability data sets are similar for both trimers (Fig. 4), with no notable differences in the defined structural regions. The only methods showing notable differences in the structures/post-translational modifications of the 2 protein molecules were RP-HPLC and CEX-HPLC. Even so, both proteins were similarly susceptible to oxidation and deamidation reactions under stressed conditions (Figs. 5-8). Thus, any differences in the prestress (i.e., control) samples do not translate into variations in whether and how chemical changes occur during accelerated stability testing. The similar outcomes of the physicochemical stability tests can be indicative of comparable storage stability profiles for the 2 SOSIP gp140 trimers under real-time conditions. 21 We suggest, therefore, that the BG505 SOSIP.v4.1-GT1.1 gp140 trimer is likely to be as stable as the prototypic SOSIP.664 gp140 trimer, while noting that long-term stability studies will be required to confirm this supposition.
The formulation, storage, and administration of vaccine antigens for an initial phase 1 clinical trial is typically designed based on stricter, more well-controlled conditions (e.g., administration by a medical professional at 1 clinical site), which would not be practical for wide distribution of commercial formulations. For example, vaccine antigens can be stored frozen as bulk drug substances to ensure long-term stability, and then thawed, formulated for fill as drug products before they are mixed with adjuvants ("bedside" mix) and immediately administered to a patient. To this end, the aggregation propensities and overall stabilities of the GT1.1 and SOSIP.664 gp140 trimers were assessed during agitation and freeze-thaw and found to be comparable when both trimers were formulated in the same simple buffer (20 mM Tris, 100 mM NaCl, pH 7.5) (Figs. 9, 10, and Supplemental Fig. S9). This outcome was observed using the orthogonal methods of SV-AUC and BLI immunoassay and confirmed by NS-EM imaging, a technique previously utilized to evaluate trimer stability after freeze-thaw cycles. 5,22 The freeze-thaw stability results are somewhat surprising given the formulation contains 2 excipients (Tris buffer and NaCl) with known incompatibilities with proteins during freeze-thaw stress. 23 First, the pH of Tris-buffered solutions is temperature-dependent, 24 which can lead to protein instability because of pH changes during freeze-thaw cycles. Thus, when a Tris buffer is adjusted to pH 7.5 at 25 C, its pH will increase to~8.1 at 5 C and to~8.25 at 0 C. 25 Despite this factor, we saw no instability on freeze-thawing, which indicates that both trimers remain physically stable under these pH conditions. Concerns over the aforementioned temperature-dependent pH shifts prompted preliminary stability studies to be performed using Histidine and HEPES buffer systems, but no notable changes in stability (and hence no improvements) were observed in these tests (data not shown). Furthermore, the resilience of SOSIP.664 gp140 to alkaline pHs has been observed previously. 5 A second factor is that sodium chloride is known to concentrate during freezing, which can increase the ionic strength of solutions. 25 But again, as we saw no physical instability in the freeze-thaw studies, we can infer that both trimers remain physically stable under these ionic strength conditions. We did perform preliminary stability studies using 10% sucrose instead of NaCl as a cryoprotectant and tonicifying agent, but again found no notable changes in stability (and hence no improvements).
In summary, engineering the native-like GT1.1 gp140 trimers to bind gl-bNAb precursors is a plausible approach to initiating the induction of bNAbs as a HIV-1 vaccine strategy. 1 However, modifications to the protein sequence (that can also lead to differences in glycosylation profile) may impair the conformational, chemical, and colloidal stability properties of a vaccine antigen. Each successive iteration of the prototypic BG505 SOSIP.664 gp140 trimer must therefore be evaluated by methodologies similar or equivalent to those described here, to assess the pharmaceutical stability of the new antigen and identify whether additional formulation development work is required for initial clinical development using a frozen liquid formulation. Here, we characterized both the BG505 SOSIP.664 gp140 trimer prototype and its new GT1.1 variant, using a battery of physicochemical methods and compared their physicochemical stability profiles versus pH, temperature, agitation, and freeze-thaw stresses. We conclude from this developability assessment that the BG505 SOSIP.v4.1-GT1.1 gp140 vaccine candidate is similar in its overall structural stability to the SOSIP.664 gp140 trimer, from which it was engineered, from a formulation development point of view. Hence, similar formulation conditions Figure 9. Subvisible (2-100 mm) particle formation (# of particle/mL) during agitation stress studies of BG505 SOSIP.664 and GT1.1 gp140 trimers as determined by MFI. Samples were diluted 10Â before analysis and presented without correction. Error bars represent standard deviation from triplicate measurements. Formulation buffer refers to 20 mM Tris, 100 mM NaCl, pH 7.5. Figure 10. Effect of freeze-thaw stress on stability profile of BG505 SOSIP.664 and GT1.1 gp140 trimers as determined by (a) molecular size distribution as measured by SV-AUC. SV-AUC plots are representative traces from duplicate measurements with y-axis zoom shown in inset. (b) PGT145 antibody binding as measured by biolayer interferometry (BLI). Error bars in panel (c) represent standard deviation from triplicate measurements. and processes can be used for the existing (SOSIP.664) and new (GT1.1) vaccine drug product candidates. 5 | v3-fos-license |
2018-12-12T07:46:04.776Z | 2008-09-12T00:00:00.000 | 92847522 | {
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} | pes2o/s2orc | Consecutive multi-component syntheses of heterocycles via palladium-copper catalyzed generation of alkynones
Alkynones are prominent three-carbon building blocks in heterocyclic chemistry. They can be generated very easily and efficiently by modified Sonogashira coupling of acid chlorides and terminal alkynes. Mild reaction conditions now set the stage for new diversity-oriented routes to heterocycles by sequential and consecutive transformations. Hence, isoxazoles, indolizines, pyrazoles, pyridimines, 1,5-benzoheteroazepines, furans, oxazoles, and tetrahydro-β -carbolines are accessible by consecutive coupling-cycloaddition or coupling-addition-cyclocondensation multi-component sequences
Introduction
The increasing demand for rapid syntheses of functional and biologically active molecules has stimulated synthetic chemists to explore and devise intelligent strategies that inevitably address the very fundamental principles of efficiency and efficacy.Besides the crucial issues of chemo-, regio-and stereoselectivity, nowadays these processes also have to consider economical and ecological aspects of green chemistry.Therefore, the intellectual challenge to invent concise, elegant and conceptually novel synthetic routes has become a steadily increasing driving force both in academia and industry.2] In particular, these diversity-oriented syntheses 3 are demanding challenges for synthetic efficiency and reaction design.Mastering unusual combinations of elementary organic reactions under similar conditions is the major conceptual challenge in crafting novel types of sequences.
In classical heterocyclic chemistry, five-, six-, and seven-membered heterocycles can be synthesized from reactive, bifunctional three-carbon building blocks such as alkynones [4][5][6][7] which react with bifunctional nucleophiles either via [3+2]-cycloaddition or via Michael additioncyclocondensation (Scheme 1).As a consequence, this general strategy has found broad application.However, standard syntheses of alkynones 8 are often harsh and require either strongly basic or strongly Lewis or Brønsted acidic conditions.Therefore, the application in onepot methodology, where delicately balanced reaction conditions are prerequisite, is largely excluded.Hence, mild reaction conditions for a catalytic generation of alkynones, which are compatible with following transformations, are highly desirable.In particular, transition metal catalysis opens many opportunities for functional group tolerant product formations and multicomponent syntheses of heterocycles. 9This account summarizes a concept developed in recent years in our group, where palladium-copper catalyzed coupling is used for the generation of alkynones and as an entry to consecutive multi-component syntheses of heterocycles.
C y c lo a d d i ti o n C y c l o c o n d e n s a ti o n
Scheme 1. Alkynones as three-carbon building blocks in heterocycle synthesis and the quest for their catalytic generation.
Upon scrutinizing the reaction conditions, we found that virtually only one equivalent of triethylamine is stoichiometrically necessary for binding hydrochloric acid, and hence, to achieve complete conversion. 12This not only reduces the amount of base but also leads to an essentially base-free reaction medium after the cross-coupling event.Furthermore, it is also possible to reduce reaction time by dielectric heating (microwave irradiation) instead of conductive heating (oil bath).With this methodological improvement in hand the stage was set for the generation of alkynones under mild conditions and in media where consecutive reactions in a one-pot fashion are readily conceivable.
Isoxazole syntheses
The 1,3-dipolar cycloaddition of aromatic nitrile oxides, a class of propargyl-type 1,3-dipoles, is a general access to isoxazoles. 13Since aromatic nitrile oxides are usually unstable compounds, it is necessary to generate them in situ by dehydrochlorination of the corresponding hydroximinoyl chlorides with a suitable base.If triethylamine is the base, this step should be fully compatible with a preceding alkynone formation.
Therefore, after reacting acid chlorides 1 with terminal alkynes 2 under modified Sonogashira conditions for 1 hour at room temperature to furnish the expected alkynones 3, subsequently, hydroximinoyl chlorides 4 and triethylamine are added.After dielectric heating for 30 minutes, the isoxazoles 5 are obtained as in moderate to excellent yields often as crystalline solids.Only one of two possible regioisomers is formed (Scheme 3). 14RKAT USA, Inc.The scope of this one-pot coupling-cycloaddition isoxazole synthesis is fairly broad and can be performed under mild conditions and with excellent chemo-and regioselectivity.As a consequence of acid chlorides as halide coupling partner, amines and hydroxy groups need to be protected prior to the reaction.The use of the acid chlorides 1 is predominantly limited to (hetero)aromatic compounds and derivatives without α-hydrogens.With one exception, cyclopropyl as substituent is tolerated in both steps of the sequence.Aliphatic as well as electron rich and electron poor aromatic alkynes can be employed.Even heterocyclic alkynes can be used as starting materials.Silylated alkynes, e. g. trimethylsilyl acetylene, also easily undergo the coupling procedure.With respect to the 1,3-dipolar nitrile oxide, electron-rich, polycyclic, electron-deficient and heterocyclic substituents are all tolerated and react readily with the alkynones 3.
Indolizine syntheses
Pyridinium ylides, allyl-type 1,3-dipolar molecules, undergo [3+2]-cycloaddition with alkynones as well.Thus, submitting (hetero)aroyl chlorides 1 and terminal alkynes 2 to the reaction conditions of the Sonogashira coupling in a mixture of THF and triethylamine at ambient temperature and after 2 h adding 1-(2-oxoethyl)pyridinium bromides 6 furnish after 14 h of stirring at room temperature indolizines 7 in 41-59 % yield as pale yellow to yellow green crystalline solids (Scheme 4). 15RKAT USA, Inc.This reaction is a novel methodological showcase for the combination of a cross-coupling and a sequential cycloaddition, giving rise to a broad variety of indolizines 7.In particular, 7-(pyridin-4-yl)-substituted representatives display pronounced fluorescence and even strong daylight fluorescence in their protonated form.The reversibility of the protonation as well as its fluorescence sensitivity in weakly acidic media render 7-(pyridin-4-yl)indolizines ideal candidates for fluorescence labeling and for studying pH-dependent and pH-alternating cellular processes.
Pyrazoles
The direct conversion of hydrazines 8 with alkynones 3 to pyrazoles by Michael additioncyclocondensation has been known for more than a century.4a, 16 However, either the regioselectivity issue has not been studied in detail or the occurrence of mixtures of regioisomers was reported. 17Despite of very few examples, 18 the regioselective formation of N-substituted pyrazoles by the alkynone pathway has remained unexplored.With respect to the interesting pharmacological and electronic properties of pyrazoles, in particular as fluorophores, and the increasing quest for tailor-made functional π-electron systems by diversity-oriented strategies, we have developed regioselective one-pot syntheses of substituted pyrazoles.
After formation of alkynones 3, hydrazines 8, and acetic acid are reacted in the same reaction vessel.Best results for the formation of pyrazoles 9 are obtained by dielectric heating in the microwave oven at 150 °C for 10 min in the presence of methanol.Pyrazoles 9 are obtained in good to excellent yields, predominantly as colorless crystalline solids (Scheme 5).Three types of hydrazines have been employed in the methodological studies, i.e. hydrazine (R 3 = H), methyl hydrazine (R 3 = CH 3 ), and aryl hydrazines (R 3 = aryl).In accordance with theory in every case only one of the two possible regioisomers, depending on the nature of the hydrazine substituent R 3 , was preferentially formed.Only traces of the other regioisomers could be detected (regioselectivity >98:2).The rapid, diversity-oriented synthetic approach to finetunable fluorophores (with fluorescence quantum yields up to 0.78) are of considerable interest for the development of tailor-made emitters in OLED applications and fluorescence labeling of biomolecules, surfaces or mesoporous materials.
Pyrimidines
As already indicated, alkynones 3 can be reacted without isolation with difunctional nucleophiles to furnish heterocycles.Amidines are bifunctional nucleophiles containing a three-atom building block and lead to the formation of six-membered heterocycles.Therefore, a consecutive threecomponent synthesis of 2-Amino pyrimidines like 11a are readily formed by condensation with guanidine as binucleophile.Interestingly, this one-pot reaction can also be applied to furnish complex ligand type pyrimidines such 11e.
An alternative catalytic three-component access to alkynones 3 can be conceived by carbonylative alkynylation of aryl iodides 12, terminal alkynes 2 and carbon monoxide. 21Upon subsequent addition of an amidinium salt 10 highly substituted pyrimidines 11 can be obtained in the sense of a four-component reaction (Scheme 7).Additionally, this approach, however, as a two step carbonylative alkynylationcyclocondensation sequence, is applicable to concise syntheses of naturally occurring and highly biologically active meridianins 11h and 11i, and variolin analogues. 22
1,5-Benzodiazepines
The expansion to seven-membered heterocycles is accomplished by reaction of ortho-phenylene diamines 13 with in situ generated alkynones 3. The corresponding products of this couplingaddition-cyclocondensation sequence are pharmacologically interesting 1,5-benzodiazepines 14 (Scheme 8). 23 1.05 equiv NEt 3, THF, 1 h, r.t.In addition, all representatives are highly fluorescent in the solid state, however, essentially nonfluorescent in solution at room temperature.Upon cooling the solutions cryo-fluorescence is observed, which can be attributed to a freezing of the ring flip and aggregation.This thermoresponsive behavior of fluorophores as a consequence of restricted conformational changes opens new avenues for the development of tailor-made emitters in thermosensors and the fluorescence labeling of biomolecules, surfaces or mesoporous materials.
3-Halo furans and trisubstituted furans
A major consequence of the application of only one stoichiometrically necessary equivalent of triethylamine in the alkynone synthesis, is the essentially base free reaction medium.This peculiar circumstance has now paved the way to subsequent steps under Lewis or Brønsted acidic conditions, yet in a one-pot fashion.Therefore, in the sense of a sequence of Sonogashira coupling of acid chlorides 1 and THP-protected propargyl alcohols 15 and acid-mediated Michael addition to the alkynone intermediate 3 with concomitant deprotection and cyclocondensation 3-halo furans 16 are obtained in moderate to good yields (Scheme 9). 24RKAT USA, Inc.This reaction is an example for a hydrohalogenation to a Michael system.Likewise, iodine monochloride as an electrophilic iodine source on its own right, opens a straightforward access to 3-chloro-4-iodo furans.24b It is noteworthy to mention that the 3-iodo furans 16 (Hal = I) can be coupled with boronic acids in a Sonogashira-addition-cyclocondensation-Suzuki sequence in a one-pot fashion, since the palladium catalyst system is still active after the acid-mediated cyclocondensation steps.24a
Oxazoles
Propargyl amine 17 is readily amidated with acid chlorides 1 under mild reaction conditions to furnish amide protected propargyl amines.Without isolation these propargylamides are reacted with acid chlorides 1', and via alkynone intermediates 3 a proton catalyzed cycloisomerization gives rise to the formation of functionalized oxazoles 18 in good yields in the sense of an amidation-coupling-cycloisomerization sequence (Scheme 10).As already discussed for the pyrimidine syntheses, this process can also be conducted in the sense of a four-component amidation-carbonylative alkynylation-cycloisomerization (ACACI) sequence.Studies addressing 1-substituted propargyl amines as substrates for the synthesis of more complex oxazoles are currently under investigation.
Tetrahydro-β-carbolines
In agreement with the fundamental principles of multi-component reactions, products of consecutive transformations are expected to contain substantial fragments of all starting materials, thus providing a high degree of atom-efficiency.Hence, β-enaminones in heterocyclic synthesis should be considered to be more than just synthetic equivalents of 1,3-dicarbonyl compounds.This aspect can be easily envisioned if one takes advantage of the unique electronically amphoteric reactivity of β-enaminones trying to conserve all atoms in the final product, including the enamino nitrogen atom.
In particular, the consecutive four-component reaction of acid chlorides 1, alkynes 2, tryptamine derivatives 19 and α,β-unsaturated acid chlorides 20 in the one-pot synthesis of tetrahydro-β-carbolines 21 most clearly demonstrates the potential of this concept and methodology for the rapid construction of highly-substituted, complex heterocycles where 5 new σ-bonds and 4 new stereocentres can be installed in a sequence of consecutive one-pot transformations (Scheme 11). 26The final key step of this sequence is an aza-annulation reaction that presumably generates an acyliminium ion which concludes the sequence by a Pictet-Spengler cyclization.
Conclusion and Outlook
Transition metal catalysis has considerably fertilized the development of diversity-oriented synthesis of heterocycles, namely by disclosing new transition metal catalyzed multi-component reactions.Besides purely insertion based domino processes, sequential and consecutive one-pot reactions have significantly expanded the playground for reaction design.Conceptually, many applications such as in natural product synthesis, in medicinal chemistry, for the design of functional fluorescent and redox active molecular materials, or in ligand syntheses for catalysis and coordination chemistry can be tackled by transition metal catalyzed multi-component reactions of heterocycles.Still many other transition metal complexes, that are known to catalyze uni-and bimolecular transformations, are waiting to be discovered for inventing new sequences.Undoubtedly, the future holds surprising processes in store. | v3-fos-license |
2018-04-03T00:11:04.320Z | 2017-05-31T00:00:00.000 | 10550110 | {
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} | pes2o/s2orc | Potent anti-cancer effects of less polar Curcumin analogues on gastric adenocarcinoma and esophageal squamous cell carcinoma cells
Curcumin and its chalcone derivatives inhibit the growth of human cancer cells. It is reported that replacement of two OH groups in curcumin with less polar groups like methoxy increases its anti-proliferative activity. In this study, we explored benzylidine cyclohexanone derivatives with non-polar groups, to see if they possess increased anti-cancer activity. Novel 2,6-bis benzylidine cyclohexanone analogues of curcumin were synthesized, and their inhibitory effects on gastric adenocarcinoma (AGS) and esophageal squamous cell carcinoma (KYSE30) cancer cells were studied using an MTT assay. Cell apoptosis was detected by EB/AO staining, and cell cycle was analyzed by flow cytometry. Real-time PCR was performed for gene expression analysis. All synthesized analogues were cytotoxic toward gastric and esophageal cancer cells and showed lower IC50 values than curcumin. Treatment with 2,6-Bis-(3-methoxy-4-propoxy-benzylidene)-cyclohexanone (BM2) was 17 times more toxic than curcumin after 48 h incubation. All novel compounds were more effective than curcumin in apoptosis induction and cell cycle arrest at G1 phase. These results suggest that less polar analogues of curcumin have potent cytotoxicity in vitro. However, they need to be investigated further, especially with animal tumor models, to confirm their chemotherapeutic activity in vivo.
reported that chalcone and bis-chalcone derivatives have inhibited the growth of the human breast and colon cancer cell lines 6 .
In this study, our purpose was to explore efficacy of benzylidine cyclohexanone derivatives with methoxy, ethoxy, alkoxy, and propoxy groups, to see if they possess increased anti-cancer activity and to explore the mechanism of action of these analogues. In order to accomplish this, we developed a new series of 2,6-bis benzylidine cyclohexanone derivatives that indicate increased activity against gastric and esophageal cancer cells in vitro, compared to treatment with unmodified curcumin.
In order to extract benzaldehydes from DMF, 100 mL water and 50 mL ethyl acetate were added to mixtures and organic phases were isolated. Then compounds were dried on sodium sulfate, and finally ethyl acetate was evaporated.
2,6-bis benzylidene cyclohexanones (4a -4e) were prepared by reacting 2 equivalents of aromatic aldehydes with 1 equivalent of cyclohexanone in the presence of ethanol and hydrochloric acid gas. Then the compounds were washed with cold ethanol and verified by TLC (Fig. 2). Finally, products were characterized and analyzed by 13 C-NMR, 1 H-NMR and FT-IR.
Details of generated 2,6-bis benzylidene cyclohexanones including formula, structure, molecular weight, melting point, color and yield are presented in Table 1.
Cell Culture and Treatment. The human gastric adenocarcinoma (AGS) and esophageal squamous cell carcinoma (KYSE30) cell lines were provided by the Pasteur Institute of Iran. All reagents, chemicals and media were prepared and used freshly.
Cancer cells were grown in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), penicillin (100 unit/ml) and streptomycin (100 μg/mL). Cells were cultured at 37 °C in a moistened atmosphere of 5% CO 2 and 95% air. Then, cells were trypsinized and plated in 96-well plates at a density of 1 × 10 4 cells per well in 150 μl medium, and incubated overnight. Next, cells were treated with a FBS-free medium containing 1 mg/ml of each compound by 1/4 serial dilutions. Then, plates were incubated for 24, 48, and 72 h. The cytotoxicity of curcumin derivatives was determined by an MTT assay.
MTT Assay. Culture media were removed 4 h before completion of the incubation time, then 200 μl of 0.25 mg/ml MTT (Merck, Germany) was added to each well. Plates were incubated again for an additional 4 h in order to complete the incubation time. The supernatants were removed and 200 μl DMSO was added to the wells, and the plates were shaken for 10 min. The absorbance was measured at 540 nm by a plate reader (Synergy HT, BioTek).
Apoptotic Cell detection by EB/AO Staining. Cells were cultured in 96-well plates at a confluence of 1 × 10 3 cells/well, incubated overnight and then treated with compounds in their 24 h specific IC 50 doses. Then plates were centrifuged for 5 min (129 g, 1,000 rpm) at 4 °C. The ethidium bromide/acridine orange (EB/AO) dye mix (100 μg/mL ethidium bromide and 100 μg/mL acridine orange) was dissolved in PBS and 20 μl of the dye mix was added to wells. Cells were counted by an inverted fluorescence microscope (IX 71, OLYMPUS). Live cells were determined by normal green color, which resulted from up-taking acridine orange (green fluorescence) and repelling ethidium bromide (red fluorescence).
Apoptotic cells display apoptotic bodies and perinuclear condensation of chromatin stained by ethidium bromide, while live cells were identified as normal nuclear chromatin stained by acridine orange. Necrotic cells were detected by uniform ethidium bromide staining of the cells 7 . Images were captured with a digital camera (DP 71, OLYMPUS) equipped microscope. Experiments were done in triplicate, quantifying a minimum of 100 cells each time.
Flow Cytometric analysis of the Cell Cycle. Cells were cultured in 6-well plates at a confluence of 1 × 10 6 cells/well. Cells were treated with compounds for 24 h with respective IC 50 values. Then, cell cycle phases and DNA content were analyzed by flow cytometry. Briefly, cells were collected and fixed with ice ethanol 70% for 2 h. Fixed cells were centrifuged (300 g, 4 °C, 5 min) and washed with cold PBS, and then stained with diamidino-2-phenylindole (DAPI, 10 μg/mL, Triton X-100 0.1% v/v in PBS). Then, cells were filtered by a nylon mesh with a pore size of 30 μm. Cell cycle analysis was done using a flow cytometer (Partec CyFlow space, Germany). The distribution of cells in different cell cycle phases was assessed by Partec FloMax software. mRNA expression analysis by qPCR. Cells were cultured in 6-well plates at a confluence of 2 × 10 5 cells/ well and kept at 37 °C in a moistened air of 5% CO2 overnight. Then, cells were treated for 48 h with respective IC 50 values.
For RNA extraction from cells, Trizol reagent (Cat. No: 15596-026, Invitrogen, CA, USA) was used according to the manufacturer's protocol. First-strand cDNA was generated from the cells' extracted RNA by the RevertAid First Strand cDNA Synthesis Kit, Fermentas (Cat No: #K1621, Maryland, USA) according to manufacturer's directions.
Relative quantities of target mRNA in test samples were measured and standardized to the housekeeping gene, RPL38 mRNA transcript level. The comparative Ct method was used to assess expression as previously described by Livak
Results
In order to assess the effect of synthesized compounds on cell proliferation, an MTT assay was conducted to test the inhibitory effect in three time points. After 24 h, all generated analogues were cytotoxic toward gastric and esophageal cancer cells and showed lower IC 50 values than curcumin. As shown in Table 3 and Fig. 3, BM2 was 4.6 times more toxic than curcumin toward gastric cancer cells. Similarly, esophageal cancer cells were more susceptible to BM2 and other synthesized compounds than curcumin (Supplementary Table S1). We observed the same pattern after 48 h, with BM2 17 times more toxic than curcumin (Table 3, and Fig. 4). Similarly, 72 h post treatment, all compounds were more effective than curcumin. Three curcumin analogues revealed IC 50 with Nanogram/mL values (Table 3, and Fig. 5). Moreover, MTT assay on KYSE-30 cells confirmed our data and showed that synthesized compounds have cytotoxicity on esophageal cancer cells as well (Supplementary Table S1 and Fig. S1). These data revealed that all synthesized analogues showed IC 50 much less than curcumin in three time points. In order to elucidate the mode of cell death, cells were stained with EB/AO, and apoptotic, necrotic, and live cells were counted. Synthetic compounds changed the morphology of treated cells to characteristic apoptotic cells. Nuclei of treated cells condensed and revealed fragmented chromatin and apoptotic bodies. As presented in Fig. 6B, treatment of AGS cells with synthesized BM2 triggered apoptotic cell death, which is characterized by fragmentation of nuclei. Quantification of treated and control cells revealed that synthesized analogues increased the number of apoptotic cells significantly compared to control cells (Fig. 6C and Fig. S2).
In order to verify that compounds trigger the apoptosis pathway, mRNA expression levels of important apoptotic factors were analyzed. Treatment with synthesized compounds elevated BAX and caspase-3 mRNA levels, and down-regulated expression of cyclin-D1, VEGFA, Bcl-2, c-myc, and Survivin ( Fig. 7 and Fig. S3).
In order to further confirm anti-proliferative effects, cell cycle distribution of the treated cells was evaluated. As displayed in Fig. 6, synthesized compounds were more potent than curcumin at arresting the cell cycle at G1 phase. BM2 and BM3 were most effective at increasing the cell population at G1 phase. Simultaneously, analogues decreased the number of cells at S phase ( Fig. 8 and Fig. S4). Table 3. IC50 values of synthetized 2,6-Bis Benzylidine cyclohexanone analogues in AGS cells that analyzed by MTT assay after 24 h, 48 h, and 72 h. Values are in µg/mL.
Discussion
To date over 100 different clinical trials have been completed with curcumin which clearly shows its safety, tolerability, and effect against numerous chronic diseases in humans 3 . Curcumin, a polyphenolic natural product, shows therapeutic function against a variety of diseases. These activities are attributed mainly to its chemical structure and unique physical, chemical, and biological properties. It is a di-feruloyl methane molecule [1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)] containing two ferulic acid residues linked by a methylene connection. It has three key functional groups: an aromatic o-methoxy phenolic group, α, β-unsaturated β-diketo moiety and a seven carbon linker 9 . In this study we designed different analogues of curcumin with non-polar and hydrophobic groups (methoxy, propoxy, ethoxy, and allyloxy), in order to test the effect of less polar synthesized analogues on cancer cell proliferation and compare them with the anti-cancer effect of curcumin. Recently, it has been reported that replacement of two OH groups in curcumin with less polar groups like methoxy (OCH 3 ) increases the anti-proliferative activity of arene-ruthenium(II) curcuminoid complexes in tumor cells. This report states that improved anti-cancer function is associated with apoptotic activity 10 . These findings are in accordance with our results: by decreasing curcumin polarity, its in vitro anti-tumor activity increases significantly. Our various experimental results on esophageal and gastric cancer cells revealed that all synthesized analogues of curcumin are more toxic than curcumin: after 48 h treatment in gastric cancer cells, BM2 was 17 times more toxic than curcumin (Fig. 4).
Very recently it is reported that curcumin triggered cell cycle arrest at G1 phase, and reduced the cell population in S phase in p53-mutated human colon adenocarcinoma cells 11 . These finding are in agreement with our data, which demonstrate that synthesized analogues increase cell population at G1 phase and decrease cell population at S phase (Fig. 8).
The same study reports that curcumin induced apoptosis in p53-mutated COLO 320DM colon adenocarcinoma cells 11 . Our novel synthesized compounds were more effective than curcumin at triggering apoptosis (Figs 6 and 7 and Supplementary Figs S2-S4).
It has been reported that curcumin induced apoptosis in leukemia cells by PARP-1 cleavage, increased level of caspase-3, apoptosis inducing factor (AIF) and down-regulation of Bcl2 12 . Another study suggested that dendrosomal curcumin (DNC) significantly increased cell population in SubG1, induced apoptosis and up-regulated p21, BAX, and Noxa in hepatocarcinoma cell lines. While the expression of Bcl-2 decreased 13 . Moreover, it has been reported that a curcumin analogue ((1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-dione) down-regulated cyclin D1 and activated Caspase 3, 8 and 9 in lung adenocarcinoma (A549), leukemia (K562) and colon cancer (SW480) cells 14 . Another study showed that curcumin suppressed VEGF secretion from tumor cells both in vitro and in vivo, and subsequently could block VEGF-VEGFR2 signaling pathways 15 . It has been evidenced that curcumin combination with resveratrol synergistically induced apoptosis in cigarette smoke condensate transformed breast epithelial cells by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3. Whereas, this combination decreased c-myc and cyclin-D1 16 . Nevertheless, it has been demonstrated that dimethoxy curcumin (DMC) as a non-polar and lipophilic analogue of curcumin down-regulating survivin and upregulating E-cadherin in colon cancer cells which significantly suppressed the growth and migration of cells 17 .
Cyclin D1 is one of the G1 phase related regulatory factors 18 ; its down-regulation in our results verifies potential effect of novel analogues on cell cycle arrest at G1 phase (Fig. 8).
Taken together, novel synthetic 2,6-bis benzylidine cyclohexanone analogues were more efficient than curcumin to inhibit cancer cell proliferation, trigger apoptosis, and arrest cell cycle at G1 phase. These data suggest that cyclohexanone analogues of curcumin could be promising anti-cancer agents to consider for more research on animal tumor models and even human clinical trials. | v3-fos-license |
2018-04-26T23:46:28.887Z | 2018-04-01T00:00:00.000 | 4811626 | {
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} | pes2o/s2orc | Scutellarin Mitigates Aβ-Induced Neurotoxicity and Improves Behavior Impairments in AD Mice
Alzheimer’s disease (AD) is pathologically characterized by excessive accumulation of amyloid-beta (Aβ) within extracellular spaces of the brain. Aggregation of Aβ has been shown to trigger oxidative stress, inflammation, and neurotoxicity resulting in cognitive dysfunction. In this study, we use models of cerebral Aβ amyloidosis to investigate anti-amyloidogenic effects of scutellarin in vitro and in vivo. Our results show that scutellarin, through binding to Aβ42, efficiently inhibits oligomerization as well as fibril formation and reduces Aβ oligomer-induced neuronal toxicity in cell line SH-SY5Y. After nine months of treatment in APP/PS1 double-transgenic mice, scutellarin significantly improves behavior, reduces soluble and insoluble Aβ levels in the brain and plasma, decreases Aβ plaque associated gliosis and levels of proinflammatory cytokines TNF-α and IL-6, attenuates neuroinflammation, displays anti-amyloidogenic effects, and highlights the beneficial effects of intervention on development or progression of AD-like neuropathology.
Introduction
Alzheimer diseases (AD) is the most common kind of neurodegeneration disease in the elderly, characterized by deterioration of cognitive functions, extracellular Aβ deposits and intracellular neurofibrillary tangles, other pathology features include the cholinergic neurons and synaptic degeneration/loss, cerebral amyloid angiopathy (CAA), neuroinflammation, and oxidative damage [1,2].
AD is so chronic and complex disease. To date, there is no current disease-modifying therapy available for the treatment of this disorder. In spite of extensive academic, pharmaceutical, and medicinal research, numerous drug candidates targeting Aβ or β-secretase have failed in clinical trials. This means drugs with single targets have less therapeutic effects or cause side effects when used to prevent or treat AD. Therefore, the new strategy towards development of safe agents with multiple targets, natural polyphenols are likely to comply with these requirements due to the advantages of multi-target effects and fewer side effects.
Flavonoids are the largest group of polyphenols available from dietary fruits and vegetables and have been found to play a neuroprotective role by inhibiting or modifying the self-assembly of the amyloid-β (Aβ) peptide into oligomers and fibrils, which are linked to the pathogenesis of Alzheimer's disease [3]. Scutellarin is a flavonoid found in the traditional Chinese herbal medicine Erigeron breviscapus (vant.), and has been widely used in clinical to treat cardiovascular diseases and cerebrovascular injury [4]). Recently, scutellarin has been proven to: promote neuroprotective effects by inhibition of microglia inflammatory activation [5], attenuate the neurotoxicity of Aβ and protects against Aβ-induced learning and memory deficits in rats [6], exert potential neuroprotective effects on AD. However, up until now the long term multiple antiamyloidogenic and fibril-destabilizing effects have not been studied. In this research, we report that scutellarin efficiently inhibits oligomerization and fibril formation through binding to Aβ42. In the meantime, scutellarin mitigates amyloid pathology and related cognitive deficits after nine months of treatment in APP/PS1 transgenic mice. This research reveals scutellarin is a potential multi-targeting agent against AD.
Aβ Preparation
Synthetic Aβ42 peptide corresponding to the human sequence was purchased from Tocris Bioscience, which was dissolved in HFIP at a concentration of 1 mg/mL and was separated into aliquots in sterile Eppendorf Tube (100 µg/tube), followed by incubation at room temperature for 24 h in the fume hood to form clear peptide film, the resulting peptide films were dried under vacuum overnight and stored at −20 • C before assaying [7].
Thioflavin T Fluorescence Assay
Thioflavin T (Th. T) dye fluorescence is used regularly to quantify the formation and inhibition of amyloid fibrils in the presence of anti-amyloidogenic compounds such as polyphenols [8].
To investigate the disaggregation effect of scutellarin on preformed Aβ fibril, 100 µg Aβ42 oligomer was pre-incubated in DMEM at 37 • C for 10 days to form Aβ fibril. Following incubation, Aβ alone, or in the presence of the concentration gradient of scutellarin (purity > 98.5%, Yunnan Plant Pharmaceutical, Yunnan, China, Figure 1) for an additional three days at 37 • C, 5 µM Thioflavin T solution was added to the samples. Emission spectra were recorded at 482 nm upon excitation at 450 nm (SpectraMax M2, Molecular Devices). Measurement was repeated three times.
Electron Microscopy (EM) Assay
In brief, copper grids were preplaced on the bottom of wells in a 24-well plate where 100 µg Aβ42 monomer were incubated with or without scutellarin at a concentration gradient for seven days at 37 • C. Following, a drop of the solution was placed on a 150 mesh Formvar coated grid for 30 s. After drying, the grid was stained with 2% (wt/vol) aqueous phosphotungstic acid for 20 min.
The observations were performed on a Hitachi7650 TEM equipped with MegaView 3 Digital Camera (Hitachi, Tokyo, Japan).
SH-SY5Y Cell Culture and Viability Assay
The SH-SY5Y cell line was obtained from BeNa Culture Collection (Shanghai, China). Cells were plated in 96-well plates containing complete medium and cultured for 24 h at 37 • C and then, the cells were treated with 1 µM Aβ42, Scutellarin (2.5, 5, or 10 µM) or 1 µM Aβ42 with Scutellarin (2.5, 5, or 10 µM) for another 24 h, followed by incubation with MTT (0.5 mg/mL) for 4 h and 10% SDS solution for another 15 min at 37 • C. The absorbance was measured at 560 nm with a microplate reader (Spectra Max M2, Molecular Devices, San Jose, CA, USA).
Animals
APP/PS1 transgenic mice and age-matching non-transgenic (WT) mice were purchased from Nanjing Biomedical Research Institute of Nanjing University and were bred in the Kunming Medical University animal house. Five or six mice were housed in each cage with free access to standard food and water and were maintained under standard laboratory conditions. The APP/PS1 transgenic mice were constructed on a C57BL/6 background and bear a chimeric mouse/human (Mo/Hu) APP695 with mutations linked to familial AD (KM 593/594 NL) and human PS1 carrying the exon-9-deleted variant associated with familial AD (PS1dE9) in one locus under control of a brain-and neuron-specific murine Thy-1 promoter element [9]. Genotypes of the offspring were determined by PCR analysis of tail DNA.
Diet Treatment
A total of 24 transgenic mice and 12 wild type littermates (C57BL/6 mice, half male and half female) were used: the transgenic mice were randomly divided into Scutellarin Tg group (N = 12, half males and half females), control Tg group (N = 12, half males and half females) and wild-type mice group (N = 12, half males and half females). The scutellarin (Tg) group were treated with scutellarin mixed food (50 mg/kg) [10]. Control (Tg) and wild-type mice group were fed with standard commercial food (Beijing KeaoXieli Feed Company, Beijing, China). Beginning at three months of age, the mice consumed both diets ad libitum for nine months. This study was carried out in strict compliance with the Guidelines for the Animal Care and Use of China. The protocols were approved by the Animal Ethics Committee of Kunming Medical University.
Behavioral Procedures
The effect of scutellarin treatment on spatial learning and memory was assessed by Morris water maze (MWM) testing at the age of 12 months, a circular plastic pool (height 40 cm, diameter 120 cm) was filled with water (plus white dye) maintained at 22 ± 1 • C. An escape platform (11 cm in diameter) 1 cm below the water surface was used. The acquisition task consisted of six consecutive days of testing with four trials per day. In each trial, the animal was put into the water at one of four starting positions of non-platform quadrants respectively, and was given 60 s to find the hidden platform. If a mouse failed to find the platform within 60 s, the training was terminated, a maximum score of 60 s was assigned, and the mouse was manually guided to the hidden platform. The escape latency, path length and swim speed were recorded semi-automatically by a video tracking system (Stoelting Co., Wood Dale, IL, USA) and analyzed by image analyzing software. On the sixth day, a probe trial was performed, and mice were placed and released opposite the site where the platform had been located, the time spent in each quadrant and target annulus crossings were recorded [11].
Tissue Processing
The mice were executed after behavioral test, the mice were deeply anesthetized, the blood was collected from the retro orbital sinus and was centrifuged immediately at 1300× g for 8 min, the supernatant from these samples was collected for future biochemical detection. The brain was perfused intracardially with chilled 0.1 M phosphate buffered saline (PBS) through the heart using a syringe and was quickly removed and placed on an ice-cold glass dish and bisected sagittally. The left-hemisphere were routinely fixed in 4% paraformaldehyde (pH 7.4) for 24 h and incubated in 30% sucrose at 4 • C for 36 h, the coronal sections were cut on a frozen microtome in 35 µm thick sections for staining. The right hemisphere was rapidly frozen in liquid nitrogen and kept at −80 • C until analysis.
Histological Staining and Imagine Analysis
The basic immunohistochemical staining was carried out following IHC kit instruction (Slide Kit Chemical international, Inc., Millipore, Burlington, MA, USA). Five sections each 210 µm apart starting from septal hippocampus were randomly selected and stained with Aβ protein deposits (Biotin-conjugated mouse anti-Aβ antibody (6E10, Serotec, Oxfordshire, UK), activated microglia (rat monoclonal anti-CD45 Chemicon, Temecula, CA, USA), and astrocyte (rabbit poly-clonal anti-glial fibrillary acidic protein, Dako, Glostrup, Denmark)). Pretreatments were: incubation in a 3% H 2 O 2 solution in distilled water for 20 min to block endogenous peroxidase; incubation in 70% formic acid for 15 min for antigen retrieval, sections were incubated with the primary antibodies overnight at +4 • C, followed by incubation with biotinylated secondary antibodies and visualization using the diaminobenzidine as chromogen. The reaction was stopped with phosphate buffer and sections were then mounted, cleared in xylene, and cover slipped with neutralized resin.
The region of neocortex and hippocampus manually was selected for analysis Aβ, microgliosis and astrocytosis burden. Stained specimens were analyzed with Olympus BX-51 microscope equipped with an Olympus DP-73 camera (Olympus, Tokyo, Japan). Images were collected at 4× magnification. Measurements were performed for a percentage of the area covered by the DAB staining.
ELISA Assay for Soluble and Insoluble Aβ
The levels of soluble and insoluble Aβ in the brain of APP/PS1 mice were quantified according to the procedures previously described [12]. Frozen brain was homogenized and sonicated in water containing 2% sodium dodecyl sulphate (SDS) and protease inhibitors (Roche, Basel, Switzerland). Homogenates were centrifuged at 100,000× g for 1 h at 4 • C, the resultant supernatant was collected, representing the TBS-soluble fraction (Aβ-TBS). The resultant pellet was suspended and sonicated in water containing 2% sodium dodecyl sulfate (SDS) and centrifuged at 100,000× g for 1 h at 4 • C, the resultant supernatant was collected, representing the SDS-soluble fraction (Aβ-SDS). The resultant pellet was extracted with 70% formic acid (FA) and the supernatant was collected, representing the SDS-insoluble fraction (Aβ-FA). Before ELISA assay, formic acid extracts were neutralized by 1:20 dilution into 1 M Trisphosphate buffer, pH 11, and then diluted in sample buffer. Then, ELISA kits were used to determine Aβ40 and Aβ42 levels in the brain and serum samples (Millipore, Burlington, MA, USA). The concentration of Aβ was expressed as picograms per milliliter (pg/mL). The concentration values of the samples fell within the linear section of the standard curve.
Quantification of Inflammatory Cytokines in the Mouse Plasma by ELISA
Plasma cytokine levels were measured using commercial assay kits according to the manufacturer's directions (Thermo Scientific, Waltham, MA, USA). The concentration of inflammatory cytokines was presented as pg/mL.
Statistical Analysis
Results for experimental groups are presented as the mean ± SEM. In cases of equal variance, statistical differences were determined using one-way analysis of variance (ANOVA) followed by post-hoc (Tukey's) tests for comparisons between groups. If homogeneity of variances was rejected, the ANOVA followed with the Dunnett's test. The threshold for statistical significance was set to p < 0.05. All statistical analyses were performed using the SPSS 20.0 software (IBM, Chicago, IL, USA)
Protective Effect of Scutellarin on the Cytotoxicity of Aβ42
Some research conclusions indicate that scutellarin is an effective compound for the prevention of AD-like neuropathology. Here, we used MTT assay to test if scutellarin can reduce cytotoxicity induced by Aβ42 in human neuroblastoma cell line (SH-SY5Y). Our results indicated the Aβ-neurotoxicity in the presence of scutellarin tended to be lower than Aβ in the absence of the drug, scutellarin at 10 uM exhibited significant protective effect against Aβ-induced cytotoxicity (p < 0.05, Figure 2). Furthermore, cell viability did not decrease after exposure to different concentrations Scutellarin (2.5, 5, 10 uM), which suggests a good safety profile (Figure 2A).
Effects of the Scutellarin On Aβ42 Oligomerization and Fibrillation
Thioflavin T (Th. T) dye fluorescence is used regularly to quantify the formation and inhibition of amyloid fibrils in the presence of anti-amyloidogenic compounds such as polyphenols [8]. Here, we used this dyeing method to detect whether scutellarin promotes the disaggregation of preformed Aβ fibrils. We have found that when scutellarin was incubated with preformed Aβ42 fibrils, the fluorescence intensity of preformed Aβ42 fibrils was dose-dependently reduced (F = 157.16, p < 0.05, Figure 3E). Moreover, transmission electron microscopy (TEM) assays visually confirmed that scutellarin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of Aβ, resulting in a reduced neurotoxicity induced by Aβ ( Figure 3A-D).
Scutellarin Improves Behavioral Impairment after Nine Months of Treatment
Morris water maze is a widely used tool to assess the spatial learning and memory capacities. After nine months of treatment, we evaluated the preventive effects of scutellarin on cognitive deficits in APP/PS1 mice. The results of the behavioral performance are shown in Figure 4. Overall, a time-dependent acquisition of platform location was observed in all groups, as evident in the reduced latency to find the platform with each successive training day in all groups (ANOVA F = 7.518, p < 0.05; Figure 4A). In addition, a significant effect of the treatment group was observed (p < 0.05). The scutellarin-treated APP/PS1 mice showed significantly shorter latencies than Tg control mice on day 2 (p < 0.05) and day 5 of training (p < 0.05, Figure 4B). Treatment with scutellarin at 50 mg/kg significantly reduced the latency to find platform (ANOVA F = 6.860, p < 0.05, Figure 4D). The effect could not be attributed to a change in swim speed because all APP/PS1 mice swam slower than the wt control group during the probe trial regardless of treatment (Dunnett's post hoc APP/PS1 vs. wt: p < 0.05; Figure 4C)
Scutellarin Reduces Aβ Burden in APP/PS1 Mice
In order to study the histopathological changes after the treatment, the Aβ plaques in the brain were stained with immunohistochemical (IHC) staining (biotin conjugated mouse anti-Aβ antibody 6E10, Serotec, USA). The statistical results show APP/PS1 mice treated with scutellarin significantly decreased 63% Aβ deposits in the brain compared to the Tg Control group (ANOVA F = 12.105, p < 0.001) ( Figure 5). Following, the levels of soluble and insoluble Aβ1-40 and Aβ1-42 in the brain homogeneates were measured by ELISA kit. Figure 6B, C show scutellarin treatment group separately reduced soluble Aβ42 and insoluble Aβ42 66% and 56% (p < 0.05), reduced the levels of soluble Aβ40 37% and insoluble Aβ40 36% when comparing with untreated Tg mice (p < 0.05). ELISA assay also showed that the scutellarin treatment group had significantly decreased levels of total Aβ 52.2% and 53% in the brain homogenates and in the serum separately (p < 0.05, Figure 6A,D). These results indicate that scutellarin is effective at reducing both cerebral parenchymal deposition of the Aβ peptide levels in the brain and the plasma, possesses the ability clearance of Aβ from brain to periphery.
Scutellarin Decreases Reactive Gliosis and Proinflammatory Cytokines in APP/PS1mice
Activated astrocytes and microglia facilitate Aβ clearance, but also mediate inflammation via cytokine production proinflammatory cytokines and immunostimulatory molecules [13]. Immunohistochemical staining for the microgliasis and astrogliosis in neocortical and hippocampal regions revealed that scutellarin treated group (Tg) had a significant lower level of microgliosis 76% and astrocytesis 66% compared to control Tg group (Figures 7 and 8). In addition, plasma levels of proinflammatory cytokines TNF-α and IL-6 were significantly decreased 42% and 64% separately when compared with control Tg group mice, but concentrations of IL-1β and IFN-γ from plasma of scutellarin treated Tg mice did not show significant differences compared to the control Tg group (ANOVA, IL-β, F = 1.407, p > 0.05, IFN-γ, F = 0.297, p > 0.05, Figure 9).
Discussion
Aβ is believed to play a critical role in the AD pathology process. Aβ peptides subsequently aggregate from monomers to oligomers, protofibrils, and fibrils, inducing cognitive deficits and a series of the deleterious cascades that exacerbate neuronal injury [14], which consequently means they have been considered as a primary toxic species in AD [15]. Therefore, developing multi-target drugs to antagonize the aggregation of various amyloid proteins and interfere with the pathological process becomes an attractive therapeutic strategy.
Here, we used a series of techniques to investigate anti-amyloidogenic effects of scutellarin on AD pathology. In vitro, we determined the effects of scutellarin on the destabilization of Aβ fibrils by thioflavin T fluorescence spectroscopy and electron microscopy. Our results present that scutellarin dependently destabilized preformed Aβ; altered the Aβ aggregation pathway to yield non-toxic, unstructured Aβ aggregates; and displayed anti-aggregation effects on Aβ. In the cell culture experiment, cell growth was remarkably inhibited by Aβ oligomers treatment. However, scutellarin reduced the Aβ-induced cytotoxicity and exerted a neuroprotective effect in the SH-SY5Y cell culture model.
Through an in vivo study, the neuroprotective effects of a scutellarin diet have been tested in APP/PS1 transgenic mouse model. Our data showed that scutellarin significantly improved behavioral impairment, decreased Aβ levels in the brain and the serum, exhibited strong cleaning capacity in the Tg mice brain and the periphery, and did not cause side effects.
Brain inflammation is one of the hallmarks of AD and originates in the central nervous system. Microglia and astrocytes are arguably the major sources of cytokines in AD. Aβ complexes interact with microglial and astrocytic expressed pattern recognition receptors that initiate innate immunity [16]. This process leads to the production of toxic and inflammatory mediators such as hydrogen peroxide, nitric oxide, and cytokines-including interleukin (IL)-1β, IL-6, TNF-a, and IFN-γ etc.-that can recruit further microglia and astrocytes to the inflammatory site [17]. The passage of cytokines through the blood-brain barrier allows for the ability to peripherally measure them. In the present study, we found scutellarin reduced neuroinflammation including Aβ plaque-associated microgliosis and activated astrocytes, cytokines levels of TNF-α and IL-6, and exerted anti-inflammatory properties through downregulation of pro-inflammatory cytokine expression, thereby contributing to improved cognition and decreased Aβ concentrations.
Obviously, these beneficial effects can be attributed to its structure of phenol, which enables it to penetrate the blood-brain barrier and helps to re-establish the redox regulation of proteins, transcription factors, and signaling cascade; successfully protects neuronal cells from oxidative damage; and potentially alleviates neuroinflammation of AD [18,19]. In addition, the interaction of scutellarin with Aβ induces conformational and structural changes in Aβ oligomers, disrupts hydrophobic and π-π interactions of Aβ peptides and prevents Aβ aggregation and toxicity. In turn, the Aβ oligomer-scutellarin interaction results in delayed fibril formation, reduces amyloid plaque load, and attenuates behavior deficits in APP/PS1 mice. A published paper has discussed the molecular mechanism of scutellarin prevention of AD pathology [20]. Yuan et al. suggest that scutellarin regulates the activation of microglia via the Notch pathway and concurrently induces morphological and functional changes in activated microglia [21]. Xu et al. propose that scutellarin can effectively upregulate the synthesis and release of NGF, glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) [22]. Chai et al. put forward that scutellarin can induce the expression of neurotrophin messenger RNAs and proteins through cyclic adenosine monophosphate response, element-binding protein (P-CREB), and p-Akt signaling and inhibit NO production in early stages of neuronal damage [23]. These conclusions indicate that scutellarin can slow down the progression of AD-like neuropathology via anti-inflammation, anti-oxidation, anti-amyloidogenic properties, consistent with ferulic acid and fisetin which have shown effects on improving behavioral impairment and Alzheimer-like pathology in transgenic mice [24,25]. Although scutellarin exerts multiple beneficial functions in mouse models of AD, the key question remains unanswered: can scutellarin's beneficial actions on animal models be translated to the human condition? In addition, due to its poor solubility and weak oral absorption, the clinical use of scutellarin is limited [26]. In further research, we need to identify bioactive metabolites and dissect their targets in AD modification.
Conclusions
In conclusion, our studies have proven that scutellarin alleviates Alzheimer's-like pathology and cognitive decline by reducing Aβ levels in the brain and plasma, decreasing Aβ plaque associated gliosis and levels of proinflammatory cytokines TNF-α and IL-6, and attenuating neuroinflammation. Our results suggest that scutellarin has potential for use as a therapeutic candidate for AD. | v3-fos-license |
2019-04-09T13:09:37.530Z | 2015-06-11T00:00:00.000 | 102547771 | {
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} | pes2o/s2orc | THE EFFECT OF SUBSTITUENTS IN THE MOLECULES OF N-, R-ALKYL AMINES ON SOME GRAM-POSITIVE STRAINS OF MICROORGANISMS
The intensive use of antibiotics in patient care institutions often without determining specificity and sensitivity to them leads to more rapid development of resistance to pathogens of nosocomial infections; that is why it is one of the urgent problems of healthcare of Ukraine. The most common gram-positive pathogens of nosocomial infections are Staphylococcus aureus, coagulase-negative staphylococci and enterococci. In order to determine dependence of the microbiological action on the nature of substituents in the molecules of N-, R-alkylamines some methyl, ethylamine, aminoalcohols, N-hydroxymethyland N-methyl-N-carboxymethylamines have been tested. In accordance with the WHO recommendations to assess the antibacterial activity of N-, R-alkylamines the gram-positive test strains – Staphylococcus aureus АТСС 25923 and Baсillus subtilis АТСС 6633 were used. The aliphatic amines and aminoalcohols studied show a weak or moderate activity in relation to strains of Staphylococcus aureus АТСС 25923 and Bacillus subtilis АТСС 6633. Compounds containing a carboxyl group and methyl radicals in the molecule exhibit the greatest antimicrobial activity in relation to the gram-positive strains of microorganisms under research.
Antibacterial agents are practically the only group of drugs which effectiveness decreases with time due to development of resistance. The intensive use of antibiotics in patient care institutions often without determining specificity and sensitivity to them leads to more rapid development of resistance to pathogens of nosocomial infections; that is why it is one of the urgent problems of healthcare of Ukraine. The main factors that contribute to the increased disease incidence are: shortage of medicines, antiseptics, detergents and disinfectants, medical instruments, linen, sterilization equipment since medical preventive institutions are forced to work in conditions of the extremely limited funding; a significant growth in the number of hospital strains that are resistant to antibiotics and disinfectants, etc. [4].
The most common gram-positive pathogens of nosocomial infections are Staphylococcus aureus, coagulase-negative staphylococci and enterococci. The results of the multicentre randomized trial SCOPE (USA) published in 2004 indicate the predominance of gram-positive cocci in the etiological structure of nosocomial bacteriemias [14].
This tendency creates significant problems since the choice of antimicrobial agents intended to combat drugresistant gram-positive microorganisms is limited. It should be also noted the fact that antibiotics are less than 5% of the drugs being currently at the stage of drug development [5], and as resistance to the medicines used develops, there is a need in both creation of new drugs, and correction of methods for using the existing ones. Therefore, the research in development of drugs, including vaccines and diagnostic agents, are of vital importance for protection of future generations.
It is known that tertiary amine salts or quaternary ammonium bases containing radicals with a large number of carbon atoms exhibit a strong bacteriostatic and bactericidal action, as well as possess pronounced disinfectant properties [1,8]. In literature there are data that compounds with the number of carbon atoms from 5 to 16 are the most effective against microorganisms [6].
The aim of this work was to determine the effect of various functional groups containing in the molecules of N-, R-alkylamines derivatives on their antibacterial activity in relation to some gram-positive strains of microorganisms.
Materials and Methods
In order to determine dependence of the microbiological action on the nature of substituents in the molecules of N-, R-alkylamines the following groups were tested: alkylamines (compounds I-VI), aminoalcohols (compounds VII-IX), N-hydroxymethyl-N-carboxymethylamines (compounds X-XII), and N-methyl-N-carboxymethylamines (compounds XIII-XV) (see Table).
The compounds under research were obtained from commercial sources or synthesized according to the synthetic schemes previously developed [2,3,6,9,[11][12][13]. Reagents were purchased from "Sigma-Aldrich" (USA) and used without further purification. 1% Aqueous solutions of compounds I-XV were tested.
In accordance with the WHO recommendations to assess the antibacterial activity of N-, R-alkylamines the gram-positive test strains -Staphylococcus aureus АТСС 25923 and Baсillus subtilis АТСС 6633 were used. The suspension of the test microorganism was prepared according to the method [10]. Standardization of the bacterial suspension of microorganisms prepared was carried out using a Densi-La-Meter device (manufactured by PLIVA-Lachema, Czech Republic). Synchronization of cultures by changing the cultivation temperature was achieved with a single effect of low temperature (4 о С). Microbial load was 10 7 microbial cells per 1 ml of the medium and set up according to McFarland standard. We worked with 18-24 hour culture of microorganisms.
For studies Mueller-Hinton agar ("HIMedia Laboratories, Pvt. Ltd India" the shelf life of the medium to ХI 2016, manufactured by India) was used.
Diffusion of the drug into the agar was conducted by the "wells" method [7]. When assessing the activity of compounds I-XV, as well as when studying antibiotic-resistant strains the following criteria were used: • the absence of inhibition zones of microorganisms around the well, the diameter of the inhibition zone to 10 mm indicates that the organism is insensitive to the drug introduced into the well or to the antibiotic concentration; • inhibition zones with the diameter of 10-15 mm indicate a low sensitivity of the culture to the given concentration of the test substance; • inhibition zones with the diameter of 15-25 mm assessed as an indicator of the sensitivity of a microorganism to the test substance; • inhibition zones, which diameter exceeds 25 mm, indicates the high sensitivity of microorganisms to the test substance.
Results and Discussion
A low sensitivity of the set of microorganisms used to the action of aliphatic amines I-VI was determined. The antimicrobial activity of aminoalcohols VII-IX was slightly higher, and its enhancement is observed with increasing the number of hydroxyethylene radicals in the molecule. Compounds X-XII containing a carboxyl and hydroxymethyl group in the molecule was more active, with the increasing number of hydroxyethylene groups the inhibition zones were 20-21 mm for Staphylococcus aureus and 17-25 mm for Baсillus subtilis.
Compounds containing a carboxyl group and methyl radicals appeared to be the most promising among the compounds tested. It is this combination of substituents that contributes to a high microbicide activity in relation to Staphylococcus aureus and Baсillus subtilis. Increase of the number of methyl radicals in the molecules of compounds XIII-XV leads to a significant increase in activity -the inhibition zones are 39-42 mm. CONCLUSIONS 1. The effect of various functional groups containing in the molecules of N-, R-alkylamines derivatives on their antibacterial activity in relation to some grampositive strains of microorganisms has been determined.
2. The aliphatic amines and aminoalcohols studied show a weak or moderate activity in relation to strains of Staphylococcus aureus АТСС 25923 and Bacillus subtilis АТСС 6633.
3. Compounds containing a carboxyl group and methyl radicals in the molecule exhibit the greatest antimicrobial activity in relation to the gram-positive strains of microorganisms under research. | v3-fos-license |
2020-09-29T13:07:17.352Z | 2020-09-29T00:00:00.000 | 221979339 | {
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} | pes2o/s2orc | Major Factors for the Persistent Folding of Hybrid α, β, γ-Hybrid Peptides Into Hairpins
Factors responsible for the persistent adoption of hairpin conformations by hybrid oligopeptides, each having a central β/α dipeptide segment flanked by aromatic γ-amino acid (γAr) residues, are probed. Our recent studies revealed that tetrapeptide 1 and 2, having central dipeptide segments consisting of β-alanine (β-Ala) and glycine (Gly), and L-β-homophenylalanine (L-β-homoPhe) and Gly residues, respectively, that are flanked by γAr residues, fold into well-defined, expanded β-turns with doubly H-bonded γAr residues. Replacing the γAr residues of 1 and 2 with L-Val and L-Leu residues results in tetrapetides 1′ and 2′ that fail to fold into defined conformations, which confirms the decisive role played by the H-bonded γAr residues in the promoting folding of 1 and 2. Attaching L-Val and L-Leu residues to the termini of 1 affords hexapeptide 1a. With an additional H-bond between its L-Val and L-Leu residues, peptide 1a folds into a hairpin with higher stability than that of 1, indicating that the expanded β-turn can nucleate and stabilize β-hairpin with longer β-strands. Attaching L-Val and L-Leu residues to the termini of 2 affords hexapeptide 2a. Substituting the L-β-homoPhe residue of 2a with a D-β-homoPhe residue gives hexapeptide 2b. Surprisingly, hexapeptide 2a fold into a hairpin showing the similar stability as those of tetrapeptides 1 and 2. Hexapeptide 2b, with its combination of a D-β-homoPhe residue and the L-Val/L-Leu pair, fold into a hairpin that is significantly more stable than the other hybrid peptides, demonstrating that a combination of hetero-chirality between the β-amino acid residue of the dipeptide loop and the α-amino acid residues of the β-strands enhances the stability of the resultant β-hairpin.
INTRODUCTION
As a major class of protein secondary structure, reverse turns provide sites of chain reversal, which results in the globular character of a protein (Smith and Pease, 1980;Milner-White and Poet, 1987). Reverse turns include the widely occurring two-residue β-turns (Wilmot and Thornton, 1988), along with the less prevalent γ-turns (Némethy and Printz, 1972) and α-turns (Pavone et al., 1996). β-Turns and β-hairpins are frequently found in hairpin loops of globular proteins and play a key role in protein folding (Marcelino and Gierasch, 2008). The design of discrete β-hairpins relies on the availability of type II ′ β-turn of D-Pro-Gly (Haque et al., 1996;Karle et al., 1996;Haque and Gellman, 1997;Espinosa and Gellman, 2000;Syud et al., 2001;Aravinda et al., 2004) segment and type I ′ β-turns of Asn-Gly (de Alba et al., 1997;Maynard and Searle, 1997;Simpson et al., 2005) and Aib-D-Ala (Aravinda et al., 2002) segments. Hairpins and reverse turns including β-turns play crucial roles in initiating the folding of peptides and proteins (Jäger et al., 2001;Rotondi and Gierasch, 2003;Du et al., 2004;Marcelino and Gierasch, 2008), and also possess in important biological functions, for example, as epitopes in protein-protein (Ripoll, 1992;Wilson and Stanfield, 1994;DeLano et al., 2000;Tyndall et al., 2005;Shukla and Sasidhar, 2015) and protein-nucleic acid (Churchill and Suzuki, 1989;Erard et al., 1990;Maynard and Searle, 1997;Shi et al., 1998;Leon et al., 2008) interactions. Our recent studies (Zhang et al., 2019;Tang et al., 2020) led to the discovery of a series of expanded β-turns sharing a β/α loop, i.e., a central dipeptide segment consisting of a β and α amino acid residue that is flanked by doubly H-bonded aromatic γ-amino acid (γAr) residues. This expanded β-turn represents a surprisingly resilient turn motif that allows the incorporation of different α and β amino acid residues (Tang et al., 2020). It was found that introducing various α amino acid residues into the β/α dipeptide loop results in β-hairpins with the same or slightly lower stabilities, while incorporating β amino acid residues enhances the stabilities of the resultant β-hairpins. In this study, we explore the role of the γAr residues in the folding of this series of hybrid peptides. The effects of additional α-amino acid residues added to the N-and C-termini of the hybrid tetrapeptides to the stabilities of the resultant folded structures. The combinations of chirality between the β-amino acid residue in the dipeptide loop and the terminal α-amino acid residues are also examined for its influence on the folding of the corresponding hybrid peptides.
Chemistry
Reagents and solvents were purchased from commercial sources and used without further purification. Column chromatography was carried out on silica gel (300∼400 mesh). 1 H NMR spectra were recorded at 400 MHz and 600 MHz on a Bruker-400 spectrometer and JEOL-400 and 600 spectrometers at ambient temperature. 13 C NMR spectra were measured at 100 MHz and 150 MHz on the same spectrometers. Chemical shifts are reported in parts per million downfield from TMS (tetramethylsilane). Coupling constants in 1 H NMR are expressed in Hertz. Electrospray ionization high resolution mass spectra (ESI-HRMS) were acquired using a waters LCT Premier XE spectrometer (Waters, Milford, MA, USA).
Computational Methods
The models for 2a and 2b were optimized with the revPBE-D3 (Zhang and Yang, 1998;Grimme et al., 2010) functional and dispersion correction using the Amsterdam Density Functional (ADF) (Fonseca Guerra et al., 1998;te Velde et al., 2001) 1 software package. The triple-zeta with polarization functions (TZP) basis set was used while keeping the core 1s electrons fixed in the oxygen, nitrogen, and carbon atoms (van Lenthe and Baerends, 2003). The revPBE-D3 functional and dispersion correction were used due to its previous treatment of similar tetrapeptides (Zhang et al., 2019).
Synthesis
The synthesis of tetrapetides 1 and 2 has been reported by us (Zhang et al., 2019;Tang et al., 2020). Peptides 1 ′ and 2 ′ were prepared based on standard amide/peptide coupling. Figure 1 shows the general steps and conditions for synthesizing hexapeptides 1a, 2a, and 2b. Coupling I and II, which were prepared by coupling the methyl ester of D-or Lβ-homo-phenyalanine with 2-isopentyloxy-5-nitrobenzoic acid, and Boc-protected glycine with the methyl ester of 5-amino-2isopentyloxybenzoic acid, results in oligomer III. Hydrolyzing the methyl ester gives IV, which is coupled with the L-leucine derived amide to give V. Subjecting V to catalytic hydrogenation results in VI, followed by coupling with acetyl-L-valine to give peptides 1a, 2a, and 2b. The detailed synthetic steps for preparing the intermediates and final products, along with the corresponding analytical data are included in the Supplementary Materials.
The Critical Role of Aromatic γ-Amino Acid Residues
Results from our recent study indicate that hybrid tetrapeptides 1 and 2, along with eight other homologous hybrid peptides (Tang et al., 2020), persistently fold into a doubly H-bonded hairpin conformation containing an expanded β-turn that is very resilient toward incorporating different α-and β-amino acid residues into the central β/α dipeptide loop. Such a turn motif is capable of accommodating a variety of β/α dipeptide sequences that otherwise could not be introduced into a β-turn. For example (Figure 2A), glycine and β-alanine, i.e., homoglycine, two conformationally most flexible α-and β-amino acid residues, are found in tetrapeptide 1 which adopts a well-defined hairpin conformation. Replacing the β-alanine residue of 1 with other β-amino acid residues having side chains results in hybrid tetrapeptides such as 2 that adopts a hairpin conformation with enhanced stability.
Each of tetrapeptides 1 and 2, like other hybrid tetrapeptides of this series, has two aromatic γ-amino acid (γAr) residues flanking the central β/α dipeptide segment. The vital role of the γAr residues in driving the folding of 1 and 2 is further demonstrated by examining the folding of tetrapeptides 1 ′ and 2 ′ (Figure 2B), which share the same β/α dipeptide segments with 1 and 2, respectively, but the latter two have two α-amino acid residues, i.e., L-Val and L-Leu, that flank the β/α dipeptide segment. If folded, tetrapeptides 1 ′ and 2 ′ could also adopt doubly H-bonded hairpin conformations as shown in Figure 2B. FIGURE 2 | (A) Hybrid tetrapeptides 1 and 2 were found to fold into a hairpin conformation as shown. (B) Hybrid tetrapeptides 1 ′ and 2 ′ are designed to probe whether folded (hairpin) conformations could also be adopted.
The 1 H NMR spectra of 1 and 1 ′ , and 2 and 2 ′ recorded at 25 mM were compared to those recorded at 1 mM in CDCl 3 . Table 1 shows the difference in the chemical shifts ( δ NH ) of the amide protons of the four peptides at the two concentrations. In the folded conformations of 1 and 2, protons a and d, like b and e, are intramolecularly H-bonded and exhibit either small upfield shifts or insignificant downfield shifts at high vs. low concentrations; while the signals of protons c, which are not intramolecularly H-bonded, shift noticeably downfield with increasing concentration (Tang et al., 2020). In contrast, all of the amide proton resonances of peptides 1 ′ and 2 ′ recorded at 25 mM show downfield shifts relative to those at 1 mM, with the signals of protons a, c, and d showing significant shifts, while those of protons b undergoing small shifts. These observations suggest that among the amide protons of 1 ′ and 2 ′ , only protons b are intramolecularly H-bonded. The fact that protons a and d of 1 ′ and 2 ′ are not intramolecularly H-bonded indicates that 1 ′ and 2 ′ do not fold into the hairpin conformation as shown in Figure 2B.
The conformations of tetrapeptides 1 ′ and 2 ′ were examined with 2D (NOESY) spectroscopy (Supplementary Figures S4, S5). Except for NOEs between protons b and c, and c and d, the spectrum of 1 ′ reveals no NOEs between protons a and d, i and l, or j and k, which would exist if a hairpin conformation existed. The spectrum of 2 ′ contains an NOE between protons b and c, with no NOEs between protons a and d, i and l, or j and k being observed. The fact that only proton b of 1 ′ or 2 ′ is intramolecularly H-bonded, along with the absence of NOEs between other remote protons, suggests that 1 ′ and 2 ′ , being derived from replacing the aromatic γ-amino acid residues 1 and 2 with L-Val and L-Leu residues, cannot fold into hairpin conformations.
The above observations indicate that the doubly H-bonded γAr residues are indispensable in driving the folding of 1 and 2 into hairpin conformations. Without the γAr residues, peptides 1 ′ and 2 ′ , although capable of forming intramolecular H-bonds involving protons a and d, fail to adopt hairpin conformations. The critical role played by the γAr residues on stabilizing these novel hairpins relies on the effective H-bonding capabilities offered by these structural units (Gong, 2007), which provides the energetic driving force for the observed persistent folding of these hybrid peptides.
Triply H-Bonded β-Hairpins: The Folding of Hexapeptide 1a Comparing tetrapeptides 1 ′ with 1, and 2 ′ with 2 revealed the critical importance of the doubly H-bonded γAr residues in ensuring the adoption of hairpin conformations by 1 and 2. Attaching additional amino acid residues to 1 or 2 results in a longer peptide that may fold into a hairpin with an enhanced stability due to the energetic contribution of added H-bond(s). Hexapeptide 1a (Figure 3), which is resulted from adding a pair of amino acid residues, L-Val and L-Leu, to the N and C termini of 1, respectively, were examined and compared to tetrapeptide 1.
The 1 H NMR spectrum of 1a recorded in CDCl 3 at 25 • C contains well-dispersed signals (Supplementary Figure 1), indicating that 1a, like 1, exists as a single discrete species with a defined conformation. At 1 mM in CDCl 3 , the signals of protons a and d of 1a appear at 10.06 and 9.53 ppm, respectively, while the same protons of 1 are found at 9.65 and 9.39 ppm. The downfield shifts of protons a and d of 1a relative to those of 1 at the same concentration indicate that the H-bonds involving protons a and d of the former are stronger than those of the latter, which suggests that, hexapeptide 1a, with one additional H-bond contributed by the L-Val/L-Leu pair, may very likely fold into a triply H-bonded conformation that is more stable than the doubly H-bonded hairpin of 1.
Comparing the difference in the chemical shifts of amide protons at 25 mM and 1 mM in CDCl 3 reveals that the amide proton resonances of 1a follow the same trend as shown by those of 1 (Supplementary Table 1). The signal of proton c of 1 or 1a undergoes the largest downfield shift (∼0.7 ppm) upon increasing the concentration from 1 to 25 mM, suggesting that proton c of 1a, like that of 1 (Tang et al., 2020), is intermolecularly H-bonded. In contrast, the signals of amide protons a, b, d, and e of 1a exhibit very small (<0.01 ppm) upfield shifts, indicating that protons a and d, like b and e, are intramolecularly H-bonded. These observations suggest that hexapeptide 1a, like tetrapeprtide 1, folds into a hairpin conformation that is stabilized by Hbonds involving protons a and d, and further reinforced by a H-bond involving proton f. Indeed, proton f undergoes an upfield shift of 0.274 ppm from 1 to 25 mM, suggesting that it is intramolecularly H-bonded.
The H-bonding interactions involving protons a and d were further examined by monitoring the chemical shifts of amide protons a and d of peptides 1 and 1a in CDCl 3 containing DMSO-d 6 . Interestingly, the signals of protons a and d shift differently with increasing ratios of DMSO (Figure 4). The resonances of protons a of both 1 and 1a first shift upfield and then move downfield with as the ratio of DMSO increases ( Figure 4A). In contrast, the signals of protons d of both 1 and 1a show overall linear downfield shifts with increasing DMSO ratio ( Figure 4B).
The different shifts of amide protons a and d toward increasing solvent polarity can be explained by the folded conformations of 1 and 1a (Figure 3). Proton d is involved in an "intraturn", N-H(i) → O=C(i −3) hydrogen bond that is part of the 11-atom, intramolecularly H-bonded ring that constitutes the expanded β-turn in the hairpin conformation of 1 or 1a. Such a H-bond, with a H•••O distance of over 2.0 Å, is slightly longer and thus weaker than typical H-bonds. As a result, proton d of 1 or 1d is more accessible to solvent molecules than proton a. With increasing proportion of DMSO, proton d becomes increasingly H-bonded with DMSO molecules, which results in the destabilization of the folded (hairpin) conformation. Such an overall destabilization of the hairpin conformation in turn weakens the H-bond involving proton a, which is reflected by the initial upfield shift of the signal of proton a. As the Hbond becomes further weakened, proton a also becomes more exposed to solvent molecules and engages in increasing Hbonding interaction with DMSO molecules, which leads to the downfield shift of its signal.
Comparing the shifts of amide protons a of 1 and 1a reveals another interesting trend. As show in Figure 4A, the upfield shift shown by the signal of proton a of 1 is reversed at ∼5% DMSO, while that of 1a is reversed at 15% DMSO, indicating that proton a of 1a is more resistant toward increasing solvent polarity than that of 1, i.e., the H-bond involving proton a in 1a is stronger than that in 1. The stronger H-bond involving proton a of 1a is mostly like due to the higher overall stability of the hairpin conformation of 1a than that of 1. The enhanced stability shown by the folded structure of 1a can be explained by the energetic contribution of H-bonding involving proton f and the terminal amide C = O group.
The folded conformation of hybrid peptide 1a is confirmed by 2D NOESY spectra. As shown in Figure 5, the NOEs observed with 1a include those between protons a and m, and h and n, which indicate the H-bonded alignment of the two aromatic γ-amino acid residues and the L-Val and L-Leu residues. In addition, NOEs between protons c and j, c and k, c and d, and d and l demonstrate the presence of a well-defined loop. These multiple NOEs confirm that hybrid peptide 1a folds into a hairpin conformation similar to those observed with hybrid tetrapeptides 1 and 2 (Tang et al., 2020).
Role of Residue Chirality on Hairpin Folding
Results from our recent study demonstrate that replacing the β-alanine residue of 1 with other β-amino acid residues such as β-homoPhe gives hybrid tetrapeptides including 2 that fold into expanded β-turns with enhanced stabilities (Tang et al., 2020). It is expected that the β-turn of 2, like that of 1, should also accommodate additional amino acid residues, resulting in longer β-hairpins. Attaching L-Val and L-Leu residues to the N and C termini of 2 results in hexapeptide 2a. Replacing the L-β-homoPhe residue of 2a with D-β-homoPhe gives hexapeptide 2b (Figure 6). By comparing the folding of 2, 2a, and 2b, and the stabilities of the folded structures, we intend to probe the compatibility of the chirality of the β-homoPhe residue in the dipeptide segment of the expanded β-turn with that of the two terminal L-α-amino acid residues. Table 2 lists the difference in the chemical shifts of amide protons a, b, c, d, and e of peptides 2, 2a, and 2b, along with the chemical shifts of amide protons f and g of 2a and 2b, measured at 25 mM and 1 mM. The amide protons of 2 and 2b show the same overall change in their chemical shifts measured at high and low concentrations, suggesting that 2b, like 2, also folds into a well-defined hairpin conformation. In contrast, the amide proton resonances of 2a show noticeable difference in that proton c does not undergo as large a downfield shift as those shown by protons c of 2 and 2b. In addition, proton d of 2a shows a significant downfield shift from 1 to 25 mM, which contrasts the negligible shifts observed with protons d of 2 and 2b. These observations imply that, compared to those of 2 and 2b, proton d of 2a is more exposed and is available for intermolecular H-bonding, which in turn suggests that the expanded β-turn of 2a involving H-bonded proton d might be partially twisted. Such a partial twisting or deformation may be resulted from the incompatibility of the L-homoPhe with the terminal L-Val and L-Leu residues of 2a.
To assess the relative stabilities of the folded conformations of 2, 2a, and 2b, the chemical shifts of protons a and d of these three hybrid peptides in CDCl 3 containing DMSO-d 6 were compared. Similar to what is observed with 1 and 1a (Figure 4), the signals of protons a of 2, 2a and 2b also first shift upfield with increasing proportion of DMSO, followed by shifting downfield as the ratio of DMSO further increases ( Figure 7A). The resonances of protons a of the three peptides, however, undergo transitions from upfield to downfield shifts at different percent of DMSO, with peptide 2 showing its transition at ∼6% DMSO, 2a at ∼8% DMSO, and 2b at ∼25% DMSO. Thus, proton a of 2b is the least responsive toward increasing solvent polarity, which indicates that the folded conformation of 2b is the most stable among those of the three peptides. The high stability of folded 2b is also demonstrated by the upfied and then down field shift of proton d of this peptide (Figure 7B), which contrasts the consistent downfield shifts observed with the resonances of protons d of the other hybrid peptides, indicating that the H-bond involving proton d is greatly enhanced in the folded conformation of 2b.
In contrast, the stability of folded hexapeptide 2a is similar to that of tetrapeptide 2 and is much less stable than those of hexapeptides 2b and 1a (Figures 4A, 7A). The strong H-bonding and high stability of 2b, and the much lower stability of 2a, suggest that D-homoPhe residue in the H-bonded loop of 2b is more compatible with the L-Val and L-Leu residues, while L-homoPhe of 2a is much less compatible. Therefore, a heterochiral combination of the β-amino acid residue in the expanded β-turn and the α-amino acid residues in the β-strands seems to favor the nucleation and stabilization of β-hairpins. For example, an expanded β-strand with a D-β-amino acid residue should promote oligopeptides of L-α-amino acids to pair into a β-sheet.
NOESY spectra of 2a and 2b provide additional insights into the folding of these two hexapeptides. As shown in Figure 8A, the NOEs observed with 2a include those between protons d and i, and h and n, which indicate the alignment of the two γAr residues and the L-Val and L-Leu residues. In addition, NOEs between protons c and j, c and k, c and d, and d and l demonstrate the presence of the H-bonded loop. The observed NOEs suggest that hybrid peptide 2a adopts an overall hairpin conformation that includes a H-bonded loop along with the expected alignment of γAr and α-amino acid residues. In comparison to those of 2a, NOEs with significantly stronger intensities are revealed by the NOESY spectrum of 2b ( Figure 8B). Strong NOEs that indicate the H-bonded alignment of the two γAr residues and the L-Val/L-Leu residues are clearly observed between protons a and m, a and n, d and i, and h and n. NOEs between protons c and j, c and k, c and d, d and j, and d and l are consistent with the presence of the 11-atom H-bonded ring that constitutes the expanded βturn. The different numbers and strengths of the NOEs detected for 2a and 2b are consistent with the above conclusion on the different stabilities of the two folded structures. The numerous strong NOEs observed with 2b is consistent with a compact, tightly folded conformation. Finally, hexapeptides 2a and 2b were computationally optimized and compared. The optimized structures are shown in Figure 9. Modeling of 2a with revPBE-D3 revealed N-H•••O hydrogen bond distances for protons a, d, and f to be 1.79, 2.08, and 1.91 Å, respectively. Measurements of the N-H•••O hydrogen bond distances in 2b show that H-bonds involving protons a, d, and f shorten to 1.78, 1.98, and 1.88 Å, respectively, which suggest strengthened H-bonds. The strengthening of the hydrogen bonds in 2b relative to 2a, assessed through the N-H•••O hydrogen bond distances, coupled with 2a having a larger buckling within the turnabout of the L-homoPhe residue relative to 2b supports the experimental findings that 2b holds a greater overall stability than 2a.
CONCLUSION
Results from this study have demonstrated that, by comparing with tetrapeptides consisting of α-amino acid residues that fail to fold into hairpin conformations, hybrid tetrapeptides sharing a general structure with a central β/α dipeptide segment flanked by doubly H-bonded γAr residues reliably fold into hairpins, which demonstrate the decisive role played by the γAr residues in driving the folding of these short peptides. Adding additional α-amino acid residues to the hybrid tetrapeptides results hexapeptides that fold into hairpins with enhanced stabilities, indicating that the expanded β-turns formed by the tetrapetides can effectively nucleate and stabilize longer hairpins. A combination of hetero-chirality between the β-amino acid residue, i.e., L-and D-homoPhe residues, in the dipeptide loop and the terminal α-amino acid residues, i.e., L-Val and L-Leu, strongly promotes the folding of the hexapeptides, based on which longer peptide strands should be aligned into defined β-sheets.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
AUTHOR CONTRIBUTIONS
The project was designed, coordinated, and supervised by BG with assistance from YZ. Synthesis of the hybrid peptides was performed by QT and YZ, under supervision of BG, Z-LL, and RL. The measurement of spectroscopic data was performed and analyzed by YZ and QT. The molecular modeling study was designed by DM and YZ, supervised by EZ and BG, and executed mainly by DM. BG analyzed and compiled the data and prepared the manuscript with support of YZ, and also of QT, DM, and EZ. The final manuscript was read and approved by all authors. | v3-fos-license |
2020-02-07T20:39:28.745Z | 2020-02-06T00:00:00.000 | 211046322 | {
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"year": 2020
} | pes2o/s2orc | Parthenolide inhibits ubiquitin-specific peptidase 7 (USP7), Wnt signaling, and colorectal cancer cell growth
It has been well-established that the deubiquitinating enzyme ubiquitin-specific peptidase 7 (USP7) supports cancer growth by up-regulating multiple cellular pathways, including Wnt/β-catenin signaling. Therefore, considerable efforts are directed at identifying and developing USP7 inhibitors. Here, we report that sesquiterpene lactone parthenolide (PTL) inhibits USP7 activity, assessed with deubiquitinating enzyme activity assays, including fluorogenic Ub-AMC/Ub-Rho110, Ub-VME/PA labeling, and Di-Ub hydrolysis assays. Further investigations using cellular thermal shift (CETSA), surface plasmon resonance (SPR), and mass spectrum (MS) assays revealed that PTL directly interacts with USP7. Consistent with the role of USP7 in stimulating Wnt signaling and carcinogenesis, PTL treatment inhibited the activity of Wnt signaling partly by destabilizing β-catenin. Moreover, using cell viability assays, we found that PTL suppresses the proliferation of colorectal cancer cells and induces apoptosis in these cells. Additionally, we examined the effects of two other sesquiterpene lactones (costunolide and α-santonin) on USP7 and Wnt signaling and found that α-methylene-γ-butyrolactone may provide a scaffold for future USP7 inhibitors. In summary, our findings reveal that PTL inhibits USP7 activity, identifying a potential mechanism by which PTL suppresses Wnt/β-catenin signaling. We further suggest that sesquiterpene lactones might represent a suitable scaffold for developing USP7 inhibitors and indicate that PTL holds promise as an anticancer agent targeting aberrant USP7/Wnt signaling.
It has been well-established that the deubiquitinating enzyme ubiquitin-specific peptidase 7 (USP7) supports cancer growth by up-regulating multiple cellular pathways, including Wnt/-catenin signaling. Therefore, considerable efforts are directed at identifying and developing USP7 inhibitors. Here, we report that sesquiterpene lactone parthenolide (PTL) inhibits USP7 activity, assessed with deubiquitinating enzyme activity assays, including fluorogenic Ub-AMC/Ub-Rho110, Ub-VME/PA labeling, and Di-Ub hydrolysis assays. Further investigations using cellular thermal shift (CETSA), surface plasmon resonance (SPR), and mass spectrum (MS) assays revealed that PTL directly interacts with USP7. Consistent with the role of USP7 in stimulating Wnt signaling and carcinogenesis, PTL treatment inhibited the activity of Wnt signaling partly by destabilizing -catenin. Moreover, using cell viability assays, we found that PTL suppresses the proliferation of colorectal cancer cells and induces apoptosis in these cells. Additionally, we examined the effects of two other sesquiterpene lactones (costunolide and ␣-santonin) on USP7 and Wnt signaling and found that ␣-methylene-␥-butyrolactone may provide a scaffold for future USP7 inhibitors. In summary, our findings reveal that PTL inhibits USP7 activity, identifying a potential mechanism by which PTL suppresses Wnt/ -catenin signaling. We further suggest that sesquiterpene lactones might represent a suitable scaffold for developing USP7 inhibitors and indicate that PTL holds promise as an anticancer agent targeting aberrant USP7/Wnt signaling.
Ubiquitination mediated by ubiquitin-proteasome system (UPS), 3 which consists of E1s, E2s, E3 ligases, and 26S proteasome, plays critical roles in control of the stability, activity, or localization of protein substrates. Oppositely, the process of ubiquitination could be reversed by deubiquitinases (DUBs) that recognize ubiquitylated proteins and remove their ubiquitin tags (1). Over the past decades, numerous studies established that dysregulation of the ubiquitin system has been involved in the pathogenesis of multiple human diseases, including various types of tumors (2). Successful approval of proteasome inhibitors (PIs) for the treatment of multiple myeloma highlights the great potential of developing inhibitors targeting UPS for anti-cancer agents (3). Because of the occurrence of clinical PI resistance and chief roles of E3s and DUBs in governing the specificity of UPS, compounds targeting these two types of enzymes therefore represent novel therapeutics with potentially enhanced specificity and reduced toxicity (4 -6).
To date, ϳ100 DUBs encoded by human genome have been identified and classified into six families. USPs are the largest subfamily of DUBs, with more than 50 family members reported (7). Among them, USP7 has been initially paid attention because of its regulating effect on oncoprotein MDM2, a major E3 ligase responsible for the proteasomal degradation of the tumor suppressor p53 (8). Beyond the MDM2-p53 axis, subsequent evidence indicates the involvement of USP7 in multiple oncogenic pathways (9 -16). For example, our and other studies recently indicated that USP7 positively regulated Wnt signaling through stabilization of the key transcriptional factor -catenin (10,11,17,18). Several other studies also demonstrated the positive role of USP7 in NF-B, Hedgehog, and Hippo signaling by deubiquitinating and stabilizing NEK2, Gli, and Yorkie/Yap, respectively (9,12,15). Additionally, multiple proteins involved in diverse cellular processes including DNA damage response, transcription, epigenetic control of gene expression, and immune response, such as Chk1, N-Myc, ASXL1, and Foxp3, have been identified as specific substrates of USP7 (19 -23). Therefore, it is not surprising that USP7 is often overexpressed and correlates with poor prognosis in diversified human malignancies including breast cancer, hepatocellular carcinoma, leukemia, and on (13,16,(23)(24)(25)(26). The inhibition of the deubiquitinating activity of USP7 thus offers a promising strategy for protein-directed therapies in the treatment of cancer.
Indeed, a number of USP7 inhibitors have been identified, and most of them exhibited in vitro and in vivo anti-cancer activity against various types of tumor (23,27). Especially, the diversified anti-cancer mechanisms of several inhibitors have been reported, which accorded with the variety of USP7 targets in cells. Two different studies pointed out that P5091, a small molecule inhibitor of USP7, down-regulated the abundance of Yap and up-regulated the level of ARF, which contributed to its cytotoxic effect on hepatocellular carcinoma (15,25). In addition, our previous study indicated that USP7 inhibition by P5091 attenuated the proliferation of colorectal cancer cells partly through destabilizing -catenin (28). In addition, P5091 has been reported to accelerate the degradation of N-Myc and Nek2 (12,20). Notably, pharmacological targeting USP7 by P217564, one derivate of P5091, led to Tip60 degradation and consequent impairment of Treg suppressive function, implicating the potential of USP7 inhibitors in future cancer immunotherapy (29). Overall, these studies demonstrated the rationality for development of USP7 inhibitors as therapeutic agents against cancer.
Natural products harbor structural diversity and are critical for screening of new drug leads. However, small molecule USP7 inhibitors from natural resources have rarely been reported. In this study, we showed that sesquiterpene lactone parthenolide (PTL), first purified from the shoots of the feverfew and used to treat migraine and arthritis for centuries, could inhibit enzymatic activity of USP7 via direct interaction. Also, PTL treatment enhanced the ubiquitination of -catenin and decreased -catenin protein levels in colorectal cancer (CRC) cells, which resulted in the inhibition of Wnt signaling and cytotoxity of CRC cells. In sum, our study suggested that sesquiterpene lactones might represent a novel scaffold for designing novel USP7 inhibitors and the potential of PTL in the treatment of cancers driven by dysregulated USP7 and Wnt signaling.
Identification of PTL as a novel USP7 inhibitor
In an effort to identify USP7 inhibitors, we performed an in vitro high-throughput screening assay against a library of natural chemicals using ubiquitin-aminomethylcoumarin (Ub-AMC) as a substrate (30), and the small molecule PTL was discovered (Fig. 1A). Detailed characterization results revealed that PTL inhibited USP7 activity in dose-and time-dependent manner ( Fig. 1B and Fig. S1A). Similar inhibitory effects of PTL on USP7 activity were also obtained using an assay based on another ubiquitin precursor, ubiquitin-rhodamine 110 (Ub-Rho110) ( Fig. 1C and Fig. S1B).
To further confirm the USP7 inhibitory capacity of PTL both in vitro and in the cellular environment, we then performed competition assays between PTL and the Ub active site probe ubiquitin-vinyl methyl ester (Ub-VME). First, purified USP7 was incubated with PTL or equivalent DMSO, followed by the addition of Ub-VME probes. As shown in Fig. 1D, recombinant USP7 incubated with Ub-VME exhibited Ub-USP7 conjugate formation, as reflected by the increase in mass of USP7 of ϳ8 kDa. In contrast, Ub-USP7 conjugate formation was dose-dependently inhibited from PTL-treated samples, with a concomitant increase in the unlabeled free form of USP7. In cellular context, HCT116, SW480, and HEK293T cells were treated with or without PTL for 2 h, and cell lysates were labeled with Ub-VME. As expected, PTL also inhibited the covalent binding of Ub-VME with USP7 ( Fig. 1, E and F), which is similar to the in vitro results. Besides, we also directly focused on cell lysates. When lysates of HEK293T cells were treated with Ub-VME in the presence or absence of PTL, a strong reduction of the labeled USP7 was observed as well (Fig. 1G). Next, the USP7 activity toward K48-linked diubiquitin (di-Ub) was investigated, and the results showed that PTL could decrease the amount of Ub hydrolyzed from di-Ub in a concentration-dependent manner (Fig. 1H).
To bring insight to the selectivity of PTL for USP7 relative to other DUBs, the Ub-Rho110 hydrolysis test against a panel of 41 DUBs was performed. As the results showed, PTL also exhibited inhibition capacity, to some extent (Ͼ25%), against other 6 DUBs including USP8, USP21, USP27X, USP30, USP35, and JOSD2 (Fig. S1C). We then utilized specific antibodies to assess the labeling efficiency for DUBs from SW480 cells. As the results showed, PTL efficiently blocked the labeling of USP7 at 40 M, but not that of USP4, USP15, and USP47 in SW480 cells (Fig. 1I). The similar findings were observed in HCT116 cells as well (Fig. S1D). We next evaluated the effect of PTL on Ub-VME labeling efficiency against several DUBs individually. For this purpose, seven DUBs, including USP4, USP7, USP15, USP25, UCHL1, and UCHL3, were purified and labeled with Ub-VME in the presence or absence of PTL. As indicated in Fig. 1J, all tested DUBs were labeled with Ub-VME indicated by mobility shift, albeit with different levels of efficiency. Particularly, PTL exhibited major inhibitory activity against USP7 and minor effects against USP25, and negligible effects against USP4, USP15, UCH-L1, and UCH-L3 (Fig. 1J). Taken together, these results confirmed the inhibitory activity of PTL against USP7 and indicated that PTL preferentially inhibited USP7 over a panel of active DUBs.
PTL interacts with USP7
Cellular thermal shift assay (CETSA) is a newly developed method to evaluate drug binding to target proteins in cells, which is based on the biophysical principle of ligand-induced thermal stabilization of target proteins (31). To evaluate whether PTL binds to USP7, CETSA was firstly employed in SW480 cell lysates. As shown in Fig. 2A, PTL treatment significantly increased the thermal stability of USP7 in supernatant. Consistent with the results, accumulation of USP7 was also markedly increased by PTL in a concentration manner (Fig. 2B). Next, to test whether PTL interact with USP7 in intact cells, we performed cell-based drug treatment before analysis by temperature shift. To this end, SW480 cells treated with PTL Inhibition effect of parthenolide on USP7 and Wnt or DMSO were collected, lysed, and then heated. Compared with DMSO treatment, PTL incubation led to an obvious thermostability of USP7 at different temperatures (Fig. 2C) and dif-ferent doses (Fig. 2D). In short, these results indicated the increased thermostability of USP7 following heat treatment in the presence of PTL, suggesting the interaction between USP7 . C, dose-dependent inhibition of USP7 activity by PTL using the Ub-Rho110 as substrate. The results are presented as mean Ϯ S.D. (n ϭ 2). D, purified FLAG-USP7 were treated with PTL, and the Ub-VME probe was added. Samples were subsequently analyzed by Western blotting using anti-USP7 antibody. E, HCT116 and SW480 cells treated with different doses of PTL were collected and lysed, and Ub-VME probes were added into the cell lysates for 30 min. Samples were subsequently analyzed by Western blotting with anti-USP7 antibody. F, HEK293T cells directly incubated with PTL were then collected and labeled with Ub-VME, followed by immunoblot analysis with anti-USP7 antibody. G, HEK293T cell lysates pretreated with or without PTL were labeled with Ub-VME and analyzed by Western blotting. H, recombinant His-USP7 was pretreated with indicated dose of PTL for 40 min and then incubated with K48-linked di-Ub for 3 h. SDS-PAGE and silver staining were employed to analyze the cleavage of di-Ub by USP7 in the presence or absence of PTL. I, SW480 cells were treated with different doses of PTL for 2 h and then labeled with Ub-PA. Individual DUBs were identified using specific antibodies. J, purified FLAG-USP7 and additional deubiquitinating enzymes (FLAG-USP4, FLAG-USP15, FLAG-USP25, FLAG-UCH-L1, and FLAG-UCH-L3) were treated with PTL for 20 min and then labeled with Ub-VME for another 20 min, followed by SDS-PAGE with FLAG-Tag antibody.
PTL promotes the ubiquitination and degradation of -catenin
Recent studies indicated that USP7 could deubiquitinate and stabilize -catenin, the key transcriptional factor of Wnt signaling pathway (10,11,17,18). The effect of PTL on the ubiquitination level of -catenin was thus explored. HEK293T cells transiently transfected with HA-Ub plasmids were treated with or without PTL, and endogenous -catenin ubiquitination was analyzed. The results showed that treatment of PTL increased the level of -catenin ubiquitination (Fig. 3A). The effect of PTL on -catenin ubiquitination was also tested in HCT116 and SW480 cells without exogenous Ub transfection. Similarly, -catenin ubiquitination was also enhanced in cells exposed to PTL (Fig. 3, B and C).
Given that PTL treatment could increase the ubiquitination level of -catenin, we next determine whether PTL promoted the degradation of -catenin. As expected, PTL treatment dose-dependently reduced -catenin levels in HCT116 and SW480 cells (Fig. 3D). Likewise, the presumably transcriptionally active form of -catenin (nonphosphorylated form of -catenin) were decreased as well (Fig. 3D). In addition, incubation of SW480 cells with PTL obviously accelerated -catenin degradation in the presence of cycloheximide, which was used to inhibit protein biosynthesis (Fig. 3E). Especially, the PTL-induced -catenin degradation was blocked in the presence of proteasome inhibitor MG132 (Fig. 3F), suggesting that -catenin degradation mediated by PTL is proteasome dependent. In line with the above-mentioned data, the results of cytoplasmic and nuclear fraction and immunofluorescence assays further indicated that PTL treatment down-regulated -catenin levels of the nuclear and cytoplasmic compartments in both HCT116 and SW480 cells (Fig. 3, G and H). In conclusion, these data suggested that PTL decreased the levels of -catenin via increasing its ubiquitination.
PTL inhibits Wnt signaling in colorectal cancer cells
We then assessed the effect of PTL on Wnt signaling through luciferase activity assay of Super TOPFlash luciferase (ST-Luc), a Wnt/-catenin signaling reporter. As shown in Fig. 4A, PTL dose-dependently inhibited the activity of ST-Luc reporter
Inhibition effect of parthenolide on USP7 and Wnt
in HEK293W cells (HEK293 cells stably co-transfected with Wnt3a, Renilla, and ST-Luc). The inhibition effect of PTL on endogenous Wnt signaling in colon cancer cells was investigated either in line with the results of HEK293W cells, the TOPFlash activity in both HCT116 and SW480 cells treated with PTL was efficiently attenuated in a dose-dependent manner (Fig. 4, B and C). The effects of PTL on the expression of known target genes of Wnt/-catenin signaling including Axin2 (32) and c-Myc (33) were further monitored. Compared with DMSO-treated HCT116 and SW480 cells, the protein and mRNA levels of Axin2 and c-Myc were both decreased in these two cell lines treated with PTL (Fig. 4, D and E).
Effect of PTL on proliferation, cell cycle, and apoptosis of colorectal cancer cells
In view of the positive role of USP7 and Wnt/-catenin signaling in CRC progression, we then employed MTS assay to assess the growth inhibitory effect of PTL in CRC cells. As illustrated in Fig. 5A, PTL exhibited stronger growth inhibition effect on CRC cells than normal colonic epithelial cells. Cell cycle and apoptosis of HCT116 and SW480 cells treated with PTL were determined by flow cytometry. Cell cycle analysis revealed that HCT116 and SW480 cells were efficiently arrested at G 2 /M phase upon PTL treatment (Fig. 5, B and C). Moreover, we evaluated the ability of PTL to induce apoptosis. Flow cytometry analysis after annexin V/PI double staining established that PTL dose-dependently Immunoprecipitation with anti--catenin antibody was performed, and ubiquitination of -catenin was analyzed by anti-HA antibody. B and C, HCT116 (B) and SW480 (C) cells were treated with DMSO or PTL (20 M) for 12 h, followed by immunoprecipitation with -catenin antibody, and -catenin ubiquitination was detected with the antibody against ubiquitin. D, treatment of HCT116 and SW480 cells with PTL for 24 h, total and active forms of -catenin were determined via Western blotting. E, SW480 cells were pretreated with DMSO or PTL (20 M) for 4 h, followed by addition of cycloheximide (CHX) (80 g/ml) for the indicated times. Cell lysates were subjected to immunoblot with antibodies against -catenin or actin. F, HCT116 and SW480 cells were treated with DMSO or PTL (15 M) for 20 h, followed by addition of DMSO or MG132 (10 M) for additional 4 h. Cells were harvested and the protein level of -catenin and actin were examined. G, PTL reduced -catenin levels in both cytoplasmic and nuclear fractions. HCT116 and SW480 cells were treated with PTL (7.5 or 15 M). Cytoplasmic and nuclear fractions were then separated and -catenin protein levels were analyzed. H, representative immunofluorescence staining images of HCT116 and SW480 cells treated with DMSO or PTL. -catenin and nucleus was recognized by anti--catenin antibody (green) or DAPI (blue), respectively. Scale bar represents 50 m. Blots for indicated protein expressions were quantified using ImageJ software.
Inhibition effect of parthenolide on USP7 and Wnt
induced accumulation of cells in early (annexin Vϩ/PIϪ) and latestage (annexin Vϩ/PIϩ) apoptosis (Fig. 5, D and E). To verify the apoptosis observed in PTL-treated cells, apoptosis-related proteins were monitored using Western blotting. As shown in Fig. 5F, treatment of HCT116 and SW480 cells with PTL-activated caspase-8 and caspase-9, accompanied with the cleavage of poly ADP-ribose polymerase in a dose-dependent manner. These results indicated the potential of PTL in the treatment of colorectal cancers mostly driven by deregulated Wnt signaling.
Discussion
DUBs have recently emerged as an attractive drug target class in cancer therapeutics (36). Inhibition of USP7 is of special interest because of its well-established connections to multiple oncogenic pathways (14,23). In this study, we demonstrate that PTL is a novel inhibitor of USP7 based on the following evidence: (a) PTL could inhibit the USP7-mediated hydrolysis of Figure 6. ␣-Methylene-␥-butyrolactone of sesquiterpene lactones is responsible for the inhibition toward USP7 and Wnt signaling. A, structure of costunolide and ␣-santonin. B, representative SPR sensorgrams of USP7 CD incubated with costunolide and ␣-santonin. Compounds were tested at a series of increasing concentrations. C, inhibition of USP7 activity by costunolide and ␣-santonin using the Ub-AMC as substrate. The results are presented as mean Ϯ S.D. (n ϭ 2). c , p Յ 0.001, difference versus DMSO. D, living SW480 cells were treated with PTL, costunolide, and ␣-santonin (40 M) for 2 h and then labeled with Ub-PA probe. Samples were subsequently analyzed by Western blotting by anti-USP7 and anti-actin antibodies. E, SW480 cells were treated with PTL, costunolide, and ␣-santonin for 24 h, -catenin were determined by Western blotting. Blots for indicated protein expressions were quantified using ImageJ software. F, effects of PTL, costunolide, and ␣-santonin on the TOPFlash reporter activity. HEK293W cells were incubated with indicated doses of compounds for 24 h. The luciferase activity was then measured and normalized to the activity of the Renilla. G, cells seeded in 96-well plates were cultured overnight and exposed to PTL, costunolide, and ␣-santonin at various doses for 48 h. Cell viability was determined by MTS assay.
Inhibition effect of parthenolide on USP7 and Wnt
Ub-AMC/Ub-Rho110 and di-Ub; (b) PTL competed with the binding of Ub-VME/Ub-PA probe to USP7; (c) CETSA, SPR, and MS analyses demonstrated that PTL directly interacted with USP7. According to previous studies, PTL exerted its biological activity through Michael addition reaction, which was based on alkylation of cysteine involving the ␣-methylene-␥butyrolactone moiety (34,35), and the MS data indicated that 13 cysteines were modified by PTL. Interestingly, the catalytic Cys-223 was not modified by PTL. Further experimental data, such as mutation analysis and co-crystal structures, are necessary to confirm the mode of PTL binding to USP7. Comparison of the efficacy of PTL with another well-known USP7 inhibitor P5091 (37) was performed side by side, and the results showed that the inhibitory activity of PTL against USP7 was a bit weaker than that of P5091 in Ub-VME labeling assay and Ub-AMC fluorescent assay (data not shown). Moreover, our study also provided a preliminary selectivity spectrum of PTL against a panel of DUBs and identified that PTL preferentially inhibited USP7. Nevertheless, the molecular basis for PTL preference against USP7 was not defined and warrant further study. As for the specificity, there are both similarities and differences in the specificity of PTL and P5091 toward DUBs. What is similar is that both PTL and P5091 did not inhibit USP2, USP5, USP15, USP20, USP28, UCHL-1, or UCHL-3 (37). However, other DUBs except USP7 targeted by PTL or P5091 were different. Our study indicated that PTL exhibited partial inhibitory activity against USP8, USP21, USP27X, USP30, USP35, and JOSD2. However, only USP47 was reported to be targeted by P5091 (38).
As a major active ingredient from feverfew, PTL exhibits multiple pharmacological activities including anti-cancer and anti-inflammatory activities via modulating various signaling pathways such as the NF-B, p53-MDM2, and STAT3 pathways. A recent study clarifying that PTL inhibits the activity of JAKs accounts for its inhibitory effect on STAT3 signaling (39). Specially, identification of PTL as a novel USP7 inhibitor here provides a rational explanation for the activation effect of PTL on p53 functions via promoting ubiquitination of MDM2 (40), which is a primary substrate of USP7 (41). In addition, USP7 was reported to interact with and deubiquitinate p65 and NEK2, leading to enhanced activity of NF-B signaling (12,42). Although several mechanisms by which PTL inhibited NF-B have been reported, identification of PTL as a USP7 inhibitor might be a further step for its strong inhibition of NF-B (43)(44)(45). Therefore, the function of PTL suppressing USP7 activity contributes to the illustration of its effect on some signaling pathways.
It should be noted that, although a couple of key signaling pathways were targeted by PTL as mentioned before, poor solubility and bioavailability of PTL leading to its weak in vivo effects are crucial limitations to impede its potential application in clinic (46). Consistently, our results showed that PTL to some extent inhibited tumor growth (ϳ30.0%) and tumor weight (ϳ22.7%) of HCT116 xenografts (data not shown). Thus, the chemistry study of PTL deserves further investigation to circumvent these limitations by constructing PTL derivatives with improved efficacy and specificity toward USP7.
Given that USP7 positively regulates Wnt signaling through mediating the deubiquitination of its major transcriptional coactivator -catenin (10,11,17,18,28), our further study indicated that PTL suppressed Wnt signaling through accelerating the ubiquitination and subsequent degradation of -catenin. Recently, Zhu et al. (47) reported that PTL potently inhibited Wnt signaling by blocking TCF4/LEF1 synthesis via targeting RPL10 without affecting -catenin stability. To be noted, destabilization of -catenin by PTL has also been validated in SW620 cells, one colorectal cancer cell line, which was consistent with our data (48). The difference in the effect of PTL on -catenin may be caused by the incubation time and dose treated. In fact, -catenin level was also decreased in SW480 cells treated with 20 M PTL in Zhu's results (47). In addition, DNMT1, one validated target of PTL, has been reported to stabilize -catenin in colorectal cancer cells (49,50). Whether other targets of PTL, such as IKK and FAK (35, 43), were involved in its effect on Wnt signaling remains unclear. Combined with previous studies, we believed that -catenin destabilization could be one of the mechanisms underlying the Wnt inhibition property of PTL.
Collectively, our study demonstrated that USP7 inhibitory activity was a novel bioactivity of PTL and elucidated another potential molecular mechanism of PTL in inhibiting the Wnt signaling pathway. These findings suggested that sesquiterpene lactones containing ␣-methylene-␥-butyrolactone might represent a novel scaffold for developing USP7 inhibitors and broadened the therapeutic application of PTL in USP7 and Wnt-aberrant cancers.
Inhibition effect of parthenolide on USP7 and Wnt
were cultured in DMEM, supplemented with 100 g/ml G418 (Sigma-Aldrich) and hygromycin B (Sigma-Aldrich), in addition to 10% FBS and 1% antibiotics. All the cells were incubated at 37°C, 5% CO 2 in a humidified atmosphere.
To confirm PTL (Selleck Chemicals, Houston, TX) as a Wnt signaling inhibitor, HEK293W cells were seeded in 96-well plates with three repeats and treated with PTL for 24 h. HCT116 and SW480 cells were seeded in 96-well plates and then transfected as follows: Wnt/-catenin signaling responsive Firefly luciferase reporter plasmid SuperTOPFlash (80 ng/well) and Renilla reporter plasmid (8 ng/well). After 3-h transfection, cells were exposed to various concentrations of PTL for 24 h and then lysed. Both Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay kit (Promega, Madison, WI). TOPFlash luciferase activities were normalized to the Renilla activities.
Labeling and competition for deubiquitinating activity with the Ub-VME/Ub-PA activity-based probe
Purified FLAG-USP7, as well as FLAG-USP4, FLAG-USP15, FLAG-USP25, FLAG-UCHL1, and FLAG-UCHL3, was incubated with PTL in 30 l labeling buffer (50 mM Tris, pH 7.6, 5 mM MgCl 2 , 0.5 mM EDTA, 2 mM DTT and 250 mM sucrose) at room temperature for 20 min. Ub-VME (to a final concentration of 5 M) (Boston Biochem, Cambridge, MA) was then added and incubated at 37°C for an additional 20 min. Samples were boiled in 5ϫ sample buffer at 70°C for 5 min and then separated by 8% SDS-PAGE.
To evaluate drug occupancy on USP7 in cell lysates, living HCT293T cells were harvested and lysed on ice with TE lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 0.5 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, and 2 mM DTT. The clarified cell lysates (40 g) were labeled with PTL at room temperature for 4 h in labeling buffer described above, and then was added Ub-VME (5 M) at 37°C for 20 min. Samples were boiled and then separated by SDS-PAGE. To evaluate drug occupancy on USP7 in intact cells, HEK293T, HCT116, as well as SW480 cells were seeded in 6-well plates with a density of 8 ϫ 10 5 cells/well and then treated with various concentrations of PTL for 2 h. Cells were harvested and lysed on ice with TE buffer. The ubiquitin-labeling reaction was initiated by adding Ub-VME or Ub-PA (5 M) in labeling buffer, and the reaction was allowed to proceed in a final volume of 30 l for 20 min. Samples were boiled and then separated by SDS-PAGE.
Cellular thermal shift assay
For a CETSA in SW480 cell lysates, cells were cultured in 10-cm dishes over 12 h. Cells were harvested and washed with PBS and then diluted in PBS supplemented with complete protease inhibitor mixture. The cell suspensions were freezethawed three times with liquid nitrogen. The lysates were separated from the cell debris by centrifugation at 20,000 ϫ g for 20 min at 4°C. The cell lysates were divided into two aliquots. One aliquot was treated with DMSO, the other was mixed with PTL (40 M). After 30 min incubation at room temperature, the respective lysates were divided into smaller (50 l) aliquots and individually heated at the designated temperatures for 3 min, followed by cooling for 3 min at room temperature. For the dose-response analysis, equal amounts of cell lysates were incubated with different concentrations of PTL and DMSO for 30 min at room temperature, followed by heating all samples at 52°C for 3 min and cooling to room temperature.
For a CETSA in living SW480 cells, cells were seeded in 6-cm dishes and exposed to PTL (40 M) or DMSO for 1 h. Following incubation, cells were harvested and lysed with PBS supplemented with complete protease inhibitor mixture. The cell lysates were then divided into smaller aliquots and heated. For the dose-response analysis, equal amounts of cells were treated with different doses of PTL for 1 h, followed by heating all samples at 55°C for 3 min.
All heated lysates were centrifuged at 20,000 ϫ g for 20 min at 4°C to separate the soluble fractions from the precipitates. All the supernatants were transferred to fresh microtubes before SDS-PAGE and Western blot analysis.
K48-linked diubiquitin gel-based assay
30 nM His 6 -USP7 (Boston Biochem) and variable concentrations of PTL were preincubated in 30 l reaction buffer (50 mM Tris-HCl, pH 7.6, 0.5 mM EDTA, 5 mM DTT, 0.01% Triton X-100, and 0.05 mg/ml serum albumin) for 40 min at room temperature. The reaction was initiated by adding 2 M K48linked diubiquition and was allowed to proceed for 3 h at 37°C, and then quenched by the addition of 2ϫ Tricine Sample Buffer (Bio-Rad). The reaction products were separated on a 16.5% denaturing SDS-PAGE gel and stained with Fast Silver Stain Kit (Beyotime Biotechnology).
Deubiquination assays
HEK293T cells were transfected with control (Ctrl) or HA-Ub for 24 h. After that, cells were treated with DMSO or PTL (20 M) for 12 h and then collected. HCT116 and SW480 cells were treated with DMSO or PTL for 12 h before harvesting. All cells were lysed in lysis buffer (150 mM NaCl, 30 mM Tris, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 0.5 mM DTT, and 1 mM PMSF) plus protease inhibitors (Roche). The cell lysates were incubated with -catenin antibody at 4°C overnight, followed by addition of protein A/G beads (Santa Cruz Biotechnology, Dallas, TX) for another 6 h. The beads were washed four times with lysis buffer and then boiled in 2ϫ sample loading buffer for 10 min. Samples were blotted and probed with the indicated antibodies.
Surface plasmon resonance
A Biacore S200 (GE Healthcare) was used for characterization of the interaction between PTL and USP7. USP7 CD protein (Boston Biochem), which was diluted to 100 ng/l in 100 l coupling buffer (50 mM acetate, pH 4.5, supplemented with 2 mM DTT and 0.5 mM EDTA), was coupled to CM5 sensor chips by amine-coupling in HBS buffer (10 mM HEPES, pH 7.4, and 150 mM NaCl), supplemented with 0.05% Pluronic 127, 2 mM DTT, and 0.5 mM EDTA. CM5 chips were activated with NHS/ EDC/ethanolamine injection according to Biacore standard methods. Cell 1 was used as blank reference surface, cell 2 was coupled with USP7 CD protein.
A series concentration of PTL solutions with 5% (v/v) DMSO were applied to both cell 1 and cell 2 in HBS buffer supplemented with 0.05% Pluronic 127, 2 mM DTT, 0.5 mM EDTA, and 5% (v/v) DMSO, with a flow rate of 30 l/min at 25°C, a contact time of 120 s, and a dissociation time of 180 s. A solvent correction curve from 4.5-5.8% (v/v) DMSO was included. Analysis was performed using the Biacore Evaluation software S200 using an affinity fit and a 1:1 binding model with constant Rmax.
Cell viability assay
Cell viability was determined by MTS assay. Briefly, 5 ϫ 10 3 cells were seeded in 96-well plates in triplicates and cultured overnight. Cells were next treated with different concentrations of PTL for 48 h. Then, 100 l culture medium mixed with 20 l CellTiter 96 ® Aqueous One Solution Reagent (Promega) was added to each sample. Cells were incubated at 37°C for 30 min to 2 h. The optical density was measured at a wavelength of 490 nm using a microplate reader (Perkin-Elmer). The IC 50 values were calculated by the relative survival curves.
Cytoplasmic and nuclear fractionation assay
Treated cells were harvested and resuspended in lysis buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, and 1 mM PMSF) containing protease inhibitor mixture, followed by incubation for 10 min on ice. After centrifugation at 3100 rpm, cells were lysed in buffer A with 0.5% Nonidet P-40 for 2 min on ice. The supernatants were collected as cytoplasmic extracts after being centrifuged for 15 min at 6000 rpm. The pellets were then washed with lysis buffer A without Nonidet P-40 three times and resuspended in lysis buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 25% glycerol, and 1 mM PMSF) with protease inhibitor mixture. After being lysed about 40 min and then centrifuged, the supernatants were collected as nuclear extracts. Samples were separated by SDS-PAGE.
Cell cycle and apoptosis analysis
Cells were seeded in 6-well plates at a density of 2 ϫ 10 5 cells/ well and exposed to PTL for 24 h. Cells were subsequently collected and washed twice with PBS. Cells were fixed with precold 70% ethanol overnight at Ϫ20°C. Fixed cells were washed with PBS and then stained with solution that contained 50 g/ml propidium iodide (Sigma-Aldrich) and 50 g/ml RNase A (Sigma-Aldrich) for 30 min in the dark at room temperature. Fluorescence intensity was measured by FACSCalibur flow cytometer (BD Biosciences). The distributions of cells in each phase of the cell cycle were determined using FlowJo 7.6.1 analysis software.
Inhibition effect of parthenolide on USP7 and Wnt
Cell apoptosis was analyzed by the annexin V-FITC/PI Apoptosis kit (BD Biosciences) according to the manufacturer's protocol. Briefly, cells were seeded in 6-well plates at a density of 1 ϫ 10 5 cells/well and cultured overnight. Cells treated with indicated concentrations of PTL for 48 h, were collected, and washed twice with cold PBS, followed by resuspending in a binding buffer containing annexin V-FITC and PI. After incubation for 15 min at room temperature in dark, the fluorescent intensity was analyzed using the FACSCalibur flow cytometer. The data were analyzed using FlowJo 7.6.1 analysis software.
Immunofluorescence analyzes
Cells seeded in 96-well plates were treated with different doses of PTL for 12 h. 4% paraformaldehyde was used for fixing cells about 20 min and then 0.1% Triton X-100 for permeating about 10 min. Samples were blocked with 3% BSA at 37°C for 30 min and then incubated with anti--catenin antibody at room temperature for 6 h. After washing with PBS three times, cells were incubated with corresponding FITC conjugated secondary antibody (Alexa Fluor ® 488) for 1 h at room temperature. Nuclei were stained by DAPI (blue) for 10 min. Cells were then observed and photographed under microscopy (Eclipse, Nikon) at 400ϫ.
RT-PCR assay
HCT116 and SW480 cells were seeded in 6-well plates at a density of 5 ϫ 10 5 cells/well. After adding PTL for 12 h, total RNA was prepared with TRIzol (Thermo Fisher) according to the manufacturer's protocol. Reverse transcription was performed using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher). For qPCR, SYBR Select Master Mix (Thermo Fisher) was used with ABI 7500 Real-Time PCR System. The values of c-Myc and Axin2 were shown against the value of GAPDH that was used as the control. Primer sequences are follows: c-Myc forward, 5Ј-CTTCTCTCCGTCCTCGG-ATTCT-3Ј and reverse, 5Ј-GAAGGTGATCCAGACTCTG-ACCTT-3Ј; Axin2 forward, 5Ј-AGCCACACCCTTCTCCA-ATC-3Ј and reverse, 5Ј-ACCGTCTCATCCTCCCAGAT-3Ј.
Statistical analysis
Two-tailed Student's t test was used to determine the statistical significance. All the results were expressed as mean Ϯ S.D., and p value of less than 0.05 was considered statistically significant. | v3-fos-license |
2019-04-10T13:11:55.762Z | 2018-09-20T00:00:00.000 | 105389220 | {
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} | pes2o/s2orc | Molecular-Level Understanding of Selectively Photocatalytic Degradation of Ammonia via Copper Ferrite/N-Doped Graphene Catalyst under Visible Near-Infrared Irradiation
: Developing photocatalysts with molecular recognition function is very interesting and desired for specific applications in the environmental field. Copper ferrite/N-doped graphene (CuFe 2 O 4 /NG) hybrid catalyst was synthesized and characterized by surface photovoltage spectroscopy, X-ray powder diffraction, transmission electron microscopy, Raman spectroscopy, UV–Vis near-infrared diffuse reflectance spectroscopy and X-ray photoelectron spectroscopy. The CuFe 2 O 4 /NG catalyst can recognize ammonia from rhodamine B (RhB) in ammonia-RhB mixed solution and selectively degrade ammonia under visible near-infrared irradiation. The degradation ratio for ammonia reached 92.6% at 6 h while the degradation ratio for RhB was only 39.3% in a mixed solution containing 100.0 mg/L NH 3 -N and 50 mg/L RhB. Raman spectra and X-ray photoelectron spectra indicated ammonia adsorbed on CuFe 2 O 4 while RhB was adsorbed on NG. The products of oxidized ammonia were detected by gas chromatography, and results showed that N 2 was formed during photocatalytic oxidization. Mechanism studies showed that photo-generated electrons flow to N-doped graphene following the Z-scheme configuration to reduce O 2 dissolved in solution, while photo-generated holes oxidize directly ammonia to nitrogen gas.
Introduction
The selectively photocatalytic oxidization of specific pollutants in practical multicomponent systems such as waters contaminated by organics and ammonia is of significance for the removal of specific pollutants. The World Health Organization recommends that the total amount of ammonia (NH 3 ) in drinking water should not exceed 1.5 mg/L [1]. Therefore, the development of the photocatalysts with the specific response to NH 3 is very vital for the removal of ammonia in water treatments, which involves the molecular recognition to NH 3 . CuFe 2 O 4 is an interesting material with the band gap of 1.7 eV due to its unique electronic configuration of valence shell (Cu 3d 10 4s 1 ) [1]. It has been widely applied in magnetic memory, high-frequency devices, sensors, drug delivery, anode materials, and catalysts owing to its advantages of environmental benignity, moisture insensitive, high dispersion, high reactivity, low price, large abundance of Cu and easy separation with an external magnet [2][3][4][5]. CuFe 2 O 4 was coupled price, large abundance of Cu and easy separation with an external magnet [2][3][4][5]. CuFe2O4 was coupled to graphene [6], TiO2 [7], AgBr [8] and Ag3PO4 [9] to fabricate composites for degrading organic pollutants. Wang and co-workers utilized CuFe2O4 as a photocatalyst to selectively degrade methylene blue in the presence of methylene orange, rhodamine B and rhodamine 6G, and the authors attributed the selective degradation to the specific interaction of active sites of catalyst with the methylene blue molecule [10]. To the best of our knowledge, however, the coupling of CuFe2O4 to nitrogen-doped graphene (NG) for the selective photocatalytic oxidization of NH3 has not been reported.
Graphene is a two-dimensional sp2-hybridized carbon material with unique properties such as excellent charge transport, outstanding transparency, huge specific surface area, high mechanical strength and superior thermal conductivity, so it was often used as a co-catalyst [11][12][13][14][15][16][17]. Moreover, molecular tailoring (nitrogen atoms were doped into graphene framework) can module its intrinsic properties to meet the rapidly increasing demand for practical applications in various fields. For example, nitrogen doping can tailor its electrical properties, open a band gap and allow it to show semiconducting properties. As a result, nitrogen doping can significantly improve the catalytic activity toward photocatalytic reactions due to the enhanced electron transportation from semiconductors to NG and the reduced recombination of the photogenerated electron-hole pairs [18][19][20]. In the work, we coupled CuFe2O4 to NG to prepare CuFe2O4/NG hybrid catalyst, it is expected that the Cu-based hybrid catalyst can recognize ammonia via coordination effect since the formation constant of Cu(NH3)4 2+ approached 1.1 × 10 13 . This large constant implies a strong trend to form a complex between NH3 molecules and Cu sites, and also capturing capacity of NH3 from aqueous solutions.
X-ray Photoelectron Spectroscopic Characterization
X-ray photoelectron spectroscopic (XPS) determination indicated the as-prepared NG sample is composed of C, N and O elements ( Figure 1A), in which C 1s, N 1s and O 1s peaks appeared at ~284.6, 400.0 and 534.0 eV, respectively. The percentages of C, N and O atoms are 83.24%, 7.84% and 8.92% in NG, respectively. The high-resolution XPS spectra of N, C, and O elements revealed the presence of the N 1s peaks at 398.7, 399.9, and 401.9 eV, which corresponded to the pyridinic, pyrrolic, and graphitic nitrogen atoms, respectively [21][22][23]. The pyridinic and pyrrolic nitrogen atoms are bonded with two carbon atoms and donate one or two p-electron to the aromatic π-system. Graphitic nitrogen, also called "quaternary nitrogen", indicates that nitrogen atoms have substituted the carbon atoms in graphene layers [24]. The further studies showed the percentage of pyridinic, pyrrolic, and graphitic nitrogen atoms was 25.06%, 68.84% and 6.10%, respectively, which is consistent with those reported [25,26]. The atom ratios of copper to iron in both pure CuFe2O4 and CuFe2O4/NG samples were also determined, the results are 1:2.17 and 1:2.31, respectively, indicating very close to the 1:2 stoichiometry. Figure 2 presented the X-ray powder diffraction (XRD) patterns of the as-prepared CuFe 2 O 4 /NG, CuFe 2 O 4 and NG samples, with CuFe 2 O 4 exhibiting the typical peaks of spinel ferrites with five prominent peaks occurring at 2θ = 30.28 • , 35.75 • , 57.80 • and 62.83 • . These diffraction peaks are indexed to Bragg planes (220), (311), (511) and (440), respectively. CuFe 2 O 4 is the major crystal phase, which is in good agreement with JCPDS 25-0283 for the cubic spinel CuFe 2 O 4 [27,28]. Peaks at 2θ = 32.90 • [plane 110] and 39.11 • [plane 111] suggests a small fraction of CuO. The diffraction peak at 26.43 • is indexed to Bragg plane (002) assigned to N-doped graphene. The peak at 30.28 • from the CuFe 2 O 4 /NG sample, as seen in curve a in Figure 2, is attributed to plane (220) of CuFe 2 O 4 [29,30]. But, the peak did not appear in the single CuFe 2 O 4 sample. It showed that NG component makes CuFe 2 O 4 more regular. In addition, plane (002) assigned to N-doped graphene did not appear in the CuFe 2 O 4 /NG sample, because the amount of NG is very small in the CuFe 2 O 4 /NG sample. The average diameters (D) of the as-prepared CuFe 2 O 4 and CuFe 2 O 4 /NG crystal sizes are 12.1 and 14.5 nm, respectively, which were determined using the Scherrer equation D = Kλ/(Wcosθ) at a diffraction angle of 35.75 • (2θ). The average size of CuFe 2 O 4 /NG crystals is enlarged by the surface self-assembly of Cu 2+ or Fe 3+ ions on NG sheets. Figure 1. A wide-scan XPS spectrum of as-synthesized NG sample (A) and the high-resolution spectrum of N 1s in NG sample (B). Figure 2 presented the X-ray powder diffraction (XRD) patterns of the as-prepared CuFe2O4/NG, CuFe2O4 and NG samples, with CuFe2O4 exhibiting the typical peaks of spinel ferrites with five prominent peaks occurring at 2θ = 30.28°, 35.75°, 57.80° and 62.83°. These diffraction peaks are indexed to Bragg planes (220), (311), (511) and (440), respectively. CuFe2O4 is the major crystal phase, which is in good agreement with JCPDS 25-0283 for the cubic spinel CuFe2O4 [27,28]. Peaks at 2θ = 32.90° [plane 110] and 39.11° [plane 111] suggests a small fraction of CuO. The diffraction peak at 26.43° is indexed to Bragg plane (002) assigned to N-doped graphene. The peak at 30.28° from the CuFe2O4/NG sample, as seen in curve a in Figure 2, is attributed to plane (220) of CuFe2O4 [29,30]. But, the peak did not appear in the single CuFe2O4 sample. It showed that NG component makes CuFe2O4 more regular. In addition, plane (002) assigned to N-doped graphene did not appear in the CuFe2O4/NG sample, because the amount of NG is very small in the CuFe2O4/NG sample. The average diameters (D) of the as-prepared CuFe2O4 and CuFe2O4/NG crystal sizes are 12.1 and 14.5 nm, respectively, which were determined using the Scherrer equation D = Kλ/(Wcosθ) at a diffraction angle of 35.75° (2θ). The average size of CuFe2O4/NG crystals is enlarged by the surface self-assembly of Cu 2+ or Fe 3+ ions on NG sheets. Figure 3A,B showed that the CuFe2O4 particles were dispersed on a layered structure of NG sheets. The high-resolution transmission electron microscopy (HRTEM) image displayed the lattice fringes. The d-spacing value between the adjacent lattice fringes is 0.25 nm ( Figure 3C), which is characteristic of (211) spinel planes [27,28]. The size of CuFe2O4 crystals looked uniform; the diameter of most crystal particles is distributed about 25-30 nm, which is in the scope of the average particle size of CuFe2O4 crystals that was estimated by XRD. Figure 3A,B showed that the CuFe 2 O 4 particles were dispersed on a layered structure of NG sheets. The high-resolution transmission electron microscopy (HRTEM) image displayed the lattice fringes. The d-spacing value between the adjacent lattice fringes is 0.25 nm ( Figure 3C), which is characteristic of (211) spinel planes [27,28]. The size of CuFe 2 O 4 crystals looked uniform; the diameter of most crystal particles is distributed about 25-30 nm, which is in the scope of the average particle size of CuFe 2 O 4 crystals that was estimated by XRD. Figure 1. A wide-scan XPS spectrum of as-synthesized NG sample (A) and the high-resolution spectrum of N 1s in NG sample (B). Figure 2 presented the X-ray powder diffraction (XRD) patterns of the as-prepared CuFe2O4/NG, CuFe2O4 and NG samples, with CuFe2O4 exhibiting the typical peaks of spinel ferrites with five prominent peaks occurring at 2θ = 30.28°, 35.75°, 57.80° and 62.83°. These diffraction peaks are indexed to Bragg planes (220), (311), (511) and (440), respectively. CuFe2O4 is the major crystal phase, which is in good agreement with JCPDS 25-0283 for the cubic spinel CuFe2O4 [27,28]. Peaks at 2θ = 32.90° [plane 110] and 39.11° [plane 111] suggests a small fraction of CuO. The diffraction peak at 26.43° is indexed to Bragg plane (002) assigned to N-doped graphene. The peak at 30.28° from the CuFe2O4/NG sample, as seen in curve a in Figure 2, is attributed to plane (220) of CuFe2O4 [29,30]. But, the peak did not appear in the single CuFe2O4 sample. It showed that NG component makes CuFe2O4 more regular. In addition, plane (002) assigned to N-doped graphene did not appear in the CuFe2O4/NG sample, because the amount of NG is very small in the CuFe2O4/NG sample. The average diameters (D) of the as-prepared CuFe2O4 and CuFe2O4/NG crystal sizes are 12.1 and 14.5 nm, respectively, which were determined using the Scherrer equation D = Kλ/(Wcosθ) at a diffraction angle of 35.75° (2θ). The average size of CuFe2O4/NG crystals is enlarged by the surface self-assembly of Cu 2+ or Fe 3+ ions on NG sheets. Figure 3A,B showed that the CuFe2O4 particles were dispersed on a layered structure of NG sheets. The high-resolution transmission electron microscopy (HRTEM) image displayed the lattice fringes. The d-spacing value between the adjacent lattice fringes is 0.25 nm ( Figure 3C), which is characteristic of (211) spinel planes [27,28]. The size of CuFe2O4 crystals looked uniform; the diameter of most crystal particles is distributed about 25-30 nm, which is in the scope of the average particle size of CuFe2O4 crystals that was estimated by XRD.
Ultraviolet-Visible Near-Infrared Diffuse Reflectance Spectroscopy
UV-visible near-infrared diffuse reflectance spectra were measured to elucidate the enhanced photocatalytic activity of the CuFe 2 O 4 /NG hybrid catalyst. Compared with curve b (CuFe 2 O 4 ) in Figure 4, curve a (CuFe 2 O 4 /NG) underwent a red-shift, indicating the CuFe 2 O 4 /NG hybrid catalyst can harvest more incident light energy.
Two Tauc-curves of the CuFe 2 O 4 /NG and CuFe 2 O 4 catalysts for the direct transition were obtained using transformation data from Figure 4 and are presented in Figure 5, respectively. Extrapolation of linear portions of the curves towards absorbance axis to zero (y = 0) gave a band gap (E g ) of direct transitions. The direct band gap estimated for the CuFe 2 O 4 sample is equal to 1.70 eV, which is in very good agreement with that reported [31]. As expected, the band gap for the CuFe 2 O 4 /NG sample shifted to 1.50 eV, the red-shift of 0.20 eV exhibited a strong electron-orbital interaction between NG and CuFe 2 O 4 and a widened absorption scope of solar irradiation. In fact, the absorption edge for the CuFe 2 O 4 /NG composite catalyst was extended to 870 nm as indicated in curve a in Figure 4. This wavelength falls in the scope of near-infrared light irradiation, which revealed the CuFe 2 O 4 /NG composite catalyst can utilize near-infrared irradiation for photocatalysis. UV-visible near-infrared diffuse reflectance spectra were measured to elucidate the enhanced photocatalytic activity of the CuFe2O4/NG hybrid catalyst. Compared with curve b (CuFe2O4) in Figure 4, curve a (CuFe2O4/NG) underwent a red-shift, indicating the CuFe2O4/NG hybrid catalyst can harvest more incident light energy.
Two Tauc-curves of the CuFe2O4/NG and CuFe2O4 catalysts for the direct transition were obtained using transformation data from Figure 4 and are presented in Figure 5, respectively. Extrapolation of linear portions of the curves towards absorbance axis to zero (y = 0) gave a band gap (Eg) of direct transitions. The direct band gap estimated for the CuFe2O4 sample is equal to 1.70 eV, which is in very good agreement with that reported [31]. As expected, the band gap for the CuFe2O4/NG sample shifted to 1.50 eV, the red-shift of 0.20 eV exhibited a strong electron-orbital interaction between NG and CuFe2O4 and a widened absorption scope of solar irradiation. In fact, the absorption edge for the CuFe2O4/NG composite catalyst was extended to 870 nm as indicated in curve a in Figure 4. This wavelength falls in the scope of near-infrared light irradiation, which revealed the CuFe2O4/NG composite catalyst can utilize near-infrared irradiation for photocatalysis. Figure 6 presented the separate degradation of ammonia and RhB using CuFe2O4/NG as the photocatalyst under visible near-infrared irradiation. The degradation ratios of 96.3% for ammonia and of 63.6% for RhB were achieved, respectively, at 5 h under visible-near-infrared irradiation in UV-visible near-infrared diffuse reflectance spectra were measured to elucidate the enhanced photocatalytic activity of the CuFe2O4/NG hybrid catalyst. Compared with curve b (CuFe2O4) in Figure 4, curve a (CuFe2O4/NG) underwent a red-shift, indicating the CuFe2O4/NG hybrid catalyst can harvest more incident light energy.
Molecular Recognition and Selective Photocatalysis
Two Tauc-curves of the CuFe2O4/NG and CuFe2O4 catalysts for the direct transition were obtained using transformation data from Figure 4 and are presented in Figure 5, respectively. Extrapolation of linear portions of the curves towards absorbance axis to zero (y = 0) gave a band gap (Eg) of direct transitions. The direct band gap estimated for the CuFe2O4 sample is equal to 1.70 eV, which is in very good agreement with that reported [31]. As expected, the band gap for the CuFe2O4/NG sample shifted to 1.50 eV, the red-shift of 0.20 eV exhibited a strong electron-orbital interaction between NG and CuFe2O4 and a widened absorption scope of solar irradiation. In fact, the absorption edge for the CuFe2O4/NG composite catalyst was extended to 870 nm as indicated in curve a in Figure 4. This wavelength falls in the scope of near-infrared light irradiation, which revealed the CuFe2O4/NG composite catalyst can utilize near-infrared irradiation for photocatalysis. Figure 6 presented the separate degradation of ammonia and RhB using CuFe2O4/NG as the photocatalyst under visible near-infrared irradiation. The degradation ratios of 96.3% for ammonia and of 63.6% for RhB were achieved, respectively, at 5 h under visible-near-infrared irradiation in 100 mg/L ammonia solution alone and 50 mg/L RhB solution alone (for the blank or controlled tests, see Figure S1 in Supplementary Materials). The facts show that the photocatalyst can degrade both ammonia and RhB in a single-component solution. The degradation for organic pollutants is consistent with the references reported by Qu and Wang [32,33]. In a mixture of ammonia and RhB with the same concentration of ammonia and RhB as that in Figure 6, however, the degradation ratio for ammonia reached 92.6% at 6 h whereas the degradation ratio for RhB decreased to 39.3% as shown in Figure 7. The high degradation ratio for ammonia but low one for RhB in the mixed solution confirms the CuFe 2 O 4 /NG photocatalyst prefers to degrade ammonia in mixed solution containing ammonia and organic compounds under visible near-infrared irradiation. The phenomenon also occurred between ammonia and methyl orange. The preference indicates that the CuFe 2 O 4 /NG photocatalyst can selectively eliminate ammonia. 100 mg/L ammonia solution alone and 50 mg/L RhB solution alone (for the blank or controlled tests, see Figure S1 in Supplementary Materials). The facts show that the photocatalyst can degrade both ammonia and RhB in a single-component solution. The degradation for organic pollutants is consistent with the references reported by Qu and Wang [32,33]. In a mixture of ammonia and RhB with the same concentration of ammonia and RhB as that in Figure 6, however, the degradation ratio for ammonia reached 92.6% at 6 h whereas the degradation ratio for RhB decreased to 39.3% as shown in Figure 7. The high degradation ratio for ammonia but low one for RhB in the mixed solution confirms the CuFe2O4/NG photocatalyst prefers to degrade ammonia in mixed solution containing ammonia and organic compounds under visible near-infrared irradiation. The phenomenon also occurred between ammonia and methyl orange. The preference indicates that the CuFe2O4/NG photocatalyst can selectively eliminate ammonia. 100 mg/L ammonia solution alone and 50 mg/L RhB solution alone (for the blank or controlled tests, see Figure S1 in Supplementary Materials). The facts show that the photocatalyst can degrade both ammonia and RhB in a single-component solution. The degradation for organic pollutants is consistent with the references reported by Qu and Wang [32,33]. In a mixture of ammonia and RhB with the same concentration of ammonia and RhB as that in Figure 6, however, the degradation ratio for ammonia reached 92.6% at 6 h whereas the degradation ratio for RhB decreased to 39.3% as shown in Figure 7. The high degradation ratio for ammonia but low one for RhB in the mixed solution confirms the CuFe2O4/NG photocatalyst prefers to degrade ammonia in mixed solution containing ammonia and organic compounds under visible near-infrared irradiation. The phenomenon also occurred between ammonia and methyl orange. The preference indicates that the CuFe2O4/NG photocatalyst can selectively eliminate ammonia.
Effect of NG Content
In order to obtain the optimal degradation conditions, the mass ratio of NG to CuFe 2 O 4 was optimized, the results were shown in Figure 8 (see detailed information in Figure S2 in Supplementary Materials), the mass percentage of 6% for NG to CuFe 2 O 4 resulted in an optimal degradation ratio (96.3%) at 5 h. Comparative studies showed that the CuFe 2 O 4 /NG catalyst possessed the highest activity for ammonia, compared with those of the CuFe 2 O 4 and CuFe 2 O 4 /rGO catalysts (see Figure S3 in Supplementary Materials). Therefore, NG can enhance the photocatalytic activity of CuFe 2 O 4 . In order to obtain the optimal degradation conditions, the mass ratio of NG to CuFe2O4 was optimized, the results were shown in Figure 8 (see detailed information in Figure S2 in Supplementary Materials), the mass percentage of 6% for NG to CuFe2O4 resulted in an optimal degradation ratio (96.3%) at 5 h. Comparative studies showed that the CuFe2O4/NG catalyst possessed the highest activity for ammonia, compared with those of the CuFe2O4 and CuFe2O4/rGO catalysts (see Figure S3 in Supplementary Materials). Therefore, NG can enhance the photocatalytic activity of CuFe2O4.
Degradation Kinetics
The different initial concentrations of ammonia were used as desired while the dosage of 0.1 g catalyst in 50.0 mL solution with pH 9.5 was kept constant. The degradation kinetic curves for the various ammonia concentrations were shown in Figure 9. The parameter ln(C0/Ct) is linearly proportional to the irradiation time t, following a pseudo-first order kinetic equation The average value of the apparent rate constant kapp can be estimated as 0.3224 h −1 , the standard error is equal to 0.01278.
Degradation Kinetics
The different initial concentrations of ammonia were used as desired while the dosage of 0.1 g catalyst in 50.0 mL solution with pH 9.5 was kept constant. The degradation kinetic curves for the various ammonia concentrations were shown in Figure 9. The parameter ln(C 0 /C t ) is linearly proportional to the irradiation time t, following a pseudo-first order kinetic equation The average value of the apparent rate constant k app can be estimated as 0.3224 h −1 , the standard error is equal to 0.01278. In order to obtain the optimal degradation conditions, the mass ratio of NG to CuFe2O4 was optimized, the results were shown in Figure 8 (see detailed information in Figure S2 in Supplementary Materials), the mass percentage of 6% for NG to CuFe2O4 resulted in an optimal degradation ratio (96.3%) at 5 h. Comparative studies showed that the CuFe2O4/NG catalyst possessed the highest activity for ammonia, compared with those of the CuFe2O4 and CuFe2O4/rGO catalysts (see Figure S3 in Supplementary Materials). Therefore, NG can enhance the photocatalytic activity of CuFe2O4.
Degradation Kinetics
The different initial concentrations of ammonia were used as desired while the dosage of 0.1 g catalyst in 50.0 mL solution with pH 9.5 was kept constant. The degradation kinetic curves for the various ammonia concentrations were shown in Figure 9. The parameter ln(C0/Ct) is linearly proportional to the irradiation time t, following a pseudo-first order kinetic equation The average value of the apparent rate constant kapp can be estimated as 0.3224 h −1 , the standard error is equal to 0.01278.
Stability of CuFe 2 O 4 /NG Catalyst
The cyclic tests were performed in order to evaluate the catalytic stability during a series of experiments. The catalyst of 0.1 g CuFe 2 O 4 /NG was tested in six consecutive experiments using the fresh ammonia solutions. The reaction time was about 5 h for each run. At the end of the previous experiment, the catalyst was collected using an external magnetite, and then separated and washed with deionized water for three times. It was observed that the sixth photocatalytic degradation ratio of ammonia using the same CuFe 2 O 4 /NG catalyst still achieved 92% (Figure 10), showing the CuFe 2 O 4 /NG catalyst is very stable. The spent catalyst was taken out after 6 runs and it was measured by TEM to confirm the stability. As seen in Figure S4 in Supplementary Materials, the nanoparticles were still distributed on NG sheets, the d-spacing value of 0.25 nm indicated that the adjacent lattice fringes of (211) spinel planes remained.
Stability of CuFe2O4/NG Catalyst
The cyclic tests were performed in order to evaluate the catalytic stability during a series of experiments. The catalyst of 0.1 g CuFe2O4/NG was tested in six consecutive experiments using the fresh ammonia solutions. The reaction time was about 5 h for each run. At the end of the previous experiment, the catalyst was collected using an external magnetite, and then separated and washed with deionized water for three times. It was observed that the sixth photocatalytic degradation ratio of ammonia using the same CuFe2O4/NG catalyst still achieved 92% (Figure 10), showing the CuFe2O4/NG catalyst is very stable. The spent catalyst was taken out after 6 runs and it was measured by TEM to confirm the stability. As seen in Figure S4 in Supplementary Materials, the nanoparticles were still distributed on NG sheets, the d-spacing value of 0.25 nm indicated that the adjacent lattice fringes of (211) spinel planes remained.
Identification of Products
According to Chio's and Butler's investigations [1,34], ammonia was oxidized into N2 and NO3 − (NO2 − ) by two paths. One is through a series of · NH2, · NH, N2Hx+y(x+y =0,1,2) intermediates, giving out N2 as a consequence. The other is to form the HONH2 and NO2 − intermediates, finally generating NO3 − . NO3 − or NO2 − ions will appear the absorption band in the wavelength range of 200 to 260 nm if there exists NO3 − or NO2 − in aqueous solution [33] (see Figures S5 and S6 in Supplementary Materials).
In our case the ultraviolet-visible spectroscopic measurements displayed that there is not any absorption band from 200 nm to 260 nm except noise wave during the photocatalytic process as shown in Figure 11, suggesting neither nitrite nor nitrate were formed during the degradation process since NO2 − and NO3 − can generate the absorption peaks at 211 nm and 206 nm, respectively [34][35][36].
Identification of Products
According to Chio's and Butler's investigations [1,34], ammonia was oxidized into N 2 and NO 3 − (NO 2 − ) by two paths. One is through a series of · NH 2 , · NH, N 2 H x+y(x+y =0,1,2) intermediates, In our case the ultraviolet-visible spectroscopic measurements displayed that there is not any absorption band from 200 nm to 260 nm except noise wave during the photocatalytic process as shown in Figure 11, suggesting neither nitrite nor nitrate were formed during the degradation process since NO 2 − and NO 3 − can generate the absorption peaks at 211 nm and 206 nm, respectively [34][35][36]. In order to confirm the product of oxidized ammonia, the detection of nitrogen gas (N2) was performed during the photocatalytic degradation of ammonia in the sealed photocatalytic reaction system mentioned previously, in which a 50 mL aqueous solution containing 100.0 mg/L NH3-N was irradiated under visible-near-infrared light, the mixed gas of oxygen and argon was cycled, and the released N2 was detected with gas chromatograph. The results were displayed in Figure 12. During the visible-near-infrared light irradiation, the peak of O2 gas in the sealed reaction system was declining with irradiation time, while the peak of N2 gas was boosting with the irradiation time, indicating the generation of N2 gas during the photocatalytic decomposition of ammonia. In order to confirm the product of oxidized ammonia, the detection of nitrogen gas (N 2 ) was performed during the photocatalytic degradation of ammonia in the sealed photocatalytic reaction system mentioned previously, in which a 50 mL aqueous solution containing 100.0 mg/L NH 3 -N was irradiated under visible-near-infrared light, the mixed gas of oxygen and argon was cycled, and the released N 2 was detected with gas chromatograph. The results were displayed in Figure 12. During the visible-near-infrared light irradiation, the peak of O 2 gas in the sealed reaction system was declining with irradiation time, while the peak of N 2 gas was boosting with the irradiation time, indicating the generation of N 2 gas during the photocatalytic decomposition of ammonia. In order to confirm the product of oxidized ammonia, the detection of nitrogen gas (N2) was performed during the photocatalytic degradation of ammonia in the sealed photocatalytic reaction system mentioned previously, in which a 50 mL aqueous solution containing 100.0 mg/L NH3-N was irradiated under visible-near-infrared light, the mixed gas of oxygen and argon was cycled, and the released N2 was detected with gas chromatograph. The results were displayed in Figure 12. During the visible-near-infrared light irradiation, the peak of O2 gas in the sealed reaction system was declining with irradiation time, while the peak of N2 gas was boosting with the irradiation time, indicating the generation of N2 gas during the photocatalytic decomposition of ammonia.
Molecular Recognition Evidence
In order to explore the mechanism for the selective degradation of ammonia, Raman spectroscopic measurements were performed before and after the CuFe 2 O 4 /NG catalyst was immersed in NH 3 solution and NH 3 -RhB mixed solution, respectively. Figure 13 displayed the Raman spectra of the CuFe 2 O 4 /NG catalyst (a) itself and NH 3 adsorbed on the CuFe 2 O 4 /NG catalyst (b) and both NH 3 and RhB adsorbed on the CuFe 2 O 4 /NG catalyst (c). As to the assignments of the Raman shifts of the CuFe 2 O 4 component in the composite catalyst, they were listed in Table 1 based on the FeO 4 symmetry [37].
Besides the Raman peaks of CuFe 2 O 4 /NG itself, a new peak at 1100 cm −1 appeared in Raman spectra both (b) and (c) after the CuFe 2 O 4 /NG catalyst was immersed in NH 3 solution and NH 3 -RhB mixed solution, respectively. This Raman shift at 1100 cm −1 was assigned to the bending vibration of NH 3 -H 2 O complex [38]. Thus, it unambiguously revealed that NH 3 was adsorbed on the CuFe 2 O 4 /NG catalyst in both cases.
For RhB Raman shifts, it is known that the spontaneous Raman shift range of RhB is from 500 to 1700 cm −1 [39], thus, the shift at 602 cm −1 from Raman spectrum (c) in Figure 13 was attributed to the C-C bending vibration of xanthene ring in the molecular structure of RhB [40,41]. For the NG component, there were two Raman shifts located at 1372 and 1580 cm −1 as seen in curves (a) and (b), respectively, which were attributed to D (defect-induced mode) and G (in-plane vibration mode) bands. The G band (1580 cm −1 ) derived from the G (1610 cm −1 ) of the pristine graphene, which also confirmed that nitrogen atoms were incorporated into graphene framework [42,43]. Compared with curve (b), the significant shifts occurred of D and G bands after the CuFe 2 O 4 /NG catalyst was immersed in NH 3 -RhB mixed solution, as seen curve (c). The D and G bands were shifted from 1372 and 1580 cm −1 to 1308 and 1560 cm −1 , respectively, which indicated the strong interaction between NG sheets and RhB, thereby RhB being adsorbed on NG sheets. It is reasonable that the π-π interaction between NG and RhB occurs because there are π bonds in their planar aromatic moieties [44,45] (for RhB structure, see Figure S7 in Supplementary Materials). However, compared with curve (a) in Figure 13, no shift of the D and G bands appeared in curve (b) after the CuFe 2 O 4 /NG catalyst was immersed in the single NH 3 component solution. It suggested that NH 3 was adsorbed preferentially on CuFe 2 O 4 moiety, but not NG, in the composite catalyst.
Molecular Recognition Evidence
In order to explore the mechanism for the selective degradation of ammonia, Raman spectroscopic measurements were performed before and after the CuFe2O4/NG catalyst was immersed in NH3 solution and NH3-RhB mixed solution, respectively. Figure 13 displayed the Raman spectra of the CuFe2O4/NG catalyst (a) itself and NH3 adsorbed on the CuFe2O4/NG catalyst (b) and both NH3 and RhB adsorbed on the CuFe2O4/NG catalyst (c). As to the assignments of the Raman shifts of the CuFe2O4 component in the composite catalyst, they were listed in Table 1 based on the FeO4 symmetry [37].
Besides the Raman peaks of CuFe2O4/NG itself, a new peak at 1100 cm −1 appeared in Raman spectra both (b) and (c) after the CuFe2O4/NG catalyst was immersed in NH3 solution and NH3-RhB mixed solution, respectively. This Raman shift at 1100 cm −1 was assigned to the bending vibration of NH3-H2O complex [38]. Thus, it unambiguously revealed that NH3 was adsorbed on the CuFe2O4/NG catalyst in both cases.
For RhB Raman shifts, it is known that the spontaneous Raman shift range of RhB is from 500 to 1700 cm −1 [39], thus, the shift at 602 cm −1 from Raman spectrum (c) in Figure 13 was attributed to the C-C bending vibration of xanthene ring in the molecular structure of RhB [40,41]. For the NG component, there were two Raman shifts located at 1372 and 1580 cm −1 as seen in curves (a) and (b), respectively, which were attributed to D (defect-induced mode) and G (in-plane vibration mode) bands. The G band (1580 cm −1 ) derived from the G (1610 cm −1 ) of the pristine graphene, which also confirmed that nitrogen atoms were incorporated into graphene framework [42,43]. Compared with curve (b), the significant shifts occurred of D and G bands after the CuFe2O4/NG catalyst was immersed in NH3-RhB mixed solution, as seen curve (c). The D and G bands were shifted from 1372 and 1580 cm −1 to 1308 and 1560 cm −1 , respectively, which indicated the strong interaction between NG sheets and RhB, thereby RhB being adsorbed on NG sheets. It is reasonable that the π-π interaction between NG and RhB occurs because there are π bonds in their planar aromatic moieties [44,45] (for RhB structure, see Figure S7 in Supplementary Materials). However, compared with curve (a) in Figure 13, no shift of the D and G bands appeared in curve (b) after the CuFe2O4/NG catalyst was immersed in the single NH3 component solution. It suggested that NH3 was adsorbed preferentially on CuFe2O4 moiety, but not NG, in the composite catalyst. ν2 ν4 ν4 ν3 ν3 ν1 a * 170 223 285 378 452~472 565 684 722 b 180 241 285 378 472 565 684 725 c -216 285 397 489 -656 In order to confirm ammonia adsorbed on CuFe 2 O 4 , the single CuFe 2 O 4 sample (to avoids interference of nitrogen from NG) was synthesized and immersed in 100 mg/L NH 3 -N solution for 4 h for adsorption, then it was taken out and washed with deionized water for three times to remove ammonia adsorbed physically. Finally, it was dried at 60 • C in a vacuum chamber for XPS measurement. For the sake of contrast, other CuFe 2 O 4 component that was not immersed in NH 3 solution was also used for XPS measurement. Figure 14 displayed the high-resolution XPS spectrum of N 1s in the CuFe 2 O 4 sample after it was immersed in a 100 mg/L NH 3 -N solution. It indicated evidently the presence of nitrogen atoms, whereas an N 1s peak was not detected out when the CuFe 2 O 4 sample was not immersed in such 100 mg/L NH 3 -N solution. The dissimilarity implies that ammonia was adsorbed on the CuFe 2 O 4 sample when it was immersed in ammonia solution. Deconvolution of the XPS spectrum of N 1s may be ascribed to the Eb of NH 3 (399.97 eV), copper-NH 3 (398.30 eV) and NO 2 − [46], which implied ammonia is partly oxidized in air atmosphere. T/L ν2 ν4 ν4 ν3 ν3 ν1 a * 170 223 285 378 452~472 565 684 722 b 180 241 285 378 472 565 684 725 c -216 285 397 489 -656 In order to confirm ammonia adsorbed on CuFe2O4, the single CuFe2O4 sample (to avoids interference of nitrogen from NG) was synthesized and immersed in 100 mg/L NH3-N solution for 4 h for adsorption, then it was taken out and washed with deionized water for three times to remove ammonia adsorbed physically. Finally, it was dried at 60 °C in a vacuum chamber for XPS measurement. For the sake of contrast, other CuFe2O4 component that was not immersed in NH3 solution was also used for XPS measurement. Figure 14 displayed the high-resolution XPS spectrum of N 1s in the CuFe2O4 sample after it was immersed in a 100 mg/L NH3-N solution. It indicated evidently the presence of nitrogen atoms, whereas an N 1s peak was not detected out when the CuFe2O4 sample was not immersed in such 100 mg/L NH3-N solution. The dissimilarity implies that ammonia was adsorbed on the CuFe2O4 sample when it was immersed in ammonia solution. Deconvolution of the XPS spectrum of N 1s may be ascribed to the Eb of NH3 (399.97 eV), copper-NH3 (398.30 eV) and NO2 − [46], which implied ammonia is partly oxidized in air atmosphere. Furthermore, the coordination between NH3 and Cu(II) or Fe(III) was identified by XPS technique. The high-resolution XPS measurements of Cu 2p and Fe 2p in the CuFe2O4 sample were conducted as shown in Figures 15 and 16 before (a) and after (b) the CuFe2O4 sample was immersed in 100 mg/L NH3-N solution, respectively. Very interestingly, the shifts of Eb at 933.10 eV for Cu 2p 3/2 (0.75 eV) and at 953.06 eV for Cu 2p 1/2 (0.50 eV) were observed [47], implying that the chemical surroundings of copper in the CuFe2O4 sample were altered after it was immersed in 100 mg/L NH3-N solution. However, the shifts of Eb at 710.56 eV for Fe 2p 3/2 and Eb at 724.10 eV for Fe 2p 1/2 were not observed as shown in Figure 15 [48]. It is well known that the formation constant of Cu(NH3)4 2+ is up to 1.1 × 10 13 . Thus, it is reasonable that the CuFe2O4/NG catalyst can recognize ammonia via a coordination effect between copper atoms in the catalyst and nitrogen atoms in ammonia; it is concluded that the coordination interaction occurred between copper, but not iron and ammonia. Furthermore, the coordination between NH 3 and Cu(II) or Fe(III) was identified by XPS technique. The high-resolution XPS measurements of Cu 2p and Fe 2p in the CuFe 2 O 4 sample were conducted as shown in Figures 15 and 16 before (a) and after (b) the CuFe 2 O 4 sample was immersed in 100 mg/L NH 3 -N solution, respectively. Very interestingly, the shifts of Eb at 933.10 eV for Cu 2p 3/2 (0.75 eV) and at 953.06 eV for Cu 2p 1/2 (0.50 eV) were observed [47], implying that the chemical surroundings of copper in the CuFe 2 O 4 sample were altered after it was immersed in 100 mg/L NH 3 -N solution. However, the shifts of Eb at 710.56 eV for Fe 2p 3/2 and Eb at 724.10 eV for Fe 2p 1/2 were not observed as shown in Figure 15 [48]. It is well known that the formation constant of Cu(NH 3 ) 4 2+ is up to 1.1 × 10 13 . Thus, it is reasonable that the CuFe 2 O 4 /NG catalyst can recognize ammonia via a coordination effect between copper atoms in the catalyst and nitrogen atoms in ammonia; it is concluded that the coordination interaction occurred between copper, but not iron and ammonia.
Reaction Mechanism
The surface photovoltage spectroscopy (SPV) was utilized to explore the transfer of the photo-generated holes and photo-generated electrons in order to elucidate the degradation mechanism of ammonia. As known, the surface photovoltage is defined as the illumination-induced change in the surface potential [49], being equal to the difference between the surface potential under illumination and the surface potential in dark given by Equation (2) SPV = Vs(ill) − Vs(dark) As far as the band-to-band transitions are concerned, a positive response of SPV means that the photo-generated holes move to the irradiation side of the sample, whereas the photo-generated electrons move into the bulk of the sample [50,51]. That is, the semiconductor material is an n-type semiconductor in this case. On the contrary, a negative response represents a p-type semiconductor. In the present case, the SPV responses measured for the CuFe2O4 sample were shown in Figure 17.
Reaction Mechanism
The surface photovoltage spectroscopy (SPV) was utilized to explore the transfer of the photo-generated holes and photo-generated electrons in order to elucidate the degradation mechanism of ammonia. As known, the surface photovoltage is defined as the illumination-induced change in the surface potential [49], being equal to the difference between the surface potential under illumination and the surface potential in dark given by Equation (2) SPV = Vs(ill) − Vs(dark) As far as the band-to-band transitions are concerned, a positive response of SPV means that the photo-generated holes move to the irradiation side of the sample, whereas the photo-generated electrons move into the bulk of the sample [50,51]. That is, the semiconductor material is an n-type semiconductor in this case. On the contrary, a negative response represents a p-type semiconductor. In the present case, the SPV responses measured for the CuFe2O4 sample were shown in Figure 17.
Reaction Mechanism
The surface photovoltage spectroscopy (SPV) was utilized to explore the transfer of the photo-generated holes and photo-generated electrons in order to elucidate the degradation mechanism of ammonia. As known, the surface photovoltage is defined as the illumination-induced change in the surface potential [49], being equal to the difference between the surface potential under illumination and the surface potential in dark given by Equation (2) SPV = Vs (ill) − Vs (dark) (2) As far as the band-to-band transitions are concerned, a positive response of SPV means that the photo-generated holes move to the irradiation side of the sample, whereas the photo-generated electrons move into the bulk of the sample [50,51]. That is, the semiconductor material is an n-type semiconductor in this case. On the contrary, a negative response represents a p-type semiconductor. In the present case, the SPV responses measured for the CuFe 2 O 4 sample were shown in Figure 17. Curve (a) in Figure 17 presents a positive response under incident light illumination, suggesting that the photo-generated holes move to the illuminated surface of the CuFe2O4 sample. Applying a positive bias of 0.1 V to the CuFe2O4 sample suppressed the holes moving to the surface, resulting in a decreased SPV response (b); whereas a negative bias of 0.1 V promoted the holes moving to the surface, leading to an increased SPV response (c). It can be concluded from these facts that the CuFe2O4 material is an n-type semiconductor in the case [52,53]. The XPS valence band spectra for the CuFe2O4 and NG semiconductor materials have been determined as shown in Figure 18A,B. The valence bands are equal to 1.7 eV and 1.4 eV for CuFe2O4 and NG [54], respectively. The conduction bands are equal to 0.2 eV and −0.1 eV for CuFe2O4 and NG, respectively, based on their measurements of band gaps. UV-visible near-infrared diffuse reflectance spectrum of NG sheets has been measured as shown in Figure 19. As seen, the absorption edge of NG sheets extended to near-infrared region, the insert indicated a direct band gap of 1.50 eV for the as-synthesized NG sheets, corresponding to 826 nm near-infrared incident light. As Eda and Chai have postulated that isolated sp2 nanodomains are likely to exhibit quantum confinement-induced semiconducting behavior [55,56]. The local energy gaps of π-π* transition then vary depending on the size, shape and fraction of these sp2 domains. The smaller this sp2 domain, the higher the outcome of the energy gap [53]. The calculated energy gap between HOMO and LUMO for a cluster of 37 rings is ∼2 eV, and this energy gap progressively increases to ∼7 eV for a single benzene ring [57]. Therefore, it is reasonable that the as-synthesized NG sheets work as a semiconductor with a band gap of 1.50 eV in the present case. The conduction Curve (a) in Figure 17 presents a positive response under incident light illumination, suggesting that the photo-generated holes move to the illuminated surface of the CuFe 2 O 4 sample. Applying a positive bias of 0.1 V to the CuFe 2 O 4 sample suppressed the holes moving to the surface, resulting in a decreased SPV response (b); whereas a negative bias of 0.1 V promoted the holes moving to the surface, leading to an increased SPV response (c). It can be concluded from these facts that the CuFe 2 O 4 material is an n-type semiconductor in the case [52,53]. The XPS valence band spectra for the CuFe 2 O 4 and NG semiconductor materials have been determined as shown in Figure 18A Curve (a) in Figure 17 presents a positive response under incident light illumination, suggesting that the photo-generated holes move to the illuminated surface of the CuFe2O4 sample. Applying a positive bias of 0.1 V to the CuFe2O4 sample suppressed the holes moving to the surface, resulting in a decreased SPV response (b); whereas a negative bias of 0.1 V promoted the holes moving to the surface, leading to an increased SPV response (c). It can be concluded from these facts that the CuFe2O4 material is an n-type semiconductor in the case [52,53]. The XPS valence band spectra for the CuFe2O4 and NG semiconductor materials have been determined as shown in Figure 18A,B. The valence bands are equal to 1.7 eV and 1.4 eV for CuFe2O4 and NG [54], respectively. The conduction bands are equal to 0.2 eV and −0.1 eV for CuFe2O4 and NG, respectively, based on their measurements of band gaps. UV-visible near-infrared diffuse reflectance spectrum of NG sheets has been measured as shown in Figure 19. As seen, the absorption edge of NG sheets extended to near-infrared region, the insert indicated a direct band gap of 1.50 eV for the as-synthesized NG sheets, corresponding to 826 nm near-infrared incident light. As Eda and Chai have postulated that isolated sp2 nanodomains are likely to exhibit quantum confinement-induced semiconducting behavior [55,56]. The local energy gaps of π-π* transition then vary depending on the size, shape and fraction of these sp2 domains. The smaller this sp2 domain, the higher the outcome of the energy gap [53]. The calculated energy gap between HOMO and LUMO for a cluster of 37 rings is ∼2 eV, and this energy gap progressively increases to ∼7 eV for a single benzene ring [57]. Therefore, it is reasonable that the as-synthesized NG sheets work as a semiconductor with a band gap of 1.50 eV in the present case. The conduction UV-visible near-infrared diffuse reflectance spectrum of NG sheets has been measured as shown in Figure 19. As seen, the absorption edge of NG sheets extended to near-infrared region, the insert indicated a direct band gap of 1.50 eV for the as-synthesized NG sheets, corresponding to 826 nm near-infrared incident light. As Eda and Chai have postulated that isolated sp2 nanodomains are likely to exhibit quantum confinement-induced semiconducting behavior [55,56]. The local energy gaps of π-π* transition then vary depending on the size, shape and fraction of these sp2 domains. The smaller this sp2 domain, the higher the outcome of the energy gap [53]. The calculated energy gap between HOMO and LUMO for a cluster of 37 rings is ∼2 eV, and this energy gap progressively increases to ∼7 eV for a single benzene ring [57]. Therefore, it is reasonable that the as-synthesized NG sheets work as a semiconductor with a band gap of 1.50 eV in the present case. The conduction band of NG sheets can thereby be estimated as −0.10 eV based on its band gap of 1.50 eV and valence band of 1.40 eV measured in Figure 18B. band of NG sheets can thereby be estimated as −0.10 eV based on its band gap of 1.50 eV and valence band of 1.40 eV measured in Figure 18B. Under consideration of the standard electrodes (E 0 for N2/NH3 redox is 0.057 V vs. NHE, E 0 for O2/H2O is 1.23 V vs. NHE), a Z-scheme mechanism can be suggested as demonstrated in Scheme 1 [2,8]. Scheme 1. Z-scheme photocatalytic mechanism of CuFe2O4/NG. The photo-generated holes leave on CuFe2O4 to oxidize NH3 to N2, while the photo-generated electrons on the conduction band of CuFe2O4 flow to NG sheets along the Z-scheme configuration to reduce O2 molecules under visible-near-infrared light irradiation.
As mentioned previously, NH3 molecules were selectively adsorbed on the CuFe2O4 component, while RhB molecules were dominantly adsorbed on the NG sheets by π-π interaction. Therefore, NH3 molecules were oxidized by photo-generated holes on the valence band of copper ferrite to N2.
Under consideration of the standard electrodes (E 0 for N 2 /NH 3 redox is 0.057 V vs. NHE, E 0 for O 2 /H 2 O is 1.23 V vs. NHE), a Z-scheme mechanism can be suggested as demonstrated in Scheme 1 [2,8]. band of NG sheets can thereby be estimated as −0.10 eV based on its band gap of 1.50 eV and valence band of 1.40 eV measured in Figure 18B. Under consideration of the standard electrodes (E 0 for N2/NH3 redox is 0.057 V vs. NHE, E 0 for O2/H2O is 1.23 V vs. NHE), a Z-scheme mechanism can be suggested as demonstrated in Scheme 1 [2,8]. Scheme 1. Z-scheme photocatalytic mechanism of CuFe2O4/NG. The photo-generated holes leave on CuFe2O4 to oxidize NH3 to N2, while the photo-generated electrons on the conduction band of CuFe2O4 flow to NG sheets along the Z-scheme configuration to reduce O2 molecules under visible-near-infrared light irradiation.
As mentioned previously, NH3 molecules were selectively adsorbed on the CuFe2O4 component, while RhB molecules were dominantly adsorbed on the NG sheets by π-π interaction. Therefore, NH3 molecules were oxidized by photo-generated holes on the valence band of copper ferrite to N2.
Synthesis of N-Doped Graphene
Hummers' method was initially adopted to synthesize graphene oxide (GO) as our group has previously synthesized [59] and NG was synthesized as the reference [25]. The as-synthesized GO (0.1000 g) was ultrasonically dispersed in 25.0 mL of deionized water. Urea (30.0000 g) was dissolved in 25.0 mL of deionized water under stirring. The urea solution was added dropwise to the GO suspension solution under stirring. Subsequently, deionized water of 10 mL was added in the mixture and ultrasonicated for 2 h. After that, the mixture was transferred to a Teflon-lined stainless-steel autoclave with a volume of 60 mL, sealed and heated to 170 • C for 12 h to form NG. Finally, the resulting product was filtered, washed and dried in a vacuum chamber for use.
Synthesis of CuFe 2 O 4 /NG
A one-step hydrothermal route has been used for preparing CuFe 2 O 4 /NG samples [60]. Cu(NO 3 ) 2 ·3H 2 O (1.2080 g, 0.005 mol) and Fe(NO 3 ) 3 ·9H 2 O (4.0400 g, 0.01 mol) were separately dissolved in 10.0 mL of deionized water. NG (0.072 g, 6.0% of the CuFe 2 O 4 mass) was dispersed in 10.0 mL of deionized water by an ultrasonic vibrator. The Cu(II) and Fe(III) solutions were added to NG suspension solution under stirring. NaOH (1.6 g, 0.04 mol) was dissolved in 10.0 mL of deionized water, and this solution was added dropwise to the mixed suspension solution described above under continuous stirring. Deionized water was also added to the suspension to obtain a final volume of 60 mL. The suspension solution was then transferred to a 100 mL Teflon-lined stainless-steel autoclave that was subsequently sealed and maintained at 180 • C for 10 h. The solution was cooled to room temperature and filtered to obtain CuFe 2 O 4 /NG precipitates. The CuFe 2 O 4 nanoparticles can be formed by reaction Equation (4). The products were rinsed thrice with water to remove excess NaOH and other electrolytes and dried at 60 • C in a vacuum chamber for use. Cu(NO 3 ) 2 + 2Fe(NO 3 ) 3 + 8NaOH = CuFe 2 O 4 + 8NaNO 3 + 4H 2 O (8)
Photovoltage Characterization
Surface photovoltage spectra were recorded using a lock-in amplifier (SR830, Stanford Research Systems, Sunnyvale, CA, USA). The measurement system was composed of a 500 W xenon lamp, a monochromator (SBP500, Zolix Instruments Co., Ltd., Beijing China), a lock-in amplifier with a light chopper (SR540, Stanford Research Systems, Sunnyvale, CA, USA), and a sample chamber. The xenon lamp emitted incident photons with various wavelengths, which then passed through the monochromator to provide monochromatic light. The light was chopped with a frequency of 23 Hz, and its intensity depended on the spectral energy distribution of the lamp. The monochromator and the lock-in amplifier were controlled by a computer. The input resistance of the lock-in amplifier was set as 10 MΩ. SPV spectra were recorded by scanning from low to high photon energy. A UV-cutoff filter (λ > 420 nm) was employed at incident photon energy hv < 2.14 eV (λ > 580 nm) to remove the frequency and double the amount of light generated by the grating monochromator (doubled frequency of λ > 580 nm). The system was calibrated by a DSI200 UV-enhanced silicon detector to eliminate possible phase shift not correlated with the SPV response; thus, any phase retardation reflected the kinetics of SPV response [61].
Molecular Recognition and Selective Photocatalysis
Photocatalytic experiments were conducted under visible-near-infrared irradiation (λ > 400 nm). A 300 W UV-visible lamp (OSRAM, Munich, Germany) was used as a light source. Photocatalytic degradation was performed in a 100 mL beaker at room temperature (25 ± 2 • C). The distance between the lamp and test solution was approximately 10 cm, and the wall of the beaker was shielded from surrounding light by aluminum foil. Visible-near-infrared light was allowed to pass through a λ > 400 nm cut-off filter covering the window of the beaker; this filter absorbed UV light and allowed visible-near-infrared light of λ > 400 nm to pass through. In a typical photocatalytic experiment, 50 mL of test solution was used. The NH 3 solutions were prepared according to the desired concentrations, and the CuFe 2 O 4 /NG catalyst of 0.1 g was used for the photocatalytic experiments. NaHCO 3 -Na 2 CO 3 (0.1 mol/L) buffer was used to control the pH of the test solutions. For the selective photocatalytic degradation, the CuFe 2 O 4 /NG catalyst of 0.1 g was immersed in NH 3 -RhB mixed solution for 2 h first, then the visible-near-infrared light source was turned on for the photocatalytic tests.
A double-beam TU-1901 spectrophotometer (PGENERAL Instrument Limited-liability Company, Beijing, China) was used to determine the concentration of NH 3 by reaction with Nessler reagent during the photocatalytic process. Nessler reagent is an alkaline solution of dipotassium tetraiodomercurate(II), this reagent was prepared by dissolving 10 g of HgI 2 and 7 g of KI in water, adding to NaOH solution (16 g NaOH in 50 mL of water), and then diluting with deionized water to 100 mL. The reagent was stored in dark bottles and diluted properly before analysis. NH 3 reacts with this reagent to yield colored solutions via Reaction (2) previously. As the absorbance of the solutions showed a maximum value at 392 nm, the absorbance was measured at the wavelength of 392 nm.
The procedures for the stability tests are follows: The CuFe 2 O 4 /NG catalyst was sunk by a magnetic field outside due to the CuFe 2 O 4 magnetism after the last test finished. And then, the supernatant solution tested was poured out, the solid catalyst was kept in. Finally, a 50 mL fresh NH 3 solution was poured into the reactor. Subsequently, the next test was carried out again.
Conclusions
The composite photocatalyst composed of copper ferrite and N-doped graphene enables to recognize ammonia from NH 3 -RhB mixed solution. The measurements of gas chromatography show that the composite photocatalyst oxidizes NH 3 selectively to non-toxic N 2 under visible near-infrared light irradiation, thereby fulfilling the removal of nitrogen, the effect solar use and purification of water. The mechanism studies indicate that the photo-generated electrons flow to N-doped graphene following the Z-scheme configuration to reduce O 2 dissolved in solution. | v3-fos-license |
2018-04-03T03:38:32.117Z | 2011-11-05T00:00:00.000 | 208886928 | {
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} | pes2o/s2orc | 4-Chloro-N-phenylbenzamide
In the title compound, C13H10ClNO, the dihedral angle between the two benzene rings is 59.6 (1)°. The crystal structure features N—H⋯O hydrogen bonds, which link the molecules into C(4) chains running along the a axis.
In the title compound, C 13 H 10 ClNO, the dihedral angle between the two benzene rings is 59.6 (1) . The crystal structure features N-HÁ Á ÁO hydrogen bonds, which link the molecules into C(4) chains running along the a axis.
Data collection: CrysAlis CCD (Oxford Diffraction, 2009); cell refinement: CrysAlis CCD; data reduction: CrysAlis RED (Oxford Diffraction, 2009); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND (Brandenburg, 2002); software used to prepare material for publication: enCIFer (Allen et al., 2004). LK and JK thank the Grant Agencies for their financial support (VEGA Grant Agency of Slovak Ministry of Education 1/0679/11; Research and Development Agency of Slovakia (APVV-0202-10) and the Structural Funds, Interreg IIIA, for financial support in purchasing the diffractometer. VZR thanks the University Grants Commission, Government of India, New Delhi, for the award of an RFSMS research fellowship.
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: BT5692).
In (I), the N-H and C=O bonds in the C-NH-C(O)-C segment are anti to each other, similar to that observed in N-(4-chlorophenyl)-benzamide (II) (Gowda et al., 2008).
The dihedral angle between the two benzene rings is 59.6 (1)°, compared to the value of 60.8 (1)° in (II).
The packing of molecules linked by N-H···O hydrogen bonds into infinite chains is shown in Fig. 2.
Experimental
The title compound was prepared according to the method described by Gowda et al. (2003). The purity of the compound was checked by determining its melting point. It was characterized by recording its infrared and NMR spectra. Rod-like colourless single crystals of the title compound were obtained by slow evaporation from an ethanol solution of the compound (0.5 g in about 30 ml of ethanol) at room temperature. | v3-fos-license |
2018-09-23T19:46:07.310Z | 2018-09-21T00:00:00.000 | 52315194 | {
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} | pes2o/s2orc | New Insight into the Chloroacetanilide Herbicide Degradation Mechanism through a Nucleophilic Attack of Hydrogen Sulfide
The nucleophilic attack of hydrogen sulfide (HS−) on six different chloroacetanilide herbicides was evaluated theoretically using the dispersion-corrected hybrid functional wB97XD and the 6-311++G(2d,2p) Pople basis sets. The six evaluated substrates were propachlor (A), alachlor (B), metolachlor (C), tioacetanilide (D), β-anilide (E), and methylene (F). Three possible mechanisms were considered: (a) bimolecular nucleophilic substitution (SN2) reaction mechanism, (b) oxygen assistance, and (c) nitrogen assistance. Mechanisms based on O- and N-assistance were discarded due to a very high activation barrier in comparison with the corresponding SN2 mechanism, with the exception of compound F. The N-assistance mechanism for compound F had a free activation energy of 23.52 kcal/mol, which was close to the value for the corresponding SN2 mechanism (23.94 kcal/mol), as these two mechanisms could occur in parallel reactions with almost 50% of each one. In compounds A to D, an important electron-withdrawing effect of the C=O and C=S groups was seen, and consequently, the activation free energies in these SN2 reactions were smaller, with a value of approximately 18 kcal/mol. Instead, compounds E and F, which have a CH2 group in the β-position, presented a higher activation free energy (≈22 kcal/mol). Good agreement was found between experimental and theoretical values for all cases, and a reaction force analysis was performed on the intrinsic reaction coordinate profile in order to gain more details about the reaction mechanism. Finally, from the natural bond orbital (NBO) analysis, it was possible to evaluate the electronic reorganization through the reaction pathway where all the transition states were early in nature in the reaction coordinate (δBav < 50%); the transition states corresponding to compounds A to D turned out to be more synchronous than those for compounds E and F.
Introduction
Among the diversity of chemical compounds, pesticides constitute a heterogeneous category used specifically for the control of pests, plant diseases, and to eradicate the unwanted weeds. The synthesis and production of pesticides represent very important fields of industry. Currently, the use of pesticides remains the most effective process for the protection of plants from pests; moreover, pesticides contribute significantly to enhancing productivity and crop yields around the world [1].
Herbicides are a class of pesticides that stand out for their expanded use around the entire world. Four of them (acetochlor, alachlor, butachlor, and metolachlor) are widely used in the production of several economically important crops. The annual production of these four compounds is about 125 million pounds per year; just metolachlor alone has an annual production of 65 million pounds per year in the United States (US) [2].
Despite the importance of the production of herbicide, one aspect is still dramatic: the carcinogenic potential of chloroacetanilide herbicides. Alachlor, acetochlor, and metolachlor were characterized by the US Environmental Protection Agency as likely or possible carcinogenic compounds. However, the carcinogenic mechanism of chloroacetanilide compounds is still unclear, despite some experiments showing evidence that the carcinogenic properties relate to the herbicides' ability to react with DNA through nucleophilic reactions [3,4].
Another important characteristic of chloroacetanilide herbicides is their resistance to natural degradation in various environments. Their water solubility and great persistence in the environment represent topics of greatest interest in the scientific community. Currently, several scientific researchers are trying not only to avoid pollution, but are also searching for methods that help mitigate the already polluted soil and water systems [4].
Even with the already known environmental persistence and the pollutant characteristic of chloroacetanilide herbicides, until now, there are few decontamination methods used to remove or degrade these compounds. Bioremediation, chemical oxidation, and physical methods are used without much success; therefore, it is imperative to search for new, safe, effective, and low-cost decontamination methods for chloroacetanilide herbicide pollution [4][5][6][7].
Recently, some reports suggested a high selectivity of chloroacetanilide herbicides in the reaction with inorganic compounds such as sulfur nucleophiles like HS − , S 2− , and S 2 O 3 2− ; moreover, several earlier works documented the great reactivity of chloroacetanilide compounds toward the thiolate group of glutathione (GSH) [8,9]. Considering the importance of GSH in the biologic activity of plants, this latter characteristic allows the widespread use of chloroacetanilide for crop production. In other words, the knowledge of the reaction mechanism for chloroacetanilide against sulfur nucleophiles has a pivotal role in understanding not only the action pathway of phytotoxicity, but it could also be an opportunity to design new chemical remediation methods [8].
Several experiments were performed in order to understand the reactivity of chloroacetanilide herbicides toward thiolate groups. Even though the nucleophilic displacement of chlorine by the thiolate group of glutathione (GSH) seems to overall follow second-order kinetics via an intermolecular bimolecular nucleophilic substitution (S N 2 ) mechanism, some reported experimental results for similar compounds do not support this mechanism; e.g., propachlor obeys a second-order law, while methylene (analog of propachlor) obeys a first-order law [9,10]. Until now, the reaction mechanism of chlorine displacement to the sulfur nucleophile remains unclear, and it seems to be a more complex mechanism than a bimolecular nucleophilic substitution (S N 2 ).
Some authors proposed different reaction pathways in order to explain the kinetic behavior of chloroacetanilide toward sulfur nucleophiles. Bordwell et al. [11] stated that the activation of chlorine atoms could proceed from the interactions of anilide moiety with the incipient electrophilic center. They argued the electronic interactions of carbonyl substituents activate alkyl halides toward S N 2 reactions with strong nucleophiles, while the carbonyl moiety is able to deactivate the reactivity toward weaker nucleophiles such as amines. In contrast, a considerable increase in the electrophilicity characteristic of the CH 2 Cl group due to the α-carbonyl moiety's electronic effects was suggested; consequently, the compounds are able to react toward softer nucleophiles such as GSH and other SH-containing moieties via an S N 2 mechanism [11-13].
Arcelli et al. [14] explained the reactivity of chloroacetanilide herbicides via anchimeric assistance provided by the ether oxygen. However, if this anchimeric assistance were the only factor responsible of reactivity, it would be expected that chloroacetanilides such as alachlor and metolachlor (which have N-alkoxyalkyl side chains) would be more reactive than propachlor (with an N-alkyl substituent). In fact, there is important evidence for this statement: propachlor is more reactive toward sulfur nucleophiles than alachlor and metolachlor; consequently, this result led us to invoke new alternative explanations.
Due to the uncertainty found with respect to the reactivity of chloroacetanilide with sulfur nucleophiles, it is necessary to explore, in more detail, the reaction mechanism of these substrates. In this sense, computational methods could be a helpful tool for this goal.
This work seeks to explore chloroacetanilide's reactivity with HS − nucleophiles in order to propose a reasonable mechanistic interpretation based on theoretical calculations. With this aim, the potential energy surfaces were examined using the density functional theory (DFT) level, and the results were analyzed and compared with the experimental data. The minimum energy structure of reactants, the transition state, and the product were calculated, taking into account all possible reaction mechanisms suggested in the literature.
The processes of rupture and bond formations were studied using natural bond orbital calculations (NBO). In addition, intrinsic coordinate path reactions (IRC) and reaction force profiles were used to gain more insight into the reaction pathway.
Results and Discussion
According to the reported products for the reaction between HS − and chloroacetanilide herbicides [15], and based on the structure of each substrate, three possible mechanisms can be suggested. The main mechanism is the well-known S N 2 displacement (Scheme 1), which is proposed for all compounds. In the case of compounds B and C, the halogen elimination carried out by an anchimeric assistance of the oxygen atom can be considered (Scheme 2). For compounds E and F, due to the length of the carbon chain in the first case, and the lack of the carbonyl group in the second, there is the possibility of an anchimeric assistance by the nitrogen atom in the chloride elimination (Scheme 3). Due to the uncertainty found with respect to the reactivity of chloroacetanilide with sulfur nucleophiles, it is necessary to explore, in more detail, the reaction mechanism of these substrates. In this sense, computational methods could be a helpful tool for this goal.
This work seeks to explore chloroacetanilide's reactivity with HS − nucleophiles in order to propose a reasonable mechanistic interpretation based on theoretical calculations. With this aim, the potential energy surfaces were examined using the density functional theory (DFT) level, and the results were analyzed and compared with the experimental data. The minimum energy structure of reactants, the transition state, and the product were calculated, taking into account all possible reaction mechanisms suggested in the literature.
The processes of rupture and bond formations were studied using natural bond orbital calculations (NBO). In addition, intrinsic coordinate path reactions (IRC) and reaction force profiles were used to gain more insight into the reaction pathway.
Results and Discussion
According to the reported products for the reaction between HS − and chloroacetanilide herbicides [15], and based on the structure of each substrate, three possible mechanisms can be suggested. The main mechanism is the well-known SN 2 displacement (Scheme 1), which is proposed for all compounds. In the case of compounds B and C, the halogen elimination carried out by an anchimeric assistance of the oxygen atom can be considered (Scheme 2). For compounds E and F, due to the length of the carbon chain in the first case, and the lack of the carbonyl group in the second, there is the possibility of an anchimeric assistance by the nitrogen atom in the chloride elimination (Scheme 3). Scheme 1. SN 2 reaction mechanisms for the six compounds: A to F. Scheme 1. S N 2 reaction mechanisms for the six compounds: A to F.
Scheme 2.
Oxygen anchimeric assistance for compounds B and C.
Scheme 3.
Nitrogen anchimeric assistance for compounds E and F.
Thermodynamic Parameters
The thermodynamic parameters for the ten reaction mechanisms described above are included in Table 1. These results are compared with the experimental values, and good agreement is found among all of them. The SN 2 mechanism is suggested as the most plausible path for these reactions, with the exception of compound F.
Scheme 2.
Oxygen anchimeric assistance for compounds B and C.
Thermodynamic Parameters
The thermodynamic parameters for the ten reaction mechanisms described above are included in Table 1. These results are compared with the experimental values, and good agreement is found among all of them. The SN 2 mechanism is suggested as the most plausible path for these reactions, with the exception of compound F. Scheme 3. Nitrogen anchimeric assistance for compounds E and F.
Thermodynamic Parameters
The thermodynamic parameters for the ten reaction mechanisms described above are included in Table 1. These results are compared with the experimental values, and good agreement is found among all of them. The S N 2 mechanism is suggested as the most plausible path for these reactions, with the exception of compound F. The results reported in Table 1 show a good agreement between experimental and theoretical reactivity trends. In all cases where the oxygen anchimeric assistance was considered, higher values on the reaction barriers were found; therefore, this possibility can be discarded for those particular substrates. With respect to the nitrogen-assistance mechanism, for the case of compound F, it is also a favored mechanism, which is in agreement with the previously experimental evidence commented about these compounds [15]. Interestingly, the values of free energy of activation for the S N 2 and N-assistance mechanism are very close, which suggest that these reactions occur in parallel with almost 50% of each case. Taking into account that the values of activation free energy of these two mechanisms are similar, these two mechanisms can be differentiated only by considering the changes in activation entropy, where the N-assistance mechanism present a small negative value (∆S ‡ = −0.23) in consonance with a unimolecular process, while the S N 2 mechanism presents a high negative activation entropy value (∆S ‡ = −21.36) due to its bimolecular nature (loss in translational degree of freedom). For the case of compound E, a small positive entropy value (4.97) is reported in the experimental work, which suggests a unimolecular process involving an anchimeric assistance through the nitrogen atom, as depicted in Scheme 3, where a possible four-membered ring can be generated as an intermediate. The theoretical thermodynamic parameters (∆G ‡ , ∆H ‡ , and ∆S ‡ ) obtained for this possible mechanism were 41.45 kcal/mol, 41.38 kcal/mol, and −0.22 cal/molK, respectively. Clearly, the activation free energy for this N-assistance in compound E is higher than that corresponding to the S N 2 mechanism; therefore, the assistance can be discarded.
Based on these results, for further analysis, the S N 2 mechanism for compounds A-F and the anchimeric assistance for compound F were considered. In this sense, in Figures 1 and 2, the IRC profiles for all the reactions studied in the present work are presented. Evidently, all the reactions are exothermic except for the case of the assistance of a nitrogen atom, which is endothermic. Compounds A, B, and C have similar values for the theoretical and experimental activation thermodynamic parameters involved in the S N 2 mechanism, which is expected because they have the same reaction center with a neighboring carbonyl group, with the rest of the molecule groups far away from the reaction center. In the case of compound D, an oxygen atom is changed by a sulfur atom, and a small effect is observed due to this change: a small increase in the activation enthalpy value accompanied by a decrease in the activation entropy; however, this effect is compensated for, and a similar free energy of activation with respect to compounds A-C is found. When compounds A-D were compared with compounds E and F, a considerable difference was found in the thermodynamic activation parameters. Clearly, the carbonyl group exerts an electro-withdrawing effect, which implies a more electrophilic carbon, and consequently, a small activation barrier of the process. On the other hand, compounds E and F have a CH 2 in the beta position instead of a CO; therefore, these compounds have higher barriers. Based on these results, further analysis is centered on the S N 2 mechanism for all compounds, and the only possible anchimeric assistance considered is the N-assistance found as favorable for compound F. In addition, in Figures 1 and 2, the reaction force (RF) profiles for all the mentioned mechanisms obtained as described in the methodology section are reported. therefore, these compounds have higher barriers. Based on these results, further analysis is centered on the SN 2 mechanism for all compounds, and the only possible anchimeric assistance considered is the N-assistance found as favorable for compound F. In addition, in Figures 1 and 2, the reaction force (RF) profiles for all the mentioned mechanisms obtained as described in the methodology section are reported. therefore, these compounds have higher barriers. Based on these results, further analysis is centered on the SN 2 mechanism for all compounds, and the only possible anchimeric assistance considered is the N-assistance found as favorable for compound F. In addition, in Figures 1 and 2, the reaction force (RF) profiles for all the mentioned mechanisms obtained as described in the methodology section are reported. From the RF profiles shown in Figures 1 and 2, a clear partition between three regions can be seen: the reactant region (R), the transition state region (TS), and the product region (P). All the reactions are concerted in nature, and it was possible to obtain the four associated values of work done (W 1 -W 4 ) based on the integration of each of these regions (Table 2). This value of work done (W i ) was used to characterize the reaction in terms of structural rearrangements (W 1 ) and electron reorganization (W 2 ) from the reactant to transition state. Considering W 3 and W 4 , the reaction energy (Er) can be also estimated. In all the reaction mechanisms evaluated, W 1 > W 2 , suggesting that these reactions are principally dominated by structural rearrangement (~70%) involving the approximation of the nucleophile, which is correlated with the high entropy values found for these reactions in Table 1. W 2 began gaining an important role for compounds E and F, which have a CH 2 group instead of a CO neighbor to the reaction center. On the other hand, for the case of anchimeric N-assistance, the electronic reorganization is more important due to the unimolecular nature of this reaction. In order to characterize, in more detail, all the stationary points considered in this work, we describe the changes in geometric parameters involved in these reactions in the next section. Table 2. Values of work done associated with each region (reactant (R), transition state (TS), and product (P)) in the reaction force (RF) profiles, given in kcal/mol. Er-reaction energy.
Geometric Parameters
In terms of geometric parameters, similar changes in the S-C and C-Cl distances were found for compounds A-D. The S-C interatomic distance corresponding to the nucleophile approximation decreased from~3.7 Å to~2.5 Å, while the C-Cl dissociation increased from~1.8 Å to~2.2 Å (Table 3). Interestingly, the C-Cl bond in compounds E and F in the reactant, as well as in the transition state, were a little longer than the same bond in compounds A-D, which are in agreement with the electro-withdrawing effect of the CO group in compounds A-D, as discussed previously. With respect to the S-C-Cl angle, compounds A-D present more linear transition states (~170 • ) when compared to compounds E and F (~160 • ). The imaginary frequency values are associated with the transition vector (TV), shown in Figure 3 for the first-order transition state found as a saddle point. In an illustrative manner, Figure 3 depicts the optimized geometries for the reactant, transition state, and product for the compound A S N 2 reaction mechanism, and the anchimeric N-assistance mechanism is described for compound F. The Cartesian coordinates for all the structures considered in this study are included in Table S1 in the Supplementary Materials. Table 3. Geometric parameters for reactants (R), transition state (TS), and products (P) at the wB97XD/6-311++g(2d,2p) level. , and products (P) for the SN 2 reaction mechanism of compound A and the N-assistance mechanism of compound F.
Natural Bond Orbital (NBO) Analysis
The evolution on the electronic density through the reaction pathway plays an important role in the reaction mechanism, in order to gain information about the changes in the atom charges involved in the reaction mechanism for the different stationary points (reactants, transition states, and x Figure 3. Optimized geometries of reactants (R), transition state (TS), and products (P) for the S N 2 reaction mechanism of compound A and the N-assistance mechanism of compound F.
Natural Bond Orbital (NBO) Analysis
The evolution on the electronic density through the reaction pathway plays an important role in the reaction mechanism, in order to gain information about the changes in the atom charges involved in the reaction mechanism for the different stationary points (reactants, transition states, and products). In this sense, the NBO charge changes from reactant to transition state, denoted by δQ x = (Q TS x − Q R x ) for the corresponding x atom, are reported in Table 4. The net charges for each atom are included in Table S2 in the Supplementary Materials. Upon inspecting these results, it is evident that for compounds E and F, higher changes in the electronic density were found when compared to compounds A-D. The positive values of δQ C and δQ S suggest a decrease in electronic density in the carbon and sulfur atoms, while the chlorine atom acquired more electronic density with a negative value. Higher changes in the charge distribution were observed in the nucleophile and leaving group almost with the same magnitude in compounds A-D (more synchronic charge distribution); however, in compounds E and F, the charge distribution was less synchronic. On the other hand, in the N-assistance mechanism, a major decrease was observed in the electronic density of the carbon atom. In order to gain more insight into the reaction mechanism, the Wiberg bond indexes for the bond involved in the transition state were determined, as listed in Table 5. The C-Cl bond represents the determining factor in the rate-determining step, which presents a bigger evolution through the reaction path with a δBi value of approximately 43%. The average value of δB av < 50%, suggests that an early transition state is involved in the reaction mechanism, which is in agreement with the IRC profiles reported in Figures 1 and 2. The synchronicity values suggest that the reactions for compounds A-D are more synchronous than the reactions for compounds E and F, which agrees with the discussion put forward in the NBO charge analysis above.
Computational Details
Based on the experimentally evidenced reactions [15], the nucleophilic attack of hydrogen sulfide (HS − ) on chloroacetanilide herbicides, with the consequent displacement of a chloride atom, was studied in a total of six substrates: propachlor (A), alachlor (B), metolachlor (C), tioacetanilide (D), β-anilide (E), and methylene (F) (Scheme 4). All these calculations were performed with the Gaussian16 suite [16] at the wB97XD/6-311++G(2d,2p) level. The long-range dispersion-corrected hybrid wB97XD functional [17][18][19] was shown to be adequate in the study of reaction mechanism [20,21]. The Pople basis set 6-311++G(2d,2p) was employed in order to adequately describe all the atom orbitals involved in the reaction, including the chloride atoms [22]. For the self-consistent field (SCF) calculations, the convergence criterion for the optimization process was set as default. To achieve the convergence in the density matrix, a value of 10 −9 atomic units was required; the maximum displacement threshold value was 0.0018 Å and the maximum force threshold value was 0.00045 Hartree/Bohr. Reactants (R) and products (P) were characterized as minimum stationary points in the reaction coordinate. On the other hand, transition states (TS) were characterized as saddle points in the reaction path, with a unique negative eigenvalue on the force constant matrix, which was obtained by a frequency calculation on the optimized structures at 298.15 K [23]. With the frequency calculation, it is possible to obtain all the thermodynamic parameters, such as the zero-point energy (ZPE), absolute enthalpy (H), free energy (G), and entropy (S) values with the corresponding temperature correction, and the basis-set superposition error (BSSE) correction was considered for the transition state geometry [24]. A bimolecular nucleophilic substitution reaction (SN 2 ) mechanism was considered for all the substrates, as well as some possible anchimeric assistance for oxygen or nitrogen atoms. The anchimeric Oassistance was considered for compounds B and C, and N-assistance for compounds E and F. The solvation effect was directly taken into account in the optimization process using the polarizable continuum model (PCM) with the solvation model density (SMD) proposed by Cramer and Truhlar, and water as a solvent [25,26].
Intrinsic reaction coordinate (IRC) profiles were constructed for the reaction mechanism departing from the transition state in the forward and reverse directions [27][28][29][30]. This method allows the verification of the connection between the reactant and product through the transition state. In order to further describe the corresponding mechanism, reaction force analysis (RF) was performed by taking the first derivative of the energy with respect to the reaction coordinate ( = ⁄ ) [31][32][33][34]. This RF profile is valuable for describing the chemical changes along the reaction pathway in terms of electronic reorganization and structural rearrangements. These two contributions can be separately analyzed from the so-called works (Wi) obtained from the integration of each part on the reaction force profile. All these calculations were performed with the Gaussian16 suite [16] at the wB97XD/ 6-311++G(2d,2p) level. The long-range dispersion-corrected hybrid wB97XD functional [17][18][19] was shown to be adequate in the study of reaction mechanism [20,21]. The Pople basis set 6-311++G(2d,2p) was employed in order to adequately describe all the atom orbitals involved in the reaction, including the chloride atoms [22]. For the self-consistent field (SCF) calculations, the convergence criterion for the optimization process was set as default. To achieve the convergence in the density matrix, a value of 10 −9 atomic units was required; the maximum displacement threshold value was 0.0018 Å and the maximum force threshold value was 0.00045 Hartree/Bohr. Reactants (R) and products (P) were characterized as minimum stationary points in the reaction coordinate. On the other hand, transition states (TS) were characterized as saddle points in the reaction path, with a unique negative eigenvalue on the force constant matrix, which was obtained by a frequency calculation on the optimized structures at 298.15 K [23]. With the frequency calculation, it is possible to obtain all the thermodynamic parameters, such as the zero-point energy (ZPE), absolute enthalpy (H), free energy (G), and entropy (S) values with the corresponding temperature correction, and the basis-set superposition error (BSSE) correction was considered for the transition state geometry [24]. A bimolecular nucleophilic substitution reaction (S N 2 ) mechanism was considered for all the substrates, as well as some possible anchimeric assistance for oxygen or nitrogen atoms. The anchimeric O-assistance was considered for compounds B and C, and N-assistance for compounds E and F. The solvation effect was directly taken into account in the optimization process using the polarizable continuum model (PCM) with the solvation model density (SMD) proposed by Cramer and Truhlar, and water as a solvent [25,26]. Intrinsic reaction coordinate (IRC) profiles were constructed for the reaction mechanism departing from the transition state in the forward and reverse directions [27][28][29][30]. This method allows the verification of the connection between the reactant and product through the transition state. In order to further describe the corresponding mechanism, reaction force analysis (RF) was performed by taking the first derivative of the energy with respect to the reaction coordinate (F(ξ) = dE/dξ) [31][32][33][34].
This RF profile is valuable for describing the chemical changes along the reaction pathway in terms of electronic reorganization and structural rearrangements. These two contributions can be separately analyzed from the so-called works (Wi) obtained from the integration of each part on the reaction force profile.
The description of the electronic nature of all the stationary points was performed by means of natural bond orbital (NBO) analysis. In this sense, natural atomic charges (Q x ) and bond orders (Wiberg indexes) were obtained for the optimized geometries of the reactants (R), transition state (TS), and product (P) (B R i , B TS i , B P i ). In Q x , the x represents an atom, and, for the evaluation of the charge changes through the reaction path, a δQ x = (Q TS x − Q R x ) value was estimated for each x atom.
With the Wiberg indexes, it was possible to estimate the evolution percent of each bond involved in the transition state using Equation (1): The evolution of each bond allows us to establish what the determining factor is in the transition state and how early or late the transition state is. These indexes are useful in determining how synchronous the transition state is in nature, as described by the synchronicity concept proposed by Moyano et al. [35], defined in Equation (2): A value of zero for Sy advises a completely asynchronous process, and a value of 1 implies a synchronous process.
Conclusions
A mechanistic detailed study for the bimolecular nucleophilic substitution reaction of HS − with chloroacetanilide compounds was performed using density functional theory at the wB97XD/6-311++G(2d,2p) level. An S N 2 reaction mechanism was found as favorable for all compounds, with activation free energy values between 17 and 24 kcal/mol. Compounds A-D, which have the presence of a neighboring carbonyl group (C=O) and C=S, possess almost the same barrier (∆G ‡ ≈ 19 kcal/mol). The C=O and C=S groups exert an electro-withdrawing effect favoring the nucleophilic attack, and consequently, a minor activation free energy was found in comparison with compounds E and F, which have a neighboring methylene group (CH 2 ) in the same position. With respect to the reaction force analysis, in all cases, W 1 > W 2 ; W 1 corresponds to the work done for geometric reorganization, corresponding to the formation of the transition state, and it represents about 70% of the total activation barrier for compounds A-D, and about 60% for compounds E and F. The charge evolution and bond-order analysis are in agreement with the reactivity trends described previously for the studied substrates: the presence of a carbonyl group reduces the electron density of the carbon atom in the CH 2 Cl group, which implies a smaller free activation energy. In compound F, the anchimeric N-assistance via a unimolecular process is also favored in almost 50% of cases, because the activation free energy of this mechanism is very close to the value found for the corresponding S N 2 mechanism. Based on the Wiberg bond index analysis, it is possible to conclude that all transition states are early in nature (δBav < 50%), and the corresponding values for compounds A to D turned out to be more synchronous than those for compounds E and F. | v3-fos-license |
2019-04-09T13:02:44.105Z | 2017-06-28T00:00:00.000 | 103102724 | {
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} | pes2o/s2orc | Siidraite, Pb2Cu(OH)2I3, from Broken Hill, New South Wales, Australia: the third halocuprate(I) mineral
Siidraite, Pb2Cu(OH)2I3, is a new mineral from the Broken Hill deposit in New South Wales, Australia. It occurs as an extremely rare secondary phase alongside marshite, other lead and copper secondaries and supergene cuprite on a single specimen, BM 84642 preserved in the collection of the Natural History Museum, London. Siidraite is yellow and occurs in crystalline grainy aggregates up to 0.3mm around relict galena. The mineral is translucent with a vitreous lustre and yellow streak, no cleavages or forms have yet been observed. It is non-fluorescent in mixed-wavelength UV light. The calculated density is 6.505 g cm . Siidraite is orthorhombic, space group Fddd, a = 16.7082(9) A , b = 20.846(1) A , c = 21.016(1) A , V = 7320.0(8) A 3 and Z= 32. The empirical formula derived from a combination of electron-microprobe analysis and structure determination is Pb2.06Cu0.89(OH)2I2.97, the ideal formula has (in wt%) 8.01 Cu2O, 50.01 PbO, 42.65 I and 2.02 H2O. The five strongest lines in the calculated X-ray powder diffraction pattern are [(h k l), dobs (A ), I/Imax (%)]: [(2 4 6), 2.746, 100], [(4 0 4), 3.270, 81], [(2 6 4), 2.738, 77], [(3 1 5), 3.312, 76], [(3 5 1), 3.296, 69]. The crystal used for structure determination had minor pseudomerohedral twinning on 1⁄20 1 1 and the structure was refined taking this into account to R1 = 0.037, wR2 = 0.052, GooF = 1.016, based upon 1368 unique reflections having I> 2s(I). The structure of siidraite is a framework comprising an alternation of two structural elements, a cubane-like [Pb4(OH)4] 4þ group and a [Cu2I6] 4 dimer of edge-sharing CuI4 tetrahedra with non-equivalent Cu. Six halocuprate groups surround each [Pb4(OH)4] 4þ nucleus, and each halocuprate group is shared between six adjacent [Pb4(OH)4] 4þ groups, five long Pb–I bonds are required to complete the co-ordination of each Pb atom. The resulting Pb(OH)3I5 polyhedra are centred on a tetrahedron of O atoms to form a Pb4(OH)4I16 cluster. Siidraite has a unique composition and structure. It is the third naturally occurring halocuprate(I) after marshite and nantokite. A compositionally similar synthetic compound Pb2Cu2(OH)2I2Br has been described that has cubane and CuI4 groups, but a very different structural topology from that of siidraite. Bideauxite, Pb2Ag(OH)FCl3, which has the [Pb4(OH)4] 4þ group, shares some topological features with siidraite. Key-words: siidraite; new mineral; halocuprate(I); [Pb4(OH)4] 4þ group; iodine; Broken Hill.
Introduction
Halocuprate(I) compounds characteristically contain monovalent copper and have been studied extensively in the form of organometallic structures, which have technologically significant properties including photoluminescence, magnetism and semiconductivity. As far as we are aware, there is only one inorganic lead-bearing halocuprate(I), Pb 2 Cu(OH) 2 BrI 2 , which occurs only as a synthetic compound (Hu et al., 2011). Here, we report the discovery and characterisation of a related new inorganic halocuprate(I) mineral, siidraite, Pb 2 Cu(OH) 2 I 3 , having a very different crystal structure from Pb 2 Cu(OH) 2 BrI 2 . It is only the third halocuprate(I) mineral after marshite CuI and nantokite CuCl, both of which are rare minerals.
Siidraite was found during investigation of iodinebearing minerals in the collection of the Natural History Museum, London (henceforth NHM). It is known from just one specimen, registered as marshite, obtained from the mineral dealer Dr. A.E. Foote of Philadelphia in 1899. The specimen has abundant orange marshite on cavityridden cuprite and is from the Broken Hill Mining Area in Australia, it is registered in the mineral collection as BM 84642. A search for further specimens in the collection of the NHM and the Museum Victoria, Melbourne, Australia was unsuccessful.
Occurrence
Sample BM 84642 is from the Broken Hill deposit in New South Wales, Australia, a classic supergene enrichment deposit. The deposit has been extensively studied for decades and a detailed description of the geology and its relevance to mineral collecting and mineral specimens was summarized in Birch (1999).
Due to the entry date of the specimen into the NHM collection, the nature of recording information at that time and the fact it came to the NHM through a mineral dealer, the specific locality within the deposit is unknown. However, it is suggested that, as it was obtained not long after Broken Hill was first mined (1885), the specimen is likely to be from the upper oxidation zone levels of the mine (S. Mills, personal communication).
The specimen is a mass of cuprite and native copper, pervaded by cavities in which cuprite has grown as wellformed octahedra. Occasionally within the cuprite and the cavities there are small relict blebs and broken shards of the characteristic Broken Hill galena-Mn-silicate ore and quartz. Some cavities host a complex suite of secondary minerals that is dominated by orange/pale-brown translucent marshite tetrahedra, blue linarite and connellite, green brochantite and tsumebite, white anglesite, traces of plumbogummite-group minerals and, occasionally, where relict galena occurs in very close proximity to marshite and cuprite, tiny yellow granular aggregates of the new mineral siidraite.
Considering the coexisting minerals present, it is suggested that siidraite formed from the secondary alteration of cuprite that was at one time part of an area of supergene enrichment within the Broken Hill deposit. Mineralising fluids were extremely rich in iodine and not strongly oxidizing. Siidraite formed only due to the local availability of Pb mobilized from the small galena "blebs" and shards present, and it is this feature that determines the scarcity of the phase.
Physical properties
Siidraite occurs on the holotype specimen in patches up to 2 mm in size, although the individual aggregates of siidraite crystals within these areas are no more than 0.3 mm across (Fig. 1). Siidraite is translucent in various shades of yellow; the variation in colour is apparently due to its small size and adjacency and/or intergrowth with nearby brochantite, anglesite, cuprite and marshite. When strongest yellow and presumably purest, the colour is most similar to HTML colour codes FFFF66 and FFFF33.
Individual crystals have not been clearly observed in hand specimen or during optical microscope work. When small areas were disaggregated for single-crystal diffraction studies, the crystal shape and any forms were still indistinct, though seemingly equant with a vitreous lustre. The largest single crystals removed were up to 0.1 mm long.
Where material was sampled from the holotype specimen, the powdered remains indicate that the streak is the same yellow colour as the mineral. No cleavages or parting were observed and fracture and tenacity were impossible to assess accurately on such small crystals.
Siidraite is non-fluorescent in both short-and long-wave UV light, the size of the crystals and overall paucity of material precluded the determination of both Mohs hardness and density by direct measurement. By analogy with similar materials (bideauxite, marshite, miersite), the hardness of siidraite is likely around 2.5-3.5 on the Mohs scale. The density calculated for the empirical formula is 6.505 g cm À3 and for the ideal formula is 6.465 g cm À3 .
Due to the nature and paucity of the material, no quantitative optical properties could be obtained. The calculated mean refractive index based on the ideal formula is 2.18.
Chemical composition
A sample containing siidraite was analysed using a Cameca SX100 electron microprobe in wavelengthdispersive mode at the NHM in London using an accelerating voltage of 20 kV, a beam current of 20 nA and a beam diameter of 0.01 mm. The elements (with relevant standard) Cu (Cu metal), Pb (Pb 5 (VO 4 ) 3 Cl), I (KI), Ag (Ag metal), Fe (Fe 2 O 3 ), Sb (Sb metal), V (Pb 5 (VO 4 ) 3 Cl), Mn (MnTiO 3 ), Cl (NaCl), Br (KBr), K (orthoclase) and S (ZnS) were sought. All elements apart from Cu, I and Pb were found to be below detection limits. It is of note that an earlier analysis of a different subsample using a Zeiss EVO 15LS scanning electron microscope operated with 20 kV accelerating voltage and 3 mA beam current, coupled with an Oxford Instruments XMax 80 (EDS), revealed traces of Br. There was not enough material on the holotype specimen to perform CHN analysis or to quantify water content directly by any means other than based on stoichiometry from the structural refinement. The average values for 10 spot electron-microprobe analyses are reported in Table 1. The empirical formula calculated on the basis of five anions (including two OH), according to the crystal-structure study, is Pb 2.08 Cu 0.90 (OH) 2 I 3 . The corresponding ideal formula Pb 2 Cu(OH) 2 I 3 has 8.01 wt% Cu 2 O, 50.01 wt% PbO, 42.65 wt% I and 2.02 wt% H 2 O.
Crystal structure
Full details of the crystal structure determination of siidraite are reported by Welch et al. (2016). However, a summary of the structure is given here. A Crystallographic Information File (CIF) containing details of the data collection and structure refinement is deposited with the journal. A list of structure factors is also deposited with the journal. They are freely available online as Supplementary Material linked to this article on the GSW website of the journal: http:// eurjmin.geoscienceworld.org/. Several small yellow transparent crystals of siidraite were examined for structure determination using an XcaliburE four-circle diffractometer, EOS detector and MoKa radiation (Rigaku Oxford Diffraction). Siidraite has orthorhombic symmetry, space group Fddd. Unit-cell parameters derived from singlecrystal X-ray diffraction are: a = 16.7082 (9) The structure topology of siidraite is unique and consists of two structural elements that alternate in a checkerboard motif: (1) a Pb 4 (OH) 4 "cubane" group and (2) a Cu 2 I 6 dimer in which a pair of CuI 4 tetrahedra share an edge. These two components are shown in Fig. 2. Despite their very similar individual geometries, the two constituent Cu(1)I 4 and Cu(2)I 4 tetrahedra of the Cu 2 I 6 dimer have very different next-nearest-neighbour environments.
Raman spectroscopy
Details of the collection of the Raman spectrum and peak assignments for a single crystal of siidraite that was part of the crystal separated from BM 84642 are reported by Welch et al. (2016). Peaks below 400 cm À1 were assigned to modes of the cubane ( Q1 Jensen, 2002) and Cu 2 I 6 dimer units, and internal modes of the CuI 4 tetrahedra (Ouslati et al., 2013). Two peaks occur in the OH-stretching region at 3443 and 3455 cm À1 and are assigned to the two nonequivalent OH groups known to be present in siidraite from the structure determination. The presence of two non-equivalent OH groups is consistent with the Fddd structure, but not the tetragonal (I4 1 /acd) structure, which has only one symmetrically non-equivalent OH group.
Powder X-ray diffraction
Due to the very small quantities of siidraite available, it was not possible to collect X-ray powder diffraction data. An attempt was made to collect a pseudo-powder pattern using a quasi-Gandolfi movement on a Rigaku Rapid II curved image-plate diffractometer ( ® Rigaku Oxford Diffraction) for the single crystal for which the structure was determined. However, diffraction from this very small crystal was so weak that no usable pattern was obtained after a 2-day collection. Consequently, X-ray powder diffraction data for siidraite were calculated from the crystal structure and are given in Table S1 in Supplementary Material.
Discussion
Siidraite is only the third halocuprate(I) mineral. A synthetic compound with similar stoichiometry Pb 2 Cu (OH) 2 I 2 Br has been described (Hu et al., 2011). This phase has the Pb 4 (OH) 4 cubane group, but has chains of cornersharing CuBr 2 I 2 tetrahedra rather than isolated edgesharing Cu 2 I 6 pairs, resulting in a very different structural topology. As far as we are aware, siidraite and synthetic Pb 2 Cu(OH) 2 I 2 Br are the only Pb-based cuprous oxyhalides. Topological variants for Pb 2 M þ (OH) 2 X 3 stoichiometry may occur that depend upon the halogen motif, which itself may be dependent upon halogen type (size). The novel structure described here hints at the possibility of synthesizing a new class of inorganic halocuprates based upon the cubane-type Pb 4 (OH) 4 cluster and CuI 4 tetrahedra having varying degrees of tetrahedral polymerization of the latter that could be engineered by modifying halogen composition. A synthetic methodology for controlling structure topology could be found through better understanding of the paragenesis of siidraite within the natural environment. With this possibility in mind, it is noteworthy that the [Pb 4 (OH) 4 ] 4þ cluster has a role in controlling Pb mobility in aqueous system, but its occurrence is limited to near and just below neutral pH conditions (Ronay & Seff, 1993;Grimes et al., 1995). The rarity of minerals containing the Pb 4 (OH) 4 cluster is likely due to the transient nature of pH conditions favouring its stabilisation, as more alkaline conditions develop and new products arise from the continued oxidation of original galena.
For maricopaite (Rouse & Peacor, 1994), a mineral with Pb 4 (OH) 4 groups incorporated into a mordenite-type alumino-silicate framework, it has been suggested that the framework crystallised around the aqueous [Pb 4 (OH) 4 ] 4þ cation from a near-neutral pH solution. Further insight might also be gained from studying bideauxite, Pb 2 Ag (OH)FCl 3 , space group Fd3m, which has some structural similarity to siidraite (Cooper et al., 1999). The structure of bideauxite is composed of a checkerboard framework of alternating AgCl 6 octahedra and Pb 4 (OH 0.5 F 0.5 ) 4 cubanetype groups. The Cl motif is different from the I motif of Pb 2 Cu(OH) 2 I 3 and results in octahedral (rather than tetrahedral) sites being occupied by the monovalent cation (Ag). The cubane group in bideauxite is bonded to 18 Cl atoms to give a Pb 4 (OH 0.5 F 0.5 ) 4 Cl 18 cluster. Two extra halogen atoms enter the coordination sphere of the cubane group of bideauxite compared with the Pb 4 (OH) 4 I 16 cluster of siidraite. Although bideauxite and siidraite are very rare minerals, their existence suggests that further cubane-like halide "cluster" compounds may occur. | v3-fos-license |
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} | pes2o/s2orc | Myofibroblast induces hepatocyte-to-ductal metaplasia via laminin–ɑvβ6 integrin in liver fibrosis
Hepatocytes undergo the metaplasia into ductal biliary epithelial cells (BECs) in response to chronic injury, and subsequently contribute to liver regeneration. The mechanism underlying hepatocyte-to-ductal metaplasia has not been explored until now. In mouse models of liver fibrosis, a florid BEC response was observed in fibrotic liver, and the depletion of myofibroblasts attenuated BEC expansion remarkably. Then, in hepatocyte fate-tracing mouse model, we demonstrated the conversion of mature hepatocytes into ductal BECs in fibrotic liver, and the depletion of myofibroblasts diminished the hepatocyte-to-ductal metaplasia. Finally, the mechanism underlying the metaplasia was investigated. Myofibroblasts secreted laminin-rich extracellular matrix, and then laminin induced hepatocyte-to-ductal metaplasia through ɑvβ6 integrin. Therefore, our results demonstrated myofibroblasts induce the conversion of mature hepatocytes into ductal BECs through laminin-ɑvβ6 integrin, which reveals that the strategy improve regeneration in fibrotic liver through the modification of specific microenvironment.
Introduction
The liver is a highly regenerative organ with the ability to restore its function after acute injury and chronic injury [1][2][3][4] . The cellular sources of regenerative hepatocytes in liver injury is a fundamental issue in liver biology [3][4][5][6][7][8] . In response to acute injury or loss of liver mass, remaining healthy liver cells proliferate to restore their functions 4,9 . During chronic injuries, liver progenitor cells (LPCs) derived from quiescent facultative stem cells expand, and differentiate to hepatocytes and cholangiocytes 4 . Recently, genetic lineage tracing by labeling a specific type of cells defines the origin of the cells in animal models. Lineage tracing studies have demonstrated that hepatocytes are regenerated by self-replication rather than derived from LPCs or myofibroblasts (MFBs) in chronic liver injuries induced by chemicals [10][11][12][13][14][15][16] . Tarlow et al. revealed hepatocytes undergo reversible ductal metaplasia to a distinctive progenitor state, and give rise to hepatocyte nuclear factor (HNF) 4ɑ and Sry HMG box protein 9 (SOX9)-double-positive (HNF4ɑ + SOX9 + ) cells during chronic liver injury; HNF4ɑ + SOX9 + cells differentiate into mature hepatocytes as well as ductal biliary epithelial cells (BECs) 17 . Joan Font-Burgada et al. identified a subpopulation of periportal hepatocytes named as hybrid hepatocytes (HybHP) express low amounts of SOX9 and normal amount of HNF4ɑ. The HybHP make major contribution to parenchymal restoration after chronic liver damage 16 . All these findings suggested mature hepatocytes in periportal area undergo reversible ductal metaplasia to distinctive ductal BECs, which contributes to liver regeneration during chronic injuries. However, the mechanism underlying hepatocyte-to-ductal metaplasia has not been explored until now.
Stem-cell populations are established in niches or specific anatomic locations which maintain and regulate stem cell homeostasis. LPC niche is composed of hepatic stellate cells (HSCs), endothelial cells, macrophages, other inflammatory cells, extracellular matrix (ECM), growth factors, and cytokines 8,18,19 . HSCs, also known as Ito cells, are located in the space of Disse. During chronic liver injury, quiescent HSCs develop into contractile myofibroblast-like cells [20][21][22] . It is well-known that activated HSCs/MFBs play an important role in liver fibrosis through promoting ECM deposition. Moreover, MFBs in LPC niche are involved in the differentiation of LPC [23][24][25] . In a choline-deficient ethionine-supplemented (CDE)-induced model of chronic liver injury, ECM deposition and HSC activation occurred as an initial phase, prior to LPC expansion 26 . Inhibition of HSC activation by 2% L-cysteine diminished LPC expansion in animal models 27 . All these revealed a critical role of myofibroblast in LPC expansion. However, the role of MFBs in hepatocyte-to-ductal metaplasia has not been investigated until now. The aim of our study is to investigate the role of MFBs in the conversion of mature hepatocytes into ductal BECs in liver fibrosis.
Materials and methods
Animals C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). R26R-EYFP mice (Rosa26 loxP-stop-loxP-EYFP ) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA; 006148). The R26R-EYFP reporter mice contain a loxPflanked STOP sequence followed by EGFP in the Rosa26 locus 28 . All animals were housed in specific pathogen-free (SPF) animal facility. The protocol of animal treatment used in this study was approved by the institutional animal care and use committee of Tongji Medical College, Huazhong University of Science and Technology.
Animal model of liver fibrosis
Mouse models of liver fibrosis (8 mice per group) were established through the administration of thioacetamide (TAA), carbon tetrachloride (CCl 4 ), N-Nitrosodiethylamine (DEN), and tetrachloride (CCl 4 ). Chemicals used were listed in Supplementary Table 1 TAA administration Male mice (6-8 weeks old) were treated three times a week intraperitoneal (i.p.) injections of 150 mg/kg TAA for 6 weeks.
CCl 4 administration
Male mice (6-8 weeks old) were injected subcutaneously with CCl 4 diluted 5:5 (v/v) ratio in olive oil at a dose of 3 ml/kg twice a week for 12 weeks 21 .
The administration of DEN and CCl 4 15-day-old mice were injected intraperitoneally (i.p.) with DEN at a dose of 25 mg/kg. At 29 days, mice were injected intraperitoneally with CCl 4 diluted 1:9 (v/v) ratio in olive oil at the dose of 5 ml/kg weekly for 12 weeks 29,30 .
Depletion of MFBs by DAPT in mouse models of liver fibrosis
To deplete MFBs, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester (DAPT , Table S1), a γ-secretase inhibitor, was administrated into mouse models of liver fibrosis 25,32 . For CCl 4 and DEN/CCl 4induced liver fibrosis, the mice received antifibrotic treatment with DAPT (50 mg/kg) after 8-week exposure of CCl 4 by intraperitoneal injection five times a week for another 4 weeks. For TAA-induced liver fibrosis, the mice received antifibrotic treatment with DAPT (50 mg/kg) after 2-week exposure of TAA by intraperitoneal injection five times a week for another 4 weeks.
Immunohistochemistry and immunofluorescent staining
The sections of formalin-fixed, paraffin-embedded liver samples were stained with hematoxylin and eosin for standard histology. For the assessment of collagen deposition, Sirius Red staining was performed using the staining assay kit according to the manufacturer's instructions. For immunohistochemical staining or immunofluorescent staining, the slides were incubated with primary antibodies (Table S1) followed by appropriate secondary antibodies (Table S1). For immunofluorescent staining,the slides were mounted with DAPIcontaining medium and the images were acquired with a Nikon-A1-si confocal microscope.
HSC isolation
Mouse HSCs were obtained by in situ perfusion with collagenase type IV, pronase E, and DNAase followed by differential centrifugation on Opti-Prep density gradients 21 .
Cell viability was assessed by trypan blue exclusion. Primary HSCs were cultured in DMEM/10% fetal bovine serum (FBS) containing penicillin and streptomycin. The purity of activated HSCs (MFBs) was revealed by immunofluorescence using anti-ɑ-smooth muscle actin (ɑSMA) antibody.
Hepatocyte isolation
Hepatocytes were isolated by a two-step perfusion technique. Mouse hepatocytes were isolated from the digested liver by centrifugation 33 . Cell viability was assessed by trypan blue exclusion. Primary hepatocytes were cultured in DMEM/10% FBS containing penicillin and streptomycin. The purity of isolated hepatocytes was demonstrated by immunofluorescence using anti-albumin antibody.
Co-culture of MFBs and hepatocytes
To determine the effect of MFBs on hepatocytes, MFBs were co-cultured with hepatocytes using cell culture inserts (0.3 µm pore size). In brief, MFBs were seeded in the upper chamber, and hepatocytes isolated from normal or fibrotic liver were grown in the bottom chamber. To determine the effect of laminin on biological characteristic of hepatocytes, hepatocytes from fibrotic liver were grown in laminin-coated culture plate. In all, 60 µg/ml laminin was added to culture plates and maintained at a final concentration of 3 μg/cm 2 at 37°C for 2 h, or blowdried on a clean bench at room temperature overnight.
RNA-sequencing
Total RNA was extracted from hepatocytes using TRIzol reagent following the manufacturer's instructions. The Libraries were generated using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina® (Vazyme), and were subsequently sequenced by an Illumina HiSeq X-ten. RNA isolation, library construction, and sequencing were performed at Shanghai Biotechnology Corporation (Shanghai, China). For data analysis, the raw reads were filtered by Seqtk before mapping to genome using Tophat (version: 2.0.9). The fragments of genes were counted using HTSeq. Significant differential expressed genes (DEGs) were identified as those with a false discovery rate (FDR) value above the threshold (Q < 0.05) and foldchange >2 using edgeR software.
Immunoprecipitation and Western blot
Lysates from cells and tissues were collected using RIPA buffer (Sigma R0278, St Louis, MO, USA), and then immunoprecipitated with primary antibodies (Table S1). Equal amounts of protein was separated on SDS-polyacrylamide gels, immunoblotted with primary antibodies, then with horseradish peroxidase-conjugated secondary antibodies. The blot was washed three times and was developed with ECL according to the manufacturer's instructions. Antibodies used are listed in Table 1.
siRNA transfection
Chemically synthesized siRNAs and the controls were transfected into primary hepatocytes by Lipofectamine TM 2000 in accordance with the manufacturer's directions. 48-72 h after transfection, transfected cells were collected for further study.
Statistical analysis
Data are expressed as mean ± SEM. Comparisons between two groups were made by Student's two tailed t-tests. P < 0.05 was considered significant. Statistics and graphing were performed using Prism 5.0.1 (GraphPad) software. All experiments were analyzed from n ≥ 3 independent experiments.
Inhibition of HSC activation diminishes the expansion of ductal BECs
To determine the role of MFBs/activated HSCs in the expansion of ductal BECs in liver fibrosis, the correlation between HSC activation and ductal BEC expansion was firstly investigated. In mouse model of TAA-induced liver fibrosis, an increased deposition of ECM was demonstrated by Sirius red staining (Figs. 1b and S1), and the conversion of quiescent HSCs into MFBs was revealed by immunostaining of ɑ-SMA (Figs. 1b and S1). Together with ECM deposition, the expansion of ductal BECs was revealed by immunostaining of CK19, SOX9, and OPN (Figs. 1b and S1). All these indicated the infiltration of ductal BECs into liver parenchyma was chaperoned by HSC activation. Furthermore, mouse models of liver fibrosis induced by CCl 4 and DEN/CCl 4 were also created, and the results (Figs. 1c-f and S1) demonstrated positive correlation between HSC activation and the expansion of ductal BECs. All these revealed that the expansion of ductal BECs was chaperoned by HSC activation in fibrotic liver. Previous study demonstrate that ECM deposition and activation of matrix-producing cells occurred as an initial phase, prior to LPC expansion 26 . Thus, the role of MFBs in the expansion of ductal BECs was determined in liver fibrosis. Animal models of liver fibrosis were administrated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-Sphenylglycinet-butylester (DAPT), a γ-secretase inhibitor. Inhibition of γ-secretase resulted in the depletion of MFBs and attenuated liver fibrosis, which was revealed by Sirius red staining and ɑ-SMA expression (Figs. 1b, d, f and S1).
In addition, immunofluorescene of ɑ-SMA demonstrated that DAPT inhibited HSCs activation in vitro (Fig. S2). Fortunately, DAPT did not change the expression of endothelial cell (CD 31), macrophage (F4/80), and T lymphocytes (CD4, CD8) (Fig. S3). Importantly, primary hepatocytes isolated from fibrotic liver were treated with DAPT, and the result demonstrated DAPT had no effect on the expression of LPC markers in chronic injured hepatocytes (Fig. S2). All these confirmed the cellular specificity of DAPT-mediated inactivation of MFBs in fibrotic liver. After attenuating liver fibrosis, the mice exhibited a blunted response of ductal BECs accompanied by the inhibition of HSC activation. All these revealed that HSC inactivation diminished the expansion of ductal BECs.
MFBs induce the conversion of mature hepatocytes into ductal BECs in vivo
To determine the conversion of mature hepatocytes into ductal BECs in fibrotic liver, the fate of mature hepatocytes should be tracked specifically in mouse models of liver fibrosis. For lineage-tracing of mature hepatocytes, the R26R-EYFP reporter mice were injected with AAV8-TBG-Cre. In Rosa YFP mice, the expression of YFP is blocked by transcriptional stop sequences flanked by loxP sites. Since the injection of AAV serotype with a high tropism (AAV2/8) for hepatocytes contained the hepatocyte-specific TBG promoter, AAV8-TBG-Cre-mediated loop out of the floxed stop codon resulted in efficient YFP expression in nearly all hepatocytes (Fig. 2). Other hepatic cells, MFBs (ɑSMA+), cholangiocytes (CK19+, SOX9+, OPN+) remained YFP-negative, revealing the specificity of this labeling strategy (Fig. 2).
To determine the effect of MFBs on ductal metaplasia of hepatocytes into ductal BECs, the models of liver fibrosis were established using the R26R-EYFP mice. Firstly, 6-week-old mice were administrated with AAV8-TBG-Cre followed by intraperitoneal injection of TAA (Fig. 3a). In fibrotic liver, some of ductal BECs (labeled as CK19+, SOX9+, OPN+) expressed YFP, indicating that these ductal BECs were derived from mature hepatocytes (Fig. 3b). Secondly, immunostaining of ductal BECs marker (CK19) and MFBs (aSMA) was performed in (see figure on previous page) Fig. 1 Inhibition of HSC activation diminished the expansion of ductal biliary epithelial cells (BECs). a Experiment design for TAA-induced liver fibrosis and DAPT-mediated inhibition of HSC activation in vivo. Vertical lines represent weekly intraperitoneal injections of TAA or TAA/DAPT. TAA thioacetamide, DAPT N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butylester. b Representative liver sections from TAA-treated mice administrated with DAPT or the control (Sirius red staining, immunohistochemical staining). Collagen deposition was determined by Sirius red staining, immunohistochemical staining showed the expression of myofibroblasts (ɑSMA) and ductal BECs (CK19, OPN, and SOX9). c Experiment design for CCl 4 -induced liver fibrosis and DAPT-mediated inhibition of HSC activation in vivo. d Representative liver sections from CCl 4 -treated mice administrated with DAPT or the control (Sirius red staining, immunohistochemical staining). e Experiment design for DEN/CCl 4 -induced liver fibrosis and DAPT-mediated inhibition of HSC activation in vivo. DEN N-Nitrosodiethylamine. f Representative liver sections from DEN/CCl 4 -treated mice administrated with DAPT or the control (Sirius red staining, immunohistochemical staining). Fig. 2 Lineage-tracing of mature hepatocytes through the administration of AAV virus into the R26R-EYFP reporter mice. a Schematic diagram of hepatocyte fate-tracing strategy through the injection of AAV8-TBG-Cre into R26R-EYFP (Rosa YFP ) mice. b Experiment design for lineage-tracing of mature hepatocytes in 6-day-old Rosa YFP mice. 6-day-old Rosa YFP mice were intraperitoneally injected with AAV8-TBG-Cre. c Immunofluorescence staining demonstrated AAV8-TBG-Cre efficiently labeled mature hepatocytes in 6-day-old Rosa YFP mice. d Experiment design for lineage-tracing of mature hepatocytes in 6-week-old Rosa YFP mice. Rosa YFP mice (6-week-old) were intravenously injected with AAV8-TBG-Cre. e Immunofluorescence staining demonstrated AAV8-TBG-Cre efficiently labeled hepatocytes in 6-week-old Rosa YFP mice.
fibrotic liver of Rosa YFP mice. The result (Fig. S4) revealed neighborhood of MFBs and hepacytes-derived ductal BECs. Thirdly, mouse model of TAA-induced liver fibrosis were treated with DAPT. Interestingly, the percentage of YFP-positive BECs (YFP + /CK19 + , YFP + / SOX9 + , YFP + /OPN + ) in BECs (CK19+, SOX9+, OPN+) was reduced in DAPT-treated mice compared with the controls (Figs. 3b and S4B). All these revealed that ductal metaplasia of hepatocytes into ductal BECs was diminished after the depletion of MFBs. To confirm this finding, a flow cytometry-based assay was performed to determine hepatocyte-derived ductal BECs. Ductal BECs were isolated with surface marker MIC1-1C3 14,35 , and the results (Fig. 4a) demonstrated 16.16 ± 1.94% of BECs (MIC1-1C3 +) were YFP+ (hepatocyte-derived ductal BECs) after 6 weeks of injury; while in the DAPT-treated group, 5.33 ± 1.61% of BECs (MIC1-1C3+) were YFP+. All these revealed that MFBs induced the conversion of hepatocytes into ductal BECs in fibrotic liver. To further confirm the findings, hepatocyte-derived ductal BECs were analyzed in CCl 4 and DEN/CCl 4 -induced liver fibrosis using immunofluorescene and flow cytometry. In CCl 4 or DEN/CCl 4induced liver fibrosis, co-immunofluorescene of YFP with ductal BECs markers (CK19, SOX9, OPN) demonstrated ductal metaplasia of hepatocytes into ductal BECs was diminished in DAPT-treated mice (Figs. 3d, f and S4B). In addition, the result of flow cytometry revealed that ductal metaplasia of hepatocytes into ductal BECs was diminished in DAPT-treated mice (Fig. 4c-f). These results indicated that the effect of DAPT on hepatocyte-to-ductal metaplasia resulted from the depletion of MFBs. In summary, all these indicated MFBs contributed to the conversion of hepatocytes into ductal BECs in cirrhotic liver.
MFBs induce the conversion of mature hepatocytes into ductal BECs in vitro
To confirm the contribution of MFBs in the conversion of hepatocytes into ductal BECs, the effect of MFBs on biological characteristic of hepatocytes was determined. Firstly, MFBs were co-cultured with hepatocytes isolated from normal mice. Upon the treatment of MFBs, upregulation of ductal BEC markers (CK19, SOX9, OPN) was observed in hepatocytes (Fig. S5A). Secondly, MFBs were co-cultured with hepatocytes treated with CCl 4 in vitro, and the results (Fig. S5B) showed similar phenotypic change in hepatocytes. Thirdly, chronic injured hepatocytes were co-cultured with MFBs since chronic injured hepatocytes isolated from fibrotic liver resembled pathological status. Upon the treatment of MFBs, upregulation of ductal BEC markers was observed in chronic injured hepatocytes (Fig. 5). All these results demonstrate the contribution of MFBs in the conversion of hepatocytes into ductal BECs.
To further explore the mechanism underlying the contribution of MFBs in the conversion of hepatocytes into ductal BECs, chronic injured hepatocytes were subjected to the analysis of RNA-sequencing. In chronic injured hepatocytes co-cultured with MFBs, a total of 216 genes were upregulated and 10 genes were downregulated (Fig. 5b). Then, pathway enrichment analysis was performed to identify pathways that were differentially active between cell subpopulations. As shown in Fig. 5c, gene sets for Toll-like receptor signaling pathway, TNF signaling pathway, TGF-β signaling pathway, NF-Kappa β signaling pathway were significantly induced. During the process of fibrogenesis, HSC activation leads to accumulation of ECM. Importantly, we found gene sets for ECM receptor interaction (laminin and integrin) were induced (Fig. 5c).
MFBs induce the conversion of mature hepatocytes into ductal BECs through the interaction of laminin-ɑvβ6 integrin
MFBs are the major source of ECM in fibrotic liver. The extracellular component of the LPC niche are rich in laminin matrix. Previous study demonstrated that laminin deposition was likely to be important prerequisites to LPCs activation and expansion 36 . Thus, we hypothesized that MFBs participated in hepatocyte-to-ductal metaplasia through laminin. To verify the hypothesis, the concentration of laminin was determined and the increase of laminin concentration was observed in culture medium of MFBs compared with the control (Fig. S6A). Additionally, a decrease in laminin concentration was observed after DAPT-mediated depletion of MFBs in vitro (Fig. S6A). Then the effect of laminin on chronic injured hepatocytes was determined. Primary hepatocytes isolated from fibrotic liver were grown on the plates containing laminin, and up-regulation of ductal BECs markers (CK19, OPN, and SOX9) and down-regulation of (see figure on previous page) Fig. 3 Myofibroblasts induce the conversion of mature hepatocytes into ductal biliary epithelial cells in vivo. a Experiment design for DAPTmediated inhibition of HSC activation in TAA-treated Rosa YFP reporter mice. b Immunofluorescent staining of fibrotic liver from TAA-treated Rosa YFP reporter mice. Immunostaining of ductal BECs marker (CK19, OPN, and SOX9) in liver of Rosa YFP mice showed that the percentage of hepatocytesderived ductal BECs was diminished after DAPT-mediated inhibition of HSC activation in TAA-treated mice. c Experiment design for DAPT-mediated inhibition of HSC activation in CCl 4 -treated Rosa YFP reporter mice. d Immunofluorescence staining of fibrotic liver from CCl 4 -treated Rosa YFP reporter mice. e Experiment design for DAPT-mediated inhibition of HSC activation in DEN/CCl 4 -treated Rosa YFP reporter mice. f Immunofluorescence staining of fibrotic liver from DEN/CCl 4 -treated Rosa YFP reporter mice. Magnification, ×600.
HNF4ɑ were observed (Figs. 6a and S6B). These indicated that MFBs were involved in the metaplasia of mature hepatocytes into ductal BECs via the secretion of lamininrich ECM.
Integrin ɑvβ6 is expressed on ductal BECs and critically regulates their function in vivo and in vitro 37,38 . Thus, we hypothesized that MFBs participated in hepatocyte toductal metaplasia through the interaction of laminin and (see figure on previous page) Fig. 4 Flow cytometry analysis demonstrated the inhibition of HSC activation diminished the conversion of mature hepatocytes into ductal BECs in vivo. a Flow cytometry analysis showed hepatocyte-derived ductal BECs diminished in TAA-treated Rosa YFP reporter mice upon DAPT-mediated inhibition of HSC activation. MIC1-1C3 was used as a surface marker of ductal BECs. b The percentage of ductal BECs derived from YFP-marked hepatocytes decreased significantly in TAA-treated Rosa YFP reporter mice upon DAPT treatment. c Flow cytometry analysis showed hepatocyte-derived ductal BECs diminished in CCl 4 -treated Rosa YFP reporter mice upon DAPT-mediate inhibition of HSC activation. d The percentage of ductal BECs derived from YFP-marked hepatocytes decreased significantly in CCl 4 -treated Rosa YFP reporter mice upon DAPT treatment. e Flow cytometry analysis showed hepatocyte-derived ductal BECs diminished in DEN/CCl 4 -treated Rosa YFP reporter mice upon DAPT-mediate inhibition of HSC activation. f The percentage of ductal BECs derived from YFP-marked hepatocytes decreased significantly in DEN/CCl 4 -treated Rosa YFP reporter mice after the treatment of DAPT. Each bar represents the mean ± SD for at least triplicate experiments and the P-value was determined by Student's t-test (***P < 0.001, **P < 0.01, *P < 0.05). Fig. 5 Myofibroblasts induce the conversion of mature hepatocytes into ductal biliary epithelial cells in vitro through co-culture of myofibroblasts and chronic injured hepatocytes. Immunofluorescence staining and RNA-sequencing were performed to determine the effect of myofibroblasts on biological characteristic of chronic injured hepatocytes. a Immunofluorescence staining showed the expression of ductal BECs marker increased in chronic injured hepatocytes upon the treatment of myofibroblasts. b Heat map of the differentially expressed mRNAs in chronic injured hepatocytes treated with myofibroblasts. c Pathway enrichment analysis was performed to identify pathways involved in the conversion of mature hepatocytes into ductal biliary epithelial cells. integrin ɑvβ6. To test the hypothesis, the effect of integrin ɑvβ6 on the behavior of chronic injured hepatocytes was determined. Firstly, the expression of integrin ɑvβ6 was examined in fibrotic liver. As shown in Fig. S6C, upregulation of integrin ɑvβ6 expression was observed in periportal area of cirrhotic liver; then immunofluorescence of liver sections demonstrated ductal BECs (CK19+) expressed integrin ɑvβ6 in fibrotic liver (Fig. S6D). To determine the involvement of integrin ɑvβ6 in ductal metaplasia of hepatocytes into ductal BECs, the expression of integrin ɑvβ6 was firstly determined in chronic injured hepatocytes upon the treatment of myofibroblast or laminin. After the treatment of MFBs or laminin, up-regulation of integrin ɑvβ6 expression was observed in chronic injured hepatocytes (Fig. 6b, c). To further determine the involvement of myofibroblast in ductal metaplasia through integrin ɑvβ6, ITGB6-siRNA was transfected into hepatocytes and the effect of integrin ɑvβ6 on the behavior of chronic injured hepatocytes were evaluated. qRT-PCR, western blot, and immunofluorescence showed siRNA suppressed ITGB6 expression efficiently after siRNA transfection (Fig. 6d). After efficient knockdown of integrin ɑvβ6 expression, immunofluorescence staining showed down-regulation of ductal BECs markers (CK19, OPN, and SOX9) and upregulation of HNF4ɑ in chronic injured hepatocytes (Fig. 6e). To determine laminin induce the hepatocyte-toductal metaplasia by binding to integrin, coimmunoprecipitation was performed. The results (Fig. S6E) demonstrated laminins exerted their effect through binding of integrin β6. In summary, MFBs induced the metaplasia of mature hepatocytes into ductal BECs via laminin-ITGB6 signal pathway during the process of liver cirrhosis.
Discussion
Recent studies demonstrated mature hepatocytes serve as the major source for hepatocyte renewal and regeneration using genetic lineage tracing of mature hepatocytes in chronic liver injury [10][11][12][14][15][16][17] . Malato Y. et al. found newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and acute injury; further, conversion of hepatocytes into BECs was not found in commonly used models of biliary injury 10 . Previous studies and our study demonstrated that hepatocytes undergo reversible ductal metaplasia in response to chronic injury 39,40 , expand and subsequently redifferentiate into functional hepatocytes 17 . The difference between Malato Y.'s study and our researches may be attributed to the time of liver injury and the type of liver injury. However, the mechanism underlying the conversion of mature hepatocytes into ductal BECs has not been investigated until now. Previous studies have demonstrated that LPC niches maintain the characteristics of LPCs and the balance between their activation, proliferation, and differentiation 18,24,26,36,41 . In this study, we demonstrated that MFBs derived from quiescent HSCs induced the conversion of mature hepatocytes into ductal BECs in mouse models of liver fibrosis. In addition, laminin-rich ECM secreted by MFBs induced hepatocyteto-ductal metaplasia through integrin ɑvβ6 signal pathway. Thus the strategies to improve regeneration in cirrhotic liver should be developed to boost regeneration through modifying LPC niche in future.
The niche in which stem cells reside is the key element to regulate stem cell homeostasis. The Canals of Hering and bile ductules localized in the portal tract and the periportal parenchyma are believed to be the LPC niche. LPC niche is composed of MFBs, macrophages, and ECM in rodent models of severe liver injury and human disease 24,41 . In mouse model of CDE-induced chronic injury, HSC activation occurred prior to LPC expansion 26 . The depletion of MFBs by gliotoxin inhibited oval cell reaction which was revealed by expression of CK19 42 . All these indicated a fundamental role of MFBs during BECs activation. To determine the role of MFBs in BECs activation, animal models containing ductal BEC expansion and HSCs activation should be created. Mouse models of TAA, CCl 4 , DEN/CCl 4 -induced chronic injury were used in our study due to extensive HSC activation and a florid BEC response; whereas, extensive activation of HSCs was not observed in DDC and CDE-induced chronic injury. Importantly, animal models for manipulating HSC expression should be created in order to determine the contribution of MFBs to BEC activation, three models to manipulate HSC expression have been established to deplete HSCs in vivo, by using gliotoxin 42,43 , gliotoxincoupled antibodies (Abs) against synaptophysin 44,45 and transgenic mice expressing the herpes simplex virus thymidine kinase gene (HSV-Tk) driven by glial fibrillary acidic protein (GFAP) promoter 46 . However, gliotoxin has broad actions in vivo and in culture, targeting not only HSCs, but also immune and endothelial cells and hepatocytes. Previous studies showed that DAPT depleted MFBs specifically in vitro and in vivo 25,32 , thus DAPT was used to deplete MFBs in our study. Our study demonstrated DAPT inhibited HSC activation specifically, which was revealed by the markers of MFBs, macrophage (F4/ 80), endothelial cells (CD31), hepatocytes (HNF4ɑ), and T lymphocytes (CD4, CD8). This strategy for the depletion of MFBs was a sample method compared with transgenic mice 46 . In addition, in vitro data demonstrated DAPT had no effect on the expression of ductal BEC markers in chronic injured hepatocytes. Thus, DAPT was used to manipulate HSC expression in our study.
Cellular signaling between ductal BECs and the surrounding ECM is an important determinant of ductal BECs behavior 36 . In chronic liver injury, there is a requirement for collagen matrix to be degraded in order for ductal BECs to be activated and regenerate the liver 36 . Failure to degrade collagen-I critically impairs HSC apoptosis and prevent the effective restoration of hepatocyte mass in liver fibrosis 36,47 . In chronic injured liver, MFBs derived from quiescent HSCs produce laminin-rich ECM. Laminin-progenitor cell interactions within the LPC niche are critical for LPC-mediated regeneration 36 . Laminin is required to maintain LPCs in an undifferentiated biliary state 24 . Thus, we determined the role of laminin in the conversion of mature hepatocytes into ductal BECs. Interestingly, we found laminin secreted by MFBs promoted the conversion of mature hepatocytes into ductal BECs in vitro. Laminins are heterotrimeric proteins that contain an α-chain, a β-chain, and a γ-chain. Combinations of these chains give rise to 16 distinct isoforms, which are expressed in tissue-specific and developmentally regulated manners 48 . Thus, we speculate specific laminin isoforms are involved in the hepatocyte-cholangiocyte conversion. The identification of a specific laminin subtype involved in the hepatocyte-cholangiocyte conversion is an important issue. Thus, further studies should be carried out in future.
Integrin as its receptor is a transmembrane dimeric protein on the cell surface composed of noncovalently associated ɑ and β subunits, and facilitate cell-ECM adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate cellular signals, such as regulation of the cell cycle, organization of the intracellular cytoskeleton, and movement of new receptors to the cell membrane 49 . Integrin ɑvβ6 has an important role in models of fibrosis, such as lungs, liver, and kidney. Connective tissue growth factor (CTGF) and integrin ɑvβ6 regulate oval cell activation and fibrosis, probably through interacting with their common matrix and signal partners, fibronectin and TGF-β1 37 . Inhibition of integrin ɑvβ6 expression through genetic disruption or selective antibodies inhibits progenitor cell responses in mouse models of chronic biliary injury 38 . Thus, the involvement of integrin ɑvβ6 in hepatocyte-to-ductal metaplasia was determined in mouse models of liver fibrosis. In our study, up-regulation of integrin ɑvβ6 in ductal BECs was shown in fibrotic liver, and siRNA-mediated inhibition of integrin ɑvβ6 expression in chronic injured hepatocytes resulted in the suppression of hepatocyte-to-ductal metaplasia.
In summary, MFBs in niche of LPCs induce the conversion of chronic injured hepatocytes into ductal BECs through laminin-ɑvβ6 integrin, which reveals that the strategy improve liver regeneration in fibrotic liver through the modification of LPC niche in future. | v3-fos-license |
2017-10-17T00:43:09.160Z | 2006-01-01T00:00:00.000 | 32786331 | {
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} | pes2o/s2orc | Two Different Modes for Copper ( II ) Ion Coordination to Quinine-Type Ligands
Três novos complexos de cobre(II) com os ligantes quinuclidina [Cu(C 7 H 13 N) 2 (OH 2 )Cl]Cl·2H 2 O (1), quinina [Cu(C 20 H 23 O 2 N 2 )(OH 2 ) 2 ]ClO 4 (2) e hidroquinidina [Cu(C 20 H 27 O 2 N 2 )(OH 2 )Cl 2 ]Cl·1⁄2H 2 O (3) foram isolados e caracterizados. Os sítios de ligação foram atribuídos com base nos resultados de espectroscopia vibracional, ressonância paramagnética eletrônica e análise térmica. A possibilidade de envolvimento do nitrogênio quinuclidínico na coordenação foi evidenciada no complexo 1, no qual o cobre(II) está coordenado a duas moléculas de quinuclidina. No caso dos ligantes análogos à quinina, se o material de partida estiver desprotonado em ambos os nitrogênios, a coordenação do cobre(II) ocorrerá através do nitrogênio quinuclidínico, como no complexo 2. Por outro lado, se o material de partida estiver protonado no nitrogênio quinuclidínico, o sítio de ligação será o nitrogênio quinolínico, como no complexo 3. Estes resultados evidenciam que ambos os nitrogênios de ligantes da família da quinina constituem sítios de ligação para íons cobre(II).
Introduction
Quinine and its derivatives are alkaloids used in the treatment of malaria due to their esquizonticide effect.2][3] Quinine and quinidine are diastereoisomers, with opposite conformations at C8 and C9.Quinidine can be hydrogenated to produce hydroquinidine (Figure 1).These drugs may act as ligands by forming a stable five-membered ring with some metals through the quinuclidinic nitrogen and the hydroxyl oxygen (bidentate site), or by binding through the quinolinic aromatic nitrogen (monodentate site).
Quinine-type drugs act in the blood stages of the disease. 4These amphiphilic weak bases enter the infected erythrocyte as free bases.Driven by a pH gradient, they accumulate in the digestive vacuole of the parasite that has a pH around 4.5.At this pH, they are protonated and trapped inside the digestive vacuole.The rapid protonation of these molecules induces a temporary alkalinization of the compartment, which is counteracted by the vacuolar proton pump. 5Their exact mechanism of action is not yet fully understood but the capacity of quinine and related drugs to interact with iron seems to be relevant.It is suggested that these drugs inhibit the polimerization of ferriprotoporphyrin IX into hemozoin, within the digestive vacuole of the parasite, during its intraerythrocytic life cycle.The formation of hemozoin, also known as "malaria pigment", avails to the parasite the digestion of hemoglobin without the formation of the toxic haemin.The main target of quinoline-type drugs seems to be the ferriprotoporphyrin IX, with which they form a complex, thus preventing its polymerization. 6,7However, the mode of this interaction remains controversial.It was proposed that quinine binds to iron(III) through the oxygen of the hydroxyl group on carbon 9, being the formation of this bond followed by a co-facial π-π interaction between aromatic moieties of quinine and protoporphyrin.][10] The solution structures of some antimalarial drug-heme complexes were analyzed by high field NMR experiments. 11,12 he authors postulated that at physiological conditions, the species with a ferriprotoporphyrin IX-to-drug stoichiometry of 2:1 dominates, in which there is no covalent bond between iron and nitrogen.Nevertheless, they suggested that covalent complexes might be formed with the ferriprotoporphyrin IX monomer.In a molecular modeling study, da Silva et al. 13 proposed that the interaction with ferriprotoporphyrin IX involves the binding of Fe III to the quinuclidinic nitrogen.
Hawley et al. 14 found a good correlation between the antimalarial activity, the drug accumulation and the inhibition of heme polymerization in Plasmodium falciparum.They have demonstrated that the more potent quinolines are those with a greater ability to accumulate within the parasite rather than those with a greater ability to inhibit polymerization.Nevertheless, other targets, as phospholipids, were also proposed for antimalarial action of quinine-type drugs. 15eports on the formation of metal complexes of quinine and analogs are scarce in the literature.Tsangaris and co-workers 16,17 have isolated Cu II , Ni II , Co III and Cr III complexes of quinine and proposed the participation of both quinolinic and quinuclidinic nitrogens in the coordination without evidencing, however, the involvement or not of the hydroxyl oxygen.A ruthenium complex of cinchonine, which had its structure determined by X-ray crystallography, 18 presented a five-membered chelate ring involving the quinuclidinic nitrogen and the hydroxyl oxygen.The structure of a zwitterionic cobalt complex of quinine was solved, in which the cobalt atom is coordinated to three chloride atoms and to the quinolinic nitrogen. 19Weselucha-Birczynska et al. 20 have characterized a copper compound of cinchonine in which two [CuCl 4 ] 2-tetrahedral anions are linked by hydrogen bonds to two doubly protonated cinchonine molecules and three water molecules.
The pharmacological action of quinine seems to be associated to its ability to form metal complexes.As copper(II) is an important metal ion present in living organisms, we have decided to study its interactions with quinine.In this study we describe the synthesis and characterization of three novel copper(II) complexes of quinine (QN), hydroquinidine (HQND) and quinuclidine (QNU).The characterization was performed by means of elemental, thermogravimetric and conductivity analyses and EPR and vibrational spectroscopies.Two modes of coordination were evidenced: one bidentate through the quinuclidinic nitrogen and the oxygen of the hydroxyl group and the other monodentate through the quinolinic nitrogen.
Spectroscopic measurements
Infrared spectra were measured over the region 200-4000 cm -1 with a Perkin Elmer 283 B spectrophotometer.The samples were examined in CsI pellets.
Electron paramagnetic resonance (EPR) spectra were obtained at room temperature (298 K) on a Bruker ESP 300E equipment with modulation frequency of 100 kHz operating at 9.5 GHz (X-band).Solid samples for EPR analysis were firmly accommodated in quartz tubes.
Elemental analysis
Carbon, nitrogen and hydrogen contents were determined on a Perkin Elmer 2400 CHN analyzer.Copper content was determined by atomic absorption spectroscopy on a Z-8200 Hitachi atomic absorption spectrophotometer.Chlorine content was determined by applying the k 0 parametric neutron activation analysis.Irradiation performed in the reactor TRIGA MARK I IPR-R1 located at CDTN/CNEN (Centro de Desenvolvimento da Tecnologia Nuclear/Comissão Nacional de Energia Nuclear, Belo Horizonte, MG, Brazil), at 100 kW, under a 6.6 x 10 11 neutrons cm -2 s -1 thermal neutron flux, produced the reaction 37 Cl (n, γ) 38 Cl.The sample was irradiated simultaneously to Au and Na standards for 5 min.After suitable decay, gamma spectroscopy was performed in an HPGe detector -ORTEC, 10175-P -15% of efficiency, resolution of 1.85 keV for the 1332 keV peak of 60 Co.The concentration was calculated based on k 0 constants and equations. 21,22 rmogravimetric analysis Thermal analyses were carried out on a Shimadzu TGA-50 thermogravimetric analyzer under air atmosphere.The temperature range varied between 25 and 900 ºC and the heating rate was 10 ºC min -1 .An intermediate copper hidroquinidine complex was obtained by heating the original complex on the thermobalance under air atmosphere, at a heating rate of 10 ºC min -1 , until 400 ºC.
The residue was analyzed by X-ray diffratometry on a Rigaku Geigerflex 2037 apparatus using a copper tube and radiation CuK α = 1.54051Å, with 2θ angle varying from 10 to 130°.
Synthesis of the copper(II) quinuclidine complex (1)
Copper(II) chloride (426 mg, 2.5 mmol) was added to 50 mL of an ethanolic solution of quinuclidine hydrochloride (369 mg, 2.5 mmol).After stirring for 1 h, a yellow solid was precipitated upon the addition of ethyl ether.The complex was then separated by filtration, washed with ethyl ether and dried.The compound presents good solubility in water and methanol, and was partially soluble in ethanol.Yield: 66%.IR (CsI) ν max /cm
Synthesis of the copper(II) quinine complex (2)
Copper(II) perchlorate (111 mg, 0.3 mmol) was added to 30 mL of an ethanolic solution of free base quinine (195 mg, 0.6 mmol).After stirring for 24 h at room temperature, a green solid was isolated by filtration, washed with ethanol and dried.Yield: 70%.IR (CsI) ν max /cm
Synthesis of the copper(II) hydroquinidine complex (3)
Copper(II) chloride (128 mg, 0.8 mmol) was added to 75 mL of an ethanolic solution of hydroquinidine hydrochloride (299 mg, 0.8 mmol).After stirring for 2 h, the mixture volume was reduced under vacuum.It was observed the precipitation of a mustard-yellow solid, which was treated with ethanol in an ultrasonic bath.The complex was separated by filtration, washed several times with ethyl ether and dried.The product obtained was soluble in water, methanol, and ethanol.Yield: 78%.IR (CsI) ν max /cm
Complex 1
The microanalysis results are in agreement with the formulation [Cu(C 7 H 13 N) 2 (OH 2 )Cl]Cl•2H 2 O.The molar conductivity value of a 10 -3 mol L -1 solution of complex 1 in nitromethane at 25 °C is Λ M = 84.92Ω -1 cm 2 mol -1 , indicating that it is an 1:1 electrolyte.The classic assay with silver nitrate was carried out using an aqueous solution of the complex.The white precipitate immediately obtained (AgCl) evidenced the presence of chloride as a counter ion.
The starting material used in the synthesis was quinuclidine hydrochloride, in which the nitrogen atom is protonated.For this reason, its vibrational spectrum shows intense characteristic bands in the region 2600-2800 cm -1 .Around 2910 cm -1 , there is a quite intense band with a shoulder at 2950 cm -1 due mainly to the C-H stretching vibrations of the methylene groups present in the quinuclidine molecule.Over 3300 cm -1 , it can be observed the presence of a set of overlapping absorptions attributed to the O-H stretching.This observation can be explained by the fact that the starting material is relatively hygroscopic in its protonated form.The previous hypothesis is confirmed by the medium intensity bands present at 1640 and 1620 cm -1 (δ O-H for the associated water molecule).At 1040 cm -1 , we found a narrow band of medium intensity associated with the C-N stretching.
Significant changes are observed in the spectrum of the complex compared to that of the proligand.The bands at 2600-2670 cm -1 disappear, indicating the deprotonation of the nitrogen atom.At 525 cm -1 , there is an absorption, which is absent in the proligand spectrum, attributed to the Cu-N stretching vibrations.
Dry quinuclidine hydrochloride decomposes in a single step in the temperature range 95-345 °C, with the inflexion point at 295 °C.The complex undergoes a three-stage decomposition process (Figure 2).Calculations based on the mass loss confirmed the stoichiometry of two quinuclidine ligands per metal.The complex does not lose mass under 164.3 °C.Thus, complexation improves the thermal stability of quinuclidine.A mass loss of 57.22% takes place between 164.3 and 330.0 °C, corresponding to the simultaneous loss of three water molecules, one quinuclidinic moiety and two chlorides (the calculated value is 57.47%).Between 330.0 and 571.3 °C, an ensuing decomposition occurs corresponding to 25.47%, which agrees with the calculated values for the other quinuclidine moiety, 27.06%.Above 580 °C the curve attains a plateau, with the formation of a stable residue corresponding to 17.31% of the total mass (the calculated value is 19.36%).This residue was identified as CuO, number 41-254 of the data bank of the ICDD (International Center for Diffraction Data), by X-ray diffractometry.
EPR spectrum of the complex, presented in Figure 3, is in agreement with a distorted tetrahedral geometry around copper.
Based on the results discussed above, we propose the structure shown in Figure 4 for the compound obtained.
Complex 2
The elemental analysis of the complex is in a good agreement with the formula [Cu(C 20 H 23 O 2 N 2 )(OH 2 ) 2 ]ClO 4 .The molar conductivity value of a 10 -3 mol L -1 solution of the complex in nitromethane at 25 ºC (Λ M =96.80 Ω -1 cm 2 mol -1 ) is in the 1:1 electrolyte range.
The IR spectrum of quinine shows a broad band centered at 3175 cm -1 (ν OH) with a shoulder at 3080 cm -1 (ν CH aromatic).In the complex the stretching vibration (ν OH) appears at 3425 cm -1 , and the ν C-H aromatic remains unchanged.Centered at 2940 cm -1 , there is an absorption band with a shoulder at approximately 2890 cm -1 , assigned to the aliphatic stretching vibration ν C-H, which decreases in intensity upon complexation.The IR spectrum of the proligand presents two absorption bands due to the -C=C-and -C=N-stretchings of the quinoline ring at 1620 and 1595 cm -1 , respectively. 23These bands remain unaltered in the complex indicating that the quinolinic nitrogen is not coordinated to the metal ion.Two new absorption bands, broad and intense, corresponding to the perchlorate stretchings appear at 1090 cm -1 (ν 3 ClO 4 -) and 625 cm -1 (ν 4 ClO 4 -).Free perchlorate ion belongs to a tetrahedral symmetry group and exhibits only these two infrared active modes.At 450 cm -1 appears an absorption band attributed to Cu-N stretchings.
The TG curves of quinoline and quinuclidine hydrochloride were recorded to help in the analysis of the thermal behavior of quinine and hydroquinidine.Quinoline decomposes between 20.0 and 200 °C in a single step.Quinuclidine hydrochloride also decomposes in a single step in the temperature range 95-345 °C.
The thermal decomposition curve of quinine is more complex, occurring in three steps.Between 165.0 and 345.0 °C there is a considerable mass loss of 47.58%.As quinoline is thermally less stable than quinuclidine, we attributed the first decomposition step to the quinoline moiety, which corresponds to 48.76% of the total mass.A minor decomposition of 6.67% takes place between 345 and 435 °C.Subsequently, between 435 and 695 °C, 45.75% of the total mass is lost.
The thermal decomposition of the complex occurs in many steps, which makes its interpretation a difficult task.The TG curve of 2, shown in Figure 2, exhibits a mass loss of 2.88% from 35 to 205 °C due to the loss of one water molecule (calculated 3.45).Between 215 and 255 °C, 31.12% of the total mass decomposes, probably related to the quinoline moiety plus one water molecule.The calculated value is 33.73%.An ensuing decomposition of 19.72% of the total mass takes place between 255 and 305 °C, corresponding to the calculated value for one perchlorate, 19.03%.This temperature range is similar to that found by Singh et al. 24 in a study about the thermal decomposition of perchlorates of some metal complexes with ethylenediamine or by Lalia-Kantouri and Tzavellas 25 in a study about perchlorates of copper(II) complexes with 1,2-diamines and β-ketoenols.Between 325 and 550 °C there is a mass loss of 30.98% probably due to the decomposition of the quinuclidinic moiety, which corresponds to 31.82%.Above 550 °C, the curve attains a plateau and the residue corresponds to 15.30% of the total mass (the calculated value is 15.23%).The residue was identified as CuO, number 41-254 of the data bank of the ICDD (International Center for Diffraction Data), by X-ray diffractometry.
An electron paramagnetic resonance study was also undertaken (Figure 3).EPR parameters are typical of a distorted tetrahedral copper complex.
These results led us to propose the structure represented in Figure 4 for the complex, in which quinine is acting as a bidentate ligand through the quinuclidinic nitrogen and the deprotonated hydroxyl group.Two water molecules complete the coordination sphere and there is a perchlorate as a counter ion.complex.The molar conductivity value of a 10 -3 mol L -1 solution in methanol at 25 °C (Λ M =111.00Ω -1 cm 2 mol -1 ) is in the 1:1 electrolyte range, thus in agreement with the proposed structure.
The IR spectrum of the proligand presents a broad band around 3417 cm -1 due to the O-H stretching vibrations, which is intensified in the complex and shifted to 3400 cm -1 .
The absorption present at 3083 cm -1 , attributed to aromatic ν C-H, shifts to 3050 cm -1 in the complex.In the 2950-2850 cm -1 region, absorptions due to the stretching vibrations of the aliphatic C-H appear.In the 2700-2350 cm -1 region, bands attributed to the N-H + stretchings are observed not only in the proligand but also in the complex spectrum, indicating that the protonated state of the quinuclidinic nitrogen remains upon complexation.The proligand spectrum shows two separate intense bands: one at 1617 cm -1 (ν C=C of the quinoline ring) and another at 1583 cm -1 (ν C=N of the quinoline ring).In the complex spectrum, these bands appear at 1615 cm -1 with a shoulder at 1600 cm -1 , which suggests the involvement of the quinoline moiety in the coordination.In the low frequency region, it can be observed a band centered at 530 cm -1 attributed to the Cu-N stretching frequency.
The TG curve of the proligand presents two major decomposition steps.The shape of the DTG curve when compared to that of quinine free base seems to be inverted, e.g., the symmetric peak attributed to the decomposition of the quinoline moiety seems to correspond to the second decomposition step in the TG curve of hydroquinidine hydrochloride (data not shown).The first major decomposition starts at 178.42 °C and, up to 358.42 °C, 46.84% of the total mass is lost in a multi-step process.It probably corresponds to the decomposition of the quinuclidinic moiety plus one chloride ion (calculated 48.14%).Between 358.42 and 418.42 °C a minor decomposition occurs, 5.97%.From 418.42 to 545.79 °C, 47.19% of the initial mass decomposes.
Thermogravimetry revealed that the complex loses water between 35 and 185 °C (Figure 2).The percentage of water calculated (5.15%) is in agreement with the value found (5.71%).Subsequently, another decomposition event takes place between 185.3 and 395.0 °C, corresponding to 30.92% and probably due to the decomposition of the quinuclidinic moiety.The calculated value for quinuclidinic moiety is 31.02%.Accordingly, the IR spectrum of the residue after this stage shows the presence of a broad absorption centered at 1600 cm -1 , characteristic of quinoline.Between 395.0 and 565.0 °C, 48.65% of the total mass decomposes.This percentage corresponds to the remainder of the hydroquinidine molecule plus three chlorides, 48.77%.Above 600 °C, a stable residue corresponding to 14.72% is formed (the calculated value is 15.17%).This residue was identified as CuO, number 41-254 of the data bank of the ICDD (International Center for Diffraction Data), by X-ray diffractometry.
The EPR spectrum of compound 3, shown in Figure 3, indicates a distorted tetrahedral geometry, in agreement with the proposed structure.
Based on the results presented, we propose the structure shown in Figure 4 for the complex.The copper atom is coordinated to the hydroquinidine molecule through the quinoline nitrogen, to two chloride ions and to a water molecule, adopting a distorted tetrahedral geometry.The quinuclidine nitrogen, in turn, is protonated and a chloride is present as counter ion.
Three new copper(II) complexes with the ligands quinine, hydroquinidine, and quinuclidine have been isolated and characterized.Quinuclidine was used as a simpler chemical model to investigate either the involvement or not of the quinuclidinic group in the coordination.
The IR studies have shown that the binding sites involved in the quinine and hydroquinidine complexes are not the same.In the IR spectrum of complex 2, the vibrational changes are similar to those observed for the quinuclidine complex, in contrast to that of the complex 3, whose main shifts correspond to the quinoline moiety.In the case of quinine, copper(II) coordination occurs through the quinuclidinic nitrogen, while in hydroquinidine the binding site is the quinolinic nitrogen.It is difficult to account for this distinct behavior based on the stereochemical differences between these molecules.They display very similar conformational behavior in solution, as evidenced in a study by NMR techniques. 26oth adopt open conformation, in which the lone pair of the quinuclidinic nitrogen points away from the quinoline ring. 26Nevertheless, the different protonation state of the quinuclidinic nitrogen in the starting materials can provide an explanation for this.In complex 2, the starting reagent was the free base.In this case, as the quinuclidinic nitrogen is deprotonated, this atom takes part in the coordination to the metal.In contrast, when the starting material presents the quinuclidinic nitrogen protonated, as in the case of hydroquinidine hydrochloride, coordination takes place through the quinolinic ring (complex 3).In the case of complex 1, in which the only potential binding site is the nitrogen, copper addition induces ligand deprotonation.
EPR parameters indicate a distorted tetrahedral geometry around copper.The ratio g || / A ⊥ can be used to predict the geometry adopted by copper complexes. 27quare planar complexes show this parameter in the range 105-135 cm, whereas for distorted tetrahedral complexes, the values fall in the range 135-258 cm.
The trend of increasing g || values and/or decreasing A ⊥ values in the order 2, 3, 1 shows that the ligand field strength decreases in the order 2 > 3 > 1. 28 In the proposed structures, quinine binds to copper in a bidentate manner while hydroquinidine hydrochloride in a monodentate one.
In very early studies, Hilliard et al. 29 synthesized a copper quinuclidine complex of stoichiometry 1:1 by reacting the proligand with copper chloride in butanol.The difference between the stoichiometries of this complex and ours can be explained by the distinct experimental conditions used.
Tsangaris and Baxevanidis 16 prepared two copper quinine complexes with the compositions 2 quinine: 3 CuCl 2 and 1 quinine: 2 CuCl 2 .The exam of their IR and UV-Visible spectra led to the proposition that copper was coordinated to both quinolinic and quinuclidinic nitrogens without a final conclusion about the participation or not of the hydroxyl oxygen in the coordination.Both coordination modes were also observed by Hubel et al. 30 in some quinine organometallic complexes with Pt II and Pd II .
Thermal analysis results are in agreement with the assignments of the coordination sites.Table 1 summarizes the TGA data obtained for all the three complexes synthesized.Calculations based on the mass loss led us to propose that if the coordination takes place by quinuclidinic moiety, the quinoline ring decomposes first.Differently, coordination at quinoline ring seems to increase the thermal stability of this part of the molecule, which starts to decompose after the quinuclidinic moiety does.
In complex 2 there are two coordinated water molecules.One of them is lost at a relatively low temperature, from 35 to 205 °C.The loss of the other one occurs overlapped with quinoline moiety between 215 and 255 °C.In complex 3, the water molecules are eliminated from 35 to 185 °C.Complex 1 does not lose water before 165 °C.
In relation to the decomposition of the quinine-type ligands, the thermal stability of the complexes increases in the order: 1 < 3 < 2. This order reflects the effect of increasing the ligand field according to EPR measurements.
Conclusions
Both nitrogens of quinine-type proligands, one in the quinolinic ring and the other in the quinuclidinic ring, constitute binding sites for copper(II) ions.If the quinuclidinic nitrogen is deprotonated, this atom takes part in the coordination to the metal but when the starting material presents the quinuclidinic nitrogen protonated coordination takes place through the quinolinic ring.Calculations made from thermogravimetric curves suggest that if the coordination takes place by quinuclidinic moiety, the quinoline ring decomposes first.Differently, coordination at quinoline ring seems to improve the thermal stability of this part of the molecule, which starts to decompose after the quinuclidinic moiety.The thermal stability of the complexes increases along with the ligand field: The mechanism of antimalarial action of quinine-type drugs is not yet fully understood but the capacity of these agents to interact with iron in vivo seems to be a determining factor.Thus, the knowledge of quinine coordination modes can be useful to better understand its behavior in vivo.
Complex 3
The results of elemental analysis, atomic absorption and neutronic activation point to [Cu(C 20 H 27 O 2 N 2 ) (OH 2 )Cl 2 ]Cl•½H 2 O as the formula of the synthesized
Table 1 .
TGA data for the copper complexes | v3-fos-license |
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} | pes2o/s2orc | Strategies to Preclude Hepatitis C Virus Entry Strategies to Preclude Hepatitis C Virus Entry
Without a preventive vaccine, hepatitis C virus (HCV) remains an important pathogen worldwide with millions of carriers at risk of end-stage liver diseases. Despite the introduction of novel direct-acting antivirals (DAAs), resistance problems, challenges with the difficult-to-treat populations and high costs limit the widespread application of these drugs. Antivirals with alternative mechanism(s) of action, such as by restricting viral entry or cell-to-cell spread, could help expand the scope of antiviral strategies for the management of hepatitis C. Transfusion-associated HCV infection remains another issue in endemic and resource-limited areas around the world. This chapter describes some of the latest developments in antiviral strategies to preclude HCV entry, such as through monoclonal antibodies and small molecules, as well as measures to enhance the safety of therapeutic plasma products in blood transfusion.
Introduction
Hepatitis C virus (HCV) is a major pathogen that predisposes about 170-300 million people worldwide to risks of end-stage liver diseases (ESLD), including cirrhosis and hepatocellular carcinoma (HCC). The hepatotropic virus remains one of the top indications for liver transplantation in treating ESLD [1]. While a preventive vaccine remains unavailable, the recent introduction of direct-acting antivirals (DAAs) has revolutionized the treatment for hepatitis C, phasing out the decade-old interferon (IFN)-based regimens. The majority of DAAs, however, focus on targeting viral replication such as via inhibition of the HCV NS3/4A protease, the NS5A cofactor, and the NS5B polymerase [2]. Although the DAAs have significantly improved the rate of sustained virological response (SVR) in the most prevalent genotype 1 patients, several challenges persist in real-world setting including high cost, drug-drug interactions, emergence of drug resistance, hard-to-treat populations (e.g., human immunodeficiency virus [HIV] coinfection, ESLD, and transplant patients), and management of DAA failures [3][4][5]. With the advent of hepatitis C treatment in larger populations and borrowing from the experience with HIV cocktail therapy, it is becoming clear that developing therapeutic strategies with different modes of action would be necessary to address the various limitations of current DAAs. In addition, HCV transmission due to transfusion of contaminated blood products remains an issue in endemic areas around the world. This is particularly the case in resource-limited countries that face inadequate supply of safe blood products or have poorly controlled blood screening practices, leading to significant risk of transfusionassociated HCV infection [6]. Measures to enhance the safety of therapeutic plasma products such as through the implementation of viral inactivation treatments are therefore a necessity to reduce such risk.
The multistep process of HCV entry makes it an attractive target since it is the foremost fundamental prerequisite in establishing an infection. Following successful entry, the viral life cycle initiates to produce more virions, and with this development the underlying disease begins its progression. Blocking HCV infection by targeting its entry therefore has important implications for both prophylactic and therapeutic purposes since it abolishes the viral life cycle. As a prophylactic treatment, it can be used to prevent infection or reinfection. This is particularly useful in liver transplant setting of hepatitis C wherein the liver allograft is inevitably reinfected [7,8]. As a therapeutic treatment, precluding HCV entry via de novo infection or cell-to-cell transmission helps to restrict viral spread in an infected person which could slow the progression of the disease. In addition, incorporation of strategies to block HCV entry into existing DAA treatments is expected to maximize the treatment response rate, even producing a synergistic effect [9], as with the experience of using multiple inhibitors in HIV cocktail therapy to concomitantly target various stages of the viral life cycle. Since more steps are being targeted in such a multipronged approach, the inclusion of entry inhibitors to existing DAAs could impose a higher genetic barrier to drug-resistance development. Such tactic not only aids in disrupting persistent HCV infection but could also help to ultimately achieve viral clearance. These aspects therefore make the development of HCV entry blocking strategies highly advantageous in both expanding the scope of antiviral treatments against hepatitis C and providing new insights into antiviral management. This chapter describes some of the latest development of strategies in precluding HCV entry for the management of hepatitis C.
Overview of HCV entry
Owing to the development of infectious HCV culture systems (e.g., cell-culture-derived HCV, HCVcc) and viral pseudoparticles bearing HCV glycoproteins (e.g., HCV pseudoparticles, HCVpp), a scenario of how HCV entry occurs has slowly emerged over the last decade of research. It is widely recognized that the HCV particle undergoes a series of intimate and wellorchestrated interactions with various receptors/coreceptors on the hepatocyte host cell surface as well as in the tight junctions, which ultimately lead to the attachment, internalization, and fusion of the virion with the cellular membrane. A number of these receptor interactions are thought to be attributed to the highly lipidated nature of the HCV virion. Specifically, HCV exists as a lipo-viro particle (LVP) with a lipid composition that includes the apolipoproteins and resembles that of very low-density lipoproteins (VLDLs) and low-density lipoproteins (LDLs) [10][11][12][13][14][15]. The association with lipids on the viral particle is thought to contribute to the shielding of HCV glycoproteins from neutralization by the host antibody-mediated response. In addition, the presence of the apolipoproteins on the virion has a large influence on the production of infectious HCV and also its tissue tropism [13,[16][17][18][19][20][21][22].
Following circulation in the blood, the HCV viral particles reach the liver and begin the interactions with molecules at the surface of the hepatocytes (Figure 1). The initial contacts are with nonspecific receptor(s) including the glycosaminoglycan (GAG) heparan sulfate moieties [23][24][25] that can be found on the transmembrane core proteins syndecans [26,27]. These early interactions facilitate the attachment of the HCV virion and its accumulation on the hepatocytes for subsequent binding to more specific receptors. Although the LDL receptor (LDLR) has also been suggested as a potential initial attachment factor [28][29][30], recent evidence suggests that it may play a more essential role in viral replication [31,32]. 46,47]. HCV binding to CD81 is proposed to induce a dynamic lateral diffusion of virus-receptor complexes toward the tight junction area for further interactions with additional entry factors and viral internalization [22,48]. Specifically, CD81 forms a coreceptor complex with the tight junction protein claudin-1 (CLDN1) [49,50] and is engaged in late events of HCV entry [51]. This re-localization and virusreceptor complex association with CLDN1 involves multiple signaling pathways (e.g., Rho GTPases, PI3K/AKT, and ERK/MAPK) [52,53], includes the activation of host cell kinases such as the epidermal growth factor receptor (EGFR) and ephrin receptor A2 (EphA2) [54,55], and is influenced by the absence of the CD81-associated partner EWI-2wint on the hepatocytes [56,57]. The EWI-2wint molecule is normally bound to CD81 on most cell type surfaces and inhibits its diffusion which is required to promote HCV entry; however, it is not expressed in the hepatocytes, and hence its absence has been suggested to contribute to the restricted tropism of the virus [56]. Following interaction with the CD81/CLDN1 complex, the HCV particle is presumed to then interact with the tight junction protein occludin (OCLN) prior to viral internalization [58]. Additional proteins that take part in influencing virion entry into the hepatocyte include the transferrin receptor 1 (TfR1) [59] and the cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) [60], although their specific role and interplay with other entry factors in the HCV entry process remain to be defined. The HCV particle finally enters the cell via clathrin-mediated endocytosis [61]. The HCV-receptor complexes then migrate to endosomal compartments [62,63] where acidification occurs to induce membrane fusion, which allows the release of viral RNA into the host cytosol.
The above sequential and multistep entry process consequently yields the successful release of the HCV genome into the host cytoplasm for direct translation and the ensuing launch of viral replication. The roles played by several of these entry factors including SR-BI, CD81, CLDN1, and OCLN not only mediate HCV entry but also presumably help to define tissue and species tropism of the virus [64][65][66][67]. The understanding of how HCV achieves viral entry has led to the possibility of antiviral targeting. From docking to virus internalization, essentially all steps are targetable to prevent HCV infection of the host cell. In addition, given the association of HCV with lipoproteins and the viral particle's interaction with lipoprotein and lipid receptors (LDLR, SR-BI, and NPC1L1), the lipidic nature of HCV virion also offers various methods of pharmacological intervention. Finally, many of the entry factors including CD81, SR-BI, CLDN1, OCLN, and NPC1L1 also play a role in mediating HCV cell-to-cell transmission between intercellular junctions [68][69][70][71], and therefore targeting these molecules could help restrict both cell-free entry and cell-to-cell spread of HCV.
3. Current development in inhibition of HCV entry 3.1. Use of monoclonal antibodies to target host cell receptors or viral antigens Recent insight into the molecular interactions of HCV at the cellular membrane has significantly enhanced the understanding of the HCV entry paradigm and revealed potential targets for drug intervention, including the use of monoclonal antibodies (mAbs) to mask HCV entry receptors/coreceptors or viral antigens. As described below and summarized in Table 1, the use of mAbs targeting CD81, SR-BI, CLDN1, or the HCV E2 has been shown to have prophylactic/therapeutic effects against HCV infection in both cell culture and animal models. . The antibody also appeared to block neutralizing antibody-resistant HCV cell-to-cell transmission and viral dissemination in a dose-dependent manner, with a less cytotoxic or antiproliferative property than JS-81 in vitro. In a recent study, another mAb K04 generated with hybridoma technique not only showed inhibitory effect against HCVpp and HCVcc infection in hepatoma cells and primary human hepatocytes (PHH), but also surprisingly blocked HCV infections in both prophylactic setting and postinfection stage in human liver-chimeric mice [77]. This is probably due to the improved intrinsic binding affinity of mAb K04 to CD81 large extracellular loop (LEL) and a different binding epitope as compared to mAb JS81. However, treatment-associated reductions in body weight and human serum albumin levels were observed in this study. Further research will be needed to determine the minimal dose of antibodies needed to provide protection and to evaluate the toxicology of anti-CD81 mAbs for long-term development.
Anti-SR-BI monoclonal antibodies
SR-BI is a member of the CD36 family primarily expressed in liver and non-placental steroidogenic tissues which facilitates selective cholesterol uptake [78]. The molecule has been proposed to be a horseshoe-like glycoprotein with a large extracellular loop anchored to the plasma membrane at both N-and C-termini with short extensions into the cytoplasm [79]. It was first identified as the alternative E2 receptor on HepG2 cells which efficiently recognize [190,192,193] Small Curcumin Decrease viral envelope fluidity; inhibit cell-to-cell spread Cell culture [107] CV-N E1/E2 glycan-binding protein Cell culture [109] Griffithsin E1/E2 glycan-binding protein Mouse model [110] MBL E1/E2 glycan-binding protein Cell culture [111] Recombinant L-ficolin E1/E2 glycan-binding protein Cell culture [112] BA-LNCs E2 glycan-binding protein Cell culture [114] Oleanolic acid E2 glycan-binding protein Cell culture [115] CD81-derived peptides Interact with E2 Cell culture [116,117] Additionally, mAb16-71, mAb8, and mAb151 all showed their ability in blocking HCV cell-tocell spread in vitro and in vivo. Human liver-chimeric mouse models challenged with serumderived HCV isolates of different genotypes revealed the anti-HCV property in vivo of the three antibodies in both prophylactic and postexposure settings. Specifically, mAb16-71 showed complete blockage of infection and intrahepatic spread of HCV isolates with a prophylactic treatment, but had no effect on chronically infected chimeric mice; mAb151, on the other hand, appeared to be effective against an HCV variant escaped from adaptive immune response in a liver transplant patient and displayed better antiviral activity in inhibiting viral spread and amplification in the postexposure setup.
Anti-CLDN1 monoclonal antibodies
The CLDN1 tight junction protein has four transmembrane domains and is highly expressed in the liver [83]. Its role in HCV entry is proposed to occur in the post-binding steps [64]. Anti-CLDN1 antibodies directed against the CLDN1 extracellular loops were found effective in neutralizing HCV infection in hepatoma cells through disrupting CD81-CLDN1 association and therefore inhibiting E2 binding to the cell surface [84]. A CLDN1 mAb OM-7D3-B3 targeting CLDN1 extracellular loop was found to be effective in inhibiting HCV isolates in vitro [85]. Further experiments in human liver-chimeric mouse models confirmed its potency in preventing HCV infection and eliminating persistent infection in vivo [86]. Pretreatment of another anti-CLDN1 mAb 3A2 targeting CLDN1 extracellular loop also showed protective effect in a chimeric mouse model [87]. Safety profiles of these antibodies were also assessed regarding the levels of human albumin, aspartate transaminase, alanine transaminase and total bilirubin, and potential side effects on the other organs and tight junction integrity. Further studies were suggested to assess potential immune-mediated adverse effects to ensure its relevance for clinical use [86,87].
Anti-HCV E2 monoclonal antibodies
Another approach to developing entry-inhibiting mAbs is to target the glycoproteins on the HCV virion surface. Albeit HCV glycoproteins exhibit high variability and are protected by glycosylation and lipids on the viral particle, neutralizing mAbs have been designed to target more conserved and accessible regions, specifically on the E2 glycoprotein [88]. Effects of E2 mAbs have been shown in vitro and in vivo [89][90][91][92][93][94]. Clinical trials have been carried out to assess the protective function of human anti-E2 mAbs HCV-Ab XTL 68 and MBL-HCV1 in liver transplant settings of HCV-positive patients. With a higher dose and daily infusion of HCV-Ab XTL 68, HCV RNA in patient serum showed transient reduction in the first week posttransplantation but not yet below the detectable limits [95]. MBL-HCV1, on the other hand, successfully suppressed the viral load from 7 to 28 days after transplantation in genotype 1ainfected patients with multiple infusions. Although the primary endpoint at day 42 was not met, the viral rebound was significantly delayed, and the magnitude of the viral load reduction was greater than the previous HCV-Ab XTL 68 therapy [96]. The result indicates that mAbs may be a promising class of entry inhibitors that adsorbs circulating virions to protect the new liver from reinfection after transplantation. A study of combination therapy with DAAs to prevent allograft HCV infection is currently underway [96].
Current obstacles to the development of mAbs as therapeutic antiviral agents include the high cost of production, storage, and administration, which can only be done by injection so far [88]. Nevertheless, the associated immune responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) may help to clear the viruses and infected cells [88,97]. Antibodies that directly block host cell entry factors are more likely to be effective for the diverse circulating viral strains; however, due to the distribution and multiple functions of such molecules, the blockage may cause potential adverse side effects [97]. As for antibodies targeting viral antigens, designing suitable candidates may be a challenging issue due to the heterogeneity of the HCV glycoproteins [98], but such antibodies may provide a safer option for the synergistic therapy with other antivirals of different modes of action to suppress the development of resistance, particularly at the early post-transplantation stage [96]. Additional neutralizing antibodies against other entry factors have also been reported to antagonize HCV infection in vitro, such as anti-TfR1 [59] and anti-NPC1L1 [60] antibodies, suggesting they could also be potentially developed for treatments against hepatitis C.
Small-molecule inhibitors of HCV entry
In addition to the mAbs, great efforts have been put into identifying small molecules with potent antiviral effects against HCV entry. The source of such entry inhibitors includes clinically approved medications, synthetic molecules, and natural product-based compounds. These small molecules could be further evaluated for development as drug candidates or drug leads. Below is a panel of small molecules that have been investigated with their activities inhibiting HCV entry ( Table 1).
Small molecules inhibiting viral attachment
The attachment step represents the primary interaction of an HCV virion with its host cell surface. Since the GAG heparan sulfate moieties dominate the capturing of HCV virions, the heparan sulfate homologue heparin and its derivatives as well as the enzyme heparinases which degrade the molecule were all shown to inhibit the viral binding to hepatoma cells [24,25]. (-)-Epigallocatechin-3-gallate (EGCG), a green tea catechin, was speculated to exert its inhibitory effect on viral attachment [99] by competing with heparan sulfate for HCV binding [100] or altering the viral shape [101]. Delphinidin, an anthocyanidin extracted from plant pigment, was also demonstrated to inactivate HCVpp by altering its shape and was particularly potent when added concurrently with the viral inoculation [101]. The natural terpenoid saikosaponin b2 (SSb2), isolated from the root of Bupleurum kaoi, was observed to specifically block HCV particle binding and early viral entry without affecting other stages of the viral life cycle [102]. SSb2 could inactivate cell-free HCV particles and was suggested to target the glycoprotein E2 in mediating its antiviral effect against HCV infection. Several other natural compounds including the gallic acid (GA) extracted from Limonium sinense [103], the hydrolyzable tannins chebulagic acid (CHLA) and punicalagin (PUG) [104], and the hepatoprotective plant Phyllanthus urinariaderived monolactone loliolide (LOD) [105] and butenolide (4R,6S)-2-dihydromenisdaurilide (DHMD) [106] were also found to efficiently inactivate cell-free HCV viral particles and impede viral attachment. Another natural compound curcumin extracted from turmeric was shown to decrease the fluidity of viral envelope and therefore prevent the binding and fusion [107], possibly by inserting into the membrane in a manner similar to cholesterol [108].
Small molecules blocking viral glycoproteins
A variety of broad-spectrum antiviral agents have exhibited their ability to interact with the glycans on viral glycoproteins. In the case of HCV, glycan-binding proteins interfere with the association between the E1/E2 heterodimer and the host cell receptor CD81. Lectins such as cyanovirin-N (CV-N) and griffithsin, isolated from cyanobacterium Nostoc ellipsosporum and the red alga Griffithsia sp., respectively, were reported to have such effect. CV-N was shown to interact with N-linked glycans of HCV glycoproteins and disrupt E1/E2 binding to CD81 [109]. The inhibitory effect of griffithsin on HCV entry was also quenched when N-linked highmannose oligosaccharides were present, indicating a pattern similar to CV-N of affecting the glycoproteins-CD81 interaction [110]; pretreatment of griffithsin was shown to delay the viral infection in chimeric mouse model. Humoral lectins of the innate immune systems including the mannan-binding lectin (MBL) and L-ficolin were also considered to have analogous effect neutralizing HCV particles. MBL [111] and recombinant oligomeric L-ficolin [112] were found to interact with the glycans on the E1/E2 heterodimer in a calcium-dependent manner, thereby inhibiting the viral entry. Notably, the MBL-associated complement system was activated upon its binding to HCV E1/E2, suggesting the use of humoral lectins as viral entry inhibitors may also help facilitate viral clearance. However, the detailed mechanism and specific target of the humoral lectins remain to be defined. The boronic acid (BA)-modified nanoparticles were also found to suppress HCV entry in a way that acted similar to lectins [113], with the incorporation of lipid nanocapsule (BA-LNC) techniques enhancing their stability and solubility [114]. Chemically modified oleanolic acid, a triterpene compound originally extracted from Dipsacus asperoides, was found able to interrupt the E2-CD81 interaction by binding to E2 [115].
Besides the glycan-binding proteins, molecules imitating HCV host entry factors or viral glycoproteins were also developed in the attempt to block the viral entry. An imidazolebased scaffold presenting CD81 helix D amino acid side chains [116] and stapled peptides based on CD81 LEL [117] were designed to antagonize the E2-CD81 interaction by mimicking the putative E2-binding region of CD81. A CLDN1-derived peptide, CL58, was also found to inhibit HCV entry in the post-attachment stage by interacting with HCV E1 and E2 [118]. As for viral glycoprotein-based molecules, an E2-derived peptide was found able to block E1/E2-mediated fusion by targeting E1 and therefore interfere with the hetero-dimerization of the glycoproteins [119].
Small molecules targeting host entry factors and CD81-triggered signaling pathway
In addition to the therapeutic antibodies mentioned in the previous section, several small molecules have been suggested to exert their inhibitory activity of HCV entry by targeting cellular receptors/coreceptors. Terfenadine, an antihistamine, was found able to prevent HCV infection by competing with the CD81 antibody JS81 binding to the LEL of CD81 protein on the hepatoma cell surface [120]. ITX 5061, a clinical stage compound originally characterized as a p38 MAPK inhibitor, was identified with its capability of antagonizing SR-BI [121] and further validated for its potency of inhibiting HCV entry at post-binding step [122]. The anti-HCV effect of ITX 5061 was found additive to synergistic in combination with several standard-of-care therapeutics, and the resistant mutant was defined on the viral glycoprotein E2 [123]. A latest phase 1b clinical trial [124] revealed that the ITX 5061-treated patients, especially the genotype 1-infected patients, had a significant reduction in HCV RNA through the first week after liver transplantation and viral evolution were restricted; however, the viral RNA levels became comparable in both ITX 5061-treated and untreated patients, suggesting the need to incorporate other antiviral agents using different modes of actions to eliminate HCV infection. Aspirin, alternatively, inhibited HCV entry by downregulating CLDN1 [125].
Since the receptor tyrosine kinases are also involved in the HCV entry process, two clinically approved protein kinase inhibitors were evaluated for their ability to abrogate the viral entry. Both erlotinib, an EGFR inhibitor, and dasatinib, an EphA2 inhibitor, could successfully block HCV entry in a dose-dependent manner as well as the cell-to-cell transmission. Specifically, erlotinib was shown to inhibit the membrane fusion of hepatoma cells overexpressing HCV glycoproteins. In vivo treatment of erlotinib resulted in a significant suppression of the viral load in PHH-chimeric mouse model with HCV infection [54]. Furthermore, inhibitors of EGFR downstream kinases Ras (tipifarnib) and Raf (sorafenib) were also assessed and found effective in blocking HCV entry [126].
Inhibitors of other entry factors were also shown to be effective in hampering the viral entry. Pretreatment of ferristatin, a TfR1 inhibitor that binds to the molecule and causes its internalization and degradation, was shown to decrease HCVcc infection in vitro [59]. The NPC1L1 internalization inhibitor ezetimibe, which is also an FDA-approved cholesterol-lowering medication, diminished HCVcc foci formation before and during the viral challenge. Daily oral administration of ezetimibe starting two weeks before infection also delayed the viral growth of a genotype 1 clinical isolate in PHH-chimeric mouse model [60]. PF-429242, an SKI-1/S1P inhibitor, potentially impeded HCV entry by downregulating NPC1L1 and LDLR expression [127].
On the other hand, phenothiazines, a group of synthesized nitrogen-and sulfur-containing tricyclic compounds, inhibited HCV fusion into the cell by modulating the host cell membrane. Insertion of phenothiazines into the cholesterol-rich membrane increased its fluidity, thus possibly decreasing the local inhomogeneity of the cell required for the viral fusion [128].
Inhibition of clathrin-mediated endocytosis and viral fusion
Since HCV fusion has been discovered to be facilitated by clathrin-mediated endocytosis and requires an acidic environment, several reagents were assessed for their effectiveness in preventing HCV entry through blocking such pathways. Chlorpromazine, an inhibitor of clathrin-coated pit formation, was shown to inhibit both HCVpp and HCVcc infection in vitro in the validation of clathrin-mediated endocytosis pathway of HCV fusion to the host cell membrane [61]. Arbidol, a broad-spectrum antiviral agent that blocks viral entry and has been licensed in some regions for influenza, was described to trap the HCV virion in clathrin-coated vesicles, thereby hindering the release of viral genome and the following infection [129]. It was also suggested that arbidol could generally cause the intracellular accumulation of clathrincoated structures and restrain the formation of clathrin-coated pits on the cell surface [129], possibly due to its tropism for lipid bilayers.
Small molecules disturbing the acidic endosomal compartments were also identified as HCV entry inhibitors in the discovery of the low pH-triggered entry. These include bafilomycin A1 and concanamycin A, which are inhibitors of vacuolar H+-ATPases [130]. Weak bases such as chloroquine and ammonium chloride were also found to inhibit the low pH-dependent conformational change required for the viral fusion, based on their ability to penetrate lysosomes and increase the pH [131]. Finally, dUY11, one of the rigid amphipathic fusion inhibitors (RAFIs), was suggested to inhibit HCV entry by interacting with the hydrophobic structures in virions and preventing the formation of negative curvature required for viral fusion [132]. Curcumin [107] is also able to affect the fusion step as previously mentioned.
Small molecules inhibiting cell-to-cell transmission
Besides inhibiting the HCV entry in de novo infection, blocking cell-to-cell spread of the viral particles is also important as this mode of transmission facilitates efficient spread of the virus in the liver escaping from neutralizing antibodies [68,69]. Ferroquine was speculated to interact with HCV glycoprotein E1 and abrogate cell-to-cell spread of the virus [133]. Triazine-based compounds indicated to be closely related to the amino acids on the glycoprotein could also selectively inhibit genotype 1 HCV entry at the post-attachment step along with cellto-cell transmission [134,135]. Several molecules also block cell-to-cell spread in addition to their activities in hindering HCV viral entry. For instance, besides impeding viral attachment, CHLA and PUG exhibit pronounced antiviral effects at the postinfection stage, especially in restricting HCV foci expansion [104]. Others include EGCG [99], curcumin [107], erlotinib, and dasatinib [54]. Silibinin, the major component of Silybum marianum that has been designated as an orphan drug for the prevention of recurrent hepatitis C in liver transplant patients [136], was also suggested to possess a prominent effect blocking transmission of the viral particles between intercellular junctions [137,138], although other studies have proposed that it may slow down clathrin-mediated endocytosis [139] as well as inhibit viral membrane fusion [140]. This could be useful since DAA-resistant HCV variants have been suggested to escape via cellto-cell transmission route [141]. Therefore, the choice of inhibitors exhibiting mechanistic effect against both HCV cell-to-cell spread and cell-free entry, or a combination of such two types of inhibitors, should facilitate viral clearance.
Additional candidate entry inhibitors
Some other molecules were found able to prevent the infection at different steps of HCV entry. The estrogen receptor modulator tamoxifen [142] and HCV infectivity inhibitor 1 (HCV II-1 or GS-563253) [143] were shown to inhibit the HCV infection at both attachment and post-binding steps. HCV II-1 was also found capable of impeding infectious virion propagation [143]. HCV entry inhibitor 1 (EI-1 or BJ486K), a flavonoid ladanein, was shown to interrupt the viral entry at post-attachment stage [144]. The exact mechanisms of these molecules require further investigations. Other compounds such as serum amyloid A [145,146], p7 ion channel-derived peptide H2-3 [147], amphipathic DNA polymers [148], lactoferrins [149], tellimagrandin I and its derivatives [150], indole derivatives [151], and imidazo[1,2α] [1,8]naphthyridine derivatives [152] were found able to inhibit HCV entry with mechanisms that remain to be clarified.
Control of HCV infection risks in human blood-derived therapeutic products
Many viruses can contaminate human blood. HCV, along with HIV and HBV are a major cause of infectious complications of blood product transfusion therapy. HCV contamination in patients by transfusion of blood components such as red blood cells, platelets or clinical plasma, as well as industrial fractionated plasma products, has been well documented. At the time of the "tainted blood scandal," numerous recipients of blood components and hemophiliacs receiving plasma-derived factor VIII concentrates were contaminated through transfusion of nonvirally inactivated products prepared from blood products that were not HCV-tested.
HCV transmission through blood transfusion is a major medical issue, as infection can lead to high risk of liver cirrhosis and eventually cancer complications.
HCV safety nets for blood components
There are now over 100 million whole blood donations collected each year in the world. Collected blood is most often separated by "blood establishments" into red blood cell concentrates, platelet concentrates, and plasma that are transfused at nearby hospitals. Plasma, which can be obtained from whole blood collection or drawn by specialized apheresis procedures, can also be used as raw material for the production of "industrial" plasma protein products. These protein drugs include immunoglobulins G (IgGs), various coagulation factors, albumin, and many others. Industrial plasma products are manufactured from pools of plasma of several thousand liters, making them statistically more susceptible to contamination by HCV and other viruses as one highly infectious donation would contaminate the whole plasma pool and potentially the derived products.
Today in developed economies benefiting from strict regulatory oversight, several measures are in place to decrease the possibility for patients to acquire HCV by transfusion. Blood transfusion HCV safety nets for blood components rely on complementary measures encompassing (a) epidemiological control of the population, (b) individual screening of candidate blood donors to defer those identified as presenting potential risk factors, and (c) individual blood donation testing to identify and eliminate donations reactive to anti-HCV antibodies and/or HCV RNA nucleic acid test (NAT) [153]. In technology-advanced countries applying such procedures, this has allowed to decrease the risk of acquiring HCV by transfusion of single blood components down to approximately 1 per 1.8 million. The remaining risk reflects the inevitable presence of "window-phase" donations for which all markers to detect donor infection by HCV, either indirect or direct, are found nonreactive [154,155]. Understandably, HCV transmission risks are substantially higher in less developed economies (a) lacking a safe blood donor base, (b) relying on paid or "replacement" donors to increase the blood supply, (c) with a deficient blood collection system, and (d) with a lack of reliable viral testing procedures [6]. The ultimate barrier to avoiding HCV transmission risks from blood products collected during the window-phase period relies on the implementation of dedicated viral reduction treatments. Those have been developed for industrial plasma protein products, plasma for transfusion, and platelet concentrates. Until now, however, no treatment is available commercially for whole blood and red blood cell concentrates.
HCV reduction treatment of industrial plasma protein products
Development and implementation of dedicated viral/HCV reduction treatments of industrial plasma protein products took place in the 1980s and early 1990s [156]. In the early 1980s, albumin, a relatively heat-stable protein, was the only plasma product subjected to specific HCV inactivation by heat treatment at 60°C for 10 h in the liquid state (a process called pasteurization), in the presence of fatty acid stabilizers. From the mid-1980s to the early 1990s, heat treatment of freeze-dried coagulation factors at 60-68°C for 24-96 h or 80°C for 72 h were developed to inactivate HIV and HCV concomitantly [156]. Although pasteurization has successfully been adapted to several plasma products (such as antithrombin and alpha 1antitrypsin), a milestone in the safety of industrial plasma products was the development of the solvent/detergent (S/D) incubation procedure at 20-37°C [157] designed to dissolve the lipid envelope of viruses, including HCV, without affecting plasma protein functions. This technique is still largely used for a wide range of industrial plasma products owing to wellproven efficacy and a safety profile established by years of industrial and clinical practices [158]. Other HCV viral inactivation treatments include low pH incubation and caprylic acid precipitation/incubation of immunoglobulin products [159]. An additional milestone to enhance plasma protein product safety is nanofiltration, a procedure of filtration of protein solutions on 15-35 nm nanopore membrane devices designed to entrap and remove viruses [160]. This dedicated virus removal methodology is well established, including for HCV, and is currently applied to most plasma products [156]. Thanks to the implementation of such reduction treatments, most often combined in a complementary manner at different stages of the manufacturing process, no case of HCV transmission by industrial plasma products has been reported since 1993 [154].
Plasma
Several viral inactivation treatments of clinical plasma are licensed in various countries [161]. The S/D technology was adapted to 100-500 l of pooled industrial plasma in the early 1990s [162] and demonstrated, prior to HCV identification, to efficiently inactivate non-A-non-B hepatitis virus [163]. The removal of the S/D agents is typically achieved by oil extraction and column hydrophobic interaction chromatography [162]. A miniaturized version of the S/D process using a different detergent (Triton X-45 instead of Triton X-100) has been developed allowing its implementation in single-use equipment, thereby facilitating its application in developing countries, such as Egypt, currently lacking industrial capacity [164]. The efficacy of such method to inactivate HCV has been specifically demonstrated using an in vitro culture assay [165].
A procedure consisting in adding methylene blue and illuminating acellular plasma was made available in the early 1990s [166]. The method leads to inactivation of free HCV particles through photochemical alteration of nucleic acids and incapacity of replication [154,167].
Two other photoinactivation procedures of plasma have been licensed more recently. One combines the addition of psoralen S-59 (amotosalen) with ultraviolet light A illumination [168]. The other is based on the addition of riboflavin followed by UV irradiation [169]. These small molecules can penetrate membranes and intercalate with helical regions of HCV nucleic acids. Subsequent UV illumination irreversibly alters nucleic acids, making HCV particles unable to replicate [154,170].
Platelets
Development of HCV inactivation methods in cellular blood products in general, and platelet concentrates in particular, has been more challenging due to the difficulty to inactivate intracellular viruses without affecting cell function for transfusion. The two photoinactivation methods applied to plasma could nevertheless be adapted to the inactivation of HCV and other viruses in platelet concentrates [170][171][172].
Cryoprecipitate
Cryoprecipitate, obtained by a freeze-thaw process of plasma, is rich in factor VIII, von Willebrand factor, and fibrinogen. This plasma fraction is still largely used in many developing countries for substitution therapy in hemophilia A, von Willebrand factor disease, or fibrinogen deficiency, respectively. The frequency of treatment of patients with congenital deficiency exposes them to a high risk of infection in countries such as Egypt with a close to 10% HCV incidence [173,174]. Similar mini-pool methods of HCV inactivation used for clinical plasma are applied to cryoprecipitates [164].
Red blood cell concentrates and whole blood
No methodology is licensed yet for HCV inactivation in red blood cell concentrates or whole blood. However, the riboflavin/UV pathogen reduction technology is being adapted to the treatment of whole blood [175] and has been shown recently to contribute to lower the risk of malaria transmission in a clinical study in Ghana [176]. It is still uncertain whether a pathogen reduction technology can be developed to substantially inactivate HCV in whole blood or red blood cell concentrates without detrimentally affecting their transfusion quality and functionality or immunogenic potential.
Therapeutic apheresis and passive immunotherapy
Additional methods of precluding HCV infection are to remove circulating virus through therapeutic apheresis or attempting to neutralize HCV infectivity by administering plasmaderived anti-HCV immunoglobulins. These strategies are aimed at reducing the infectious viral load and have been explored in clinical trials.
Therapeutic apheresis for the removal of HCV virions
Therapeutic apheresis is the process of transiently circulating the blood outside the body and removing the components causing particular diseases by membrane separation and adsorption separation technologies. In the case of HCV, immunoadsorption apheresis was first applied to treat the chronic hepatitis C-related cryoglobulinemia that causes autoimmune symptoms [177]. The technique of heparin-induced extracorporeal LDL precipitation (HELP) apheresis, which could eliminate apolipoprotein B-containing lipoproteins, was then discovered to reduce HCV viral load [178]; however, the decline was found not correlated with LDL reduction in plasma and appeared to be transient due to the high turnover rate of HCV [179]. Studies using combination therapy of antiviral agents and double-filtration plasmapheresis (DFPP) that selectively removes substances with high molecular weight including HCV particles and therefore, happened to display better effects of suppressing the viral kinetics and therefore have been substantially explored during the past decade. Patients who underwent the prophylactic combination treatment of low-dose IFN, ribavirin, and DFPP had no evidence of HCV recurrence or fibrosing cholestatic hepatitis exacerbation for more than 1 year after liver transplantation [180]. Combination of DFPP and IFN also achieved impressive SVR in difficult-to-treat patients (i.e., relapsed, nonresponder, or HIVcoinfected patients) [181][182][183][184] and may also be safe for the elderly population [185]. However, the approach of apheresis for decreasing HCV viral load requires specialty equipment and possesses potential risk of adverse events (e.g., blood pressure lowering, puncture site hematoma, or infection) [181,185].
Passive immunotherapy using plasma-derived polyclonal HCV immunoglobulins
Passive immunotherapy, also known as antibody therapy, is a very well-established treatment based on the administration of polyclonal hyperimmune immunoglobulins extracted from plasma or mAbs prepared by genetic engineering technologies. One application of passive immunity is to prevent or treat infections due to viruses or to reduce the pathologies associated with bacterial or venom toxins. Human immunoglobulins for passive immunotherapy are fractionated from the plasma of immunized donors having high-titer antibodies against a particular organism or antigen. For the fractionation process, plasma donations from hundreds or thousands of donors are pooled and subjected to various purification and viral inactivation steps, as described in this chapter, to isolate an essentially pure Ig preparation [159,186]. Current human plasma-derived hyperimmune globulin products are used for the prophylaxis and treatment of viral diseases due to hepatitis B virus (HBV), rabies virus, cytomegalovirus, hepatitis A virus, or respiratory syncytial virus [187]. Human plasma-derived polyclonal hepatitis B immunoglobulin for intravenous use has been made available commercially for over 20 years in some countries. These licensed preparations are efficacious to predictably prevent HBV recurrence after liver transplantation and vertical HBV transmission from mother to child and are used as prophylactic treatment to prevent infection following contact with HBV-contaminated body fluids [188].
The possibility to use polyclonal HCV immunoglobulin to treat or prevent HCV infection has been proposed for many years [189], but no commercial preparation is available yet as it is not proven whether such immunoglobulin can prevent HCV infection or control viremia in infected patients. The rationale in polyclonal HCV immunoglobulins made from large pool of plasma units is to have a preparation that contains neutralizing antibodies to various strains of HCV [189]. However, the presence of neutralizing antibodies has been unclear initially as their presence in plasma was just considered to reflect the occurrence of an infection. Data have suggested that HCV-neutralizing antibodies exist in anti-HCV-positive plasma, but the anti-HCV antibody titer does not correlate with neutralizing capacity [190]. In vitro and animal experiments in a mice model have nevertheless suggested the presence of neutralizing antibodies in polyclonal IgG from a patient with a long-standing HCV infection [191]. A clinical study was initiated in the USA to evaluate the capacity of polyclonal plasma-derived HCV immunoglobulins to "prevent post-transplantation HCV infection of the liver graft and related progression of HCV-related liver disease." This clinical trial was "designed to evaluate a polyclonal human hepatitis C immune globulin given during and post liver transplantation for preventing or reducing the impact of recurrent HCV infection" [192]. However the trial was terminated in 2012 after treatment of seven patients (five receiving the immunoglobulins and two standard-of-care treatment alone) and no data reported. A new trial has begun in 2013 and was recently completed [193]. It is unclear whether plasma-derived polyclonal HCV immunoglobulin will be developed. If this occurs, clear donor screening and donation testing criteria should be defined to determine the specifications of the plasma donations suitable for fractionation, as well as the fractionation methodology itself to exclude any infectious risks from the fractionation of plasma donations. It should be noted that several mAbs for clinical use in HCV-infected patients have been proposed and one has undergone a clinical trial [190]. The future will indicate whether any HCV immunoglobulin, either polyclonal or monoclonal, has a role to play in the control of HCV infection.
Prospects of targeting HCV entry in clinical setting
Treatment options against hepatitis C have significantly improved owing to recent advances in the development of anti-HCV therapeutics. Nevertheless, there is still much room for improvement due to potential drug resistance and possibility of viral rebound, which usually require long periods of monitoring and analysis to uncover. More importantly, there is currently no immunization or prophylactic treatment against hepatitis C. Introducing novel antivirals with a different mode of action, such as targeting viral entry using mAbs or small molecules, not only helps expand the spectrum of anti-HCV drugs but also in developing novel treatment modalities. Many of the mAbs targeting HCV receptors/coreceptors as well as small-molecule inhibitors of HCV entry impede both viral attachment and cell-to-cell transmission; this is useful in providing protection against de novo infection and at the same time in helping restrict viral spread. The inclusion of viral entry inhibitors to current DAAs has already been shown to produce synergistic treatment effect [9]. Furthermore, taking a multistep targeting approach would help elevate the genetic barrier against selection of resistant variants, thus facilitating viral clearance. Finally, the advantage of developing entry inhibitors is its potential prophylactic application against hepatitis C, which is particularly useful in protecting liver allografts from recurrent HCV infection. Other protective measures of hepatitis C transmission in clinical scenarios include implementation of viral inactivation methods for the removal of HCV infectivity in therapeutic plasma products [165].
In addition, therapeutic apheresis [180] and protective anti-HCV immunoglobulins [192,193] have also been suggested for prevention of HCV reinfection in liver transplant patients. In the absence of an approved hepatitis C vaccine, these approaches could be explored as preventive and prophylactic measures against HCV infection. With the above-described strategies to preclude HCV entry, it is foreseeable, in a not-too-distant future, that these tactics under development will help provide a better management of chronic and recurrent hepatitis C, particularly in liver transplant setting. | v3-fos-license |
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} | pes2o/s2orc | Characterization of novel dual tandem CD19/BCMA chimeric antigen receptor T cells to potentially treat multiple myeloma
Treatment with chimeric antigen receptor (CAR)-engineered T cells directed against the B-cell maturation antigen (BCMA) promoted transient recovery from multiple myeloma (MM). However, the absence of this antigen on immature plasma cells may limit the efficacy of this modality and facilitate relapse. The purpose of this study is to characterize a novel CAR that includes both a single-chain variable fragment (scFv)-BCMA and an scFv-CD19 in tandem orientation (tan-CAR) in an attempt to target both BCMA and CD19 expression on MM cells.
The scFv sequences from the anti-CD19 antibody FMC63 and the anti-BCMA antibody C11D5.3 were ligated in tandem with transmembrane and T-cell signaling domains to generate the tan-CAR construct. Specificity and efficacy of activated tan-CAR T cells were analyzed using in vitro proliferation, cytokine release, and cytolysis assays. We also evaluated the in vivo efficacy with a xenograft mouse model that included target tumor cells that expressed CD19 or BCMA and compared the results to those obtained with conventional CAR T cells.
The in vitro studies revealed specific activation of tan-CAR T cells by K562 cells that overexpressed CD19 and/or BCMA. Cell proliferation, cytokine release, and cytolytic activity were all comparable to the responses of single scFv CAR T cells. Importantly, in vivo studies of tan-CAR T cells revealed specific inhibition of tumor growth in the mouse xenograft model that included cells expressing both CD19 and BCMA. Systemic administration of tan-CAR T cells resulted in complete tumor remission, in contrast to the reduced efficacies of BCMA-CAR T and CD19-CAR T alone in this setting.
We report the successful design and execution of novel tan-CAR T cells that promote significant anti-tumor efficacy against both CD19 and BCMA antigen-positive tumor cells in vitro and in vivo. The data from this study reveal a novel strategy that may help to reduce the rate of relapse in the treatment with single scFv-CAR T cells.
Introduction
Multiple myeloma (MM) is a malignant neoplasm in which uncontrolled expansion and proliferation of clonal plasma cells leads to osteolytic lesions and bone marrow failure in association with end-organ damage [1]. Several new drugs and drug regimens have recently been introduced in an effort to improve treatment for MM. Although these regimens are overall safer than previous therapies, only a limited number patients respond completely and effectively [2][3][4]. As such, we need to consider more innovative strategies with the aim of generating a more significant and long-lasting therapeutic effect.
Cellular immunotherapy is a novel and evolving treatment strategy in which cytotoxic T cells are engineered to promote recognition of specific tumor antigens. Adoptive transfer of chimeric antigen receptor (CAR)-engineered autologous T cells has met with unprecedented success for the treatment of hematological malignancies [5][6][7]. In parallel, several diverse immunotherapeutic approaches currently under investigation have utilized this approach and focus on engineering target antigen specificity and Tcell activation [8]. The CAR T-cell approach for the treatment of MM has shown considerable promise and has been associated with manageable toxicities. Notably, several efforts have focused on B-cell maturation antigen (BCMA) due to its preferential expression on plasma cells [9][10][11]. To date, early phase clinical trials that explore the impact of single-chain fragment variable (scFv) anti-BCMA-modified CAR T cells have shown undeniably high response rates. Unfortunately, the responses are often transient with frequent relapse [12]. One of the reasons of relapse might due to a group of residual malignant CD19 + plasma cells which can be detected among the tumor cells; these cells can drive self-renewal, myeloma propagation, and resistance to chemotherapy and can be considered to be cancer stem cells [13]. Furthermore, sustained remission was observed with advanced MM in one patient who received anti-CD19 CAR T cells in conjunction with an autologous stem cell transplantation [14]. Thus, CD19 might be the potential target for multiple myeloma treatment. Moreover, sequential delivery of BCMA-CAR and CD19-CAR T cells resulted in a strong therapeutic outcome; preliminary data suggested that amplification of CD19-CAR T cells might be critically associated with this response and even the absence of even minimal residual disease [15]. However, it is critical to note that patients diagnosed with associated lymphocytopenia may not have enough T cells for the production of two CAR T products; high manufacturing costs are also a key limitation to be considered. We also note that sequential delivery of two independent CAR T products might be associated with limited efficacy of the second infusion [16]. Previous study proved bi-specific CAR capable of preventing antigen escape in vivo by post-mortem analysis which revealed the outgrowth of CD19− mutants in the mixed-Raji xenograft [17]. Taken together, these results suggest that we might employ CAR T cells that simultaneously recognize both CD19 and BCMA for effective treatment of MM and reduce the risk of relapse.
Here, we describe a novel CAR lentiviral construct with tandem alignment of a dual scFv (tan-CAR) targeting both CD19 and BCMA antigens. To the best of our knowledge, this is the first time this approach has been considered. Among our results, we found that tan-CAR T cells targeting one or both antigens promote equivalent cytotoxic effects in vitro as do conventional CAR T cells with only a single scFv. Interestingly, the results of our in vivo studies suggested that tan-CAR T cells were capable of eradicating a mix of malignant cells that express CD19 or BCMA, ultimately resulting in complete remission. As such, our results suggest that the tandemdual antigen targeting strategy will represent effective anti-neoplastic therapy may ultimately prevent relapse secondary to absence or loss of BCMA expression on the malignant MM cells.
Plasmid construction and production of recombinant lentiviral vectors
The tandem-CAR construct is a second-generation vector consisting of the following components in-frame from the 5′ end to the 3′ end: the CD8 signal peptide sequence, anti-BCMA-scFv (C11D5.3) [18] , anti-CD19 scFv (FMC63AA 1-267, GenBank ID: HM852952.1), the hinge and transmembrane regions of the CD8α molecule, the cytoplasmic domain of CD28, and the CD3 zeta signaling domain. The sequence was synthesized by Tsingke Biological Technology (Shanghai, China) and cloned into the pUT plasmid backbone (Unicar-Therapy Biomedicine Technology Co., Ltd., Shanghai, China). The newly-constructed lentiviral vector is referred to as tan-CAR. We also prepared the scFv domain CAR vectors CD19-CAR and BCMA-CAR with the CD8 signal peptide sequence anti-CD19 scFv (FMC63AA 1-267, GenBank ID: HM852952.1) or anti-BCMA-scFv (C11D5.3), the hinge and transmembrane regions of the CD8α molecule and cytoplasmic domain of CD28 and the CD3 zeta signaling domain. Lentiviruses were generated from these constructs via transient transfection of HEK293T cells.
Cell lines
All the cell lines were purchased from the American Tissue Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Medical Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA). K562 cells were stably transduced with the lentiviral constructs encoding CD19 or BCMA and luciferase. Following transduction, single luciferase-positive cells were selected for clonal expansion. K562-CD19-luc and K562-BCMA-luc stable cell lines were generated by this method. Myeloma cell line 8226 were transduced with the lentiviral constructs encoding CD19 to obtain the tumor cells expressing both CD19 and BCMA.
Preparation of CAR T cells
Healthy donor-derived peripheral blood mononuclear cells were isolated from blood by gradient centrifugation using Lymphoprep™ (Oriental Hua Hui, Beijing, China) followed by CD3 + T-cell enrichment by positive selection using a magnetic bead separation method (Miltenyi Biotec, Bergisch Gladbach, Germany). CD3 + T cells were cultured and activated in vitro using anti-CD3/CD28 monoclonal antibodies (Miltenyi Biotec) in a 5% CO 2 atmosphere at 37°C for 18-24 h. The activated T cells were then transduced with lentivirus (CD19-CAR, BCMA-CAR and tandem CAR) for 48 h. We also tansduced the BCMA CAR (D1) followed by CD19-CAR (D2) to obtain the T cells expressed two scFv by transduced two lentivirus. After transduction, the CAR T cells were cultured and expanded in a 5% CO 2 atmosphere at 37°C for 14 days in AIM-V medium (Gibco, Grand Island, NY, USA), supplemented with 100 IU/mL recombinant human interleukin-2 (IL-2; Peprotech, Rocky Hill, NJ, USA), 5 ng/ml recombinant human IL-7 (Peprotech), 5 ng/mL recombinant human IL-15 (Peprotech) and 10% autologous plasma [19].
Flow cytometry
For the flow cytometry assays, the cells were harvested and washed twice with 1 mL of phosphate-buffered saline (PBS) containing 2% FBS (Gibco). To determine transduction efficiency and the CD4/CD8 ratio, the CAR T cells were labeled with the recombinant protein L-FITC (ACRO Biosystems, Beijing, China), anti-CD4-PE-Cy7 (eBioscience, San Diego, CA), and anti-CD8-APC (eBioscience) for 45 min at 4°C in the dark. For detection of the CD19 CAR-expressing T cells, the CAR T cells were incubated with human CD19 protein-FITC (ACRO) for 45 min at 4°C in the dark. For detection of the BCMA-CAR T cells, the CAR T cells were labeled with human BCMA protein-FITC (ACRO) for 45 min at 4°C in the dark. The cells were washed twice before analysis by Attune NxT flow cytometer (Thermo Fisher, Waltham, USA).
T-cell activation assay
T-cell activation was evaluated by measuring CD69 expression on tan-CAR T cells in response to 24-h coculture with target cells. Un-transduced (NC) T cells were used as negative controls, and the T cells transduced with CD19-CAR or BCMA-CAR served as positive controls. After co-culture, the cells were harvested and washed twice with 1 mL of PBS containing 2% FBS and then labeled with CD69-PE (Biolegend, San Diego, CA, USA), CD3-FITC (Biolegend), and protein L-FITC (ACRO) for 20 min at room temperature in the dark. CD69 expression in CAR T cells as detected by flow cytometry was used as a marker of CAR T-cell activation.
Quantitation of T-cell proliferation
Cell proliferation assays were performed using a Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) assay kit (Abcam, Cambridge, UK) following the manufacturer's instructions. In brief, the CAR T cells were labeled with 2.5 μM CFSE and then co-cultured with Raji cells which treated with mitomycin before to stop the division, at a stimulator to responder ratio of 2:1 (10 6 CAR T cells/mL) for 5 days in 96-well plates in 200 μL serum-free AIM-V (Gibco) medium per well. Flow cytometry was performed using an Attune NxT flow cytometer (Thermo Fisher) to detect changes in CFSE intensity. FlowJo V10 software (TreeStar, San Carlos, CA, USA) was used for data analysis.
Cytotoxicity assays
Cytotoxicity was determined via quantitation of lactate dehydrogenase activity in the supernatants of effector and target cell co-cultures using the Cytotoxicity Detection Kit (Promega, Madison, WI, USA) following the manufacturer's protocol. All the transduced CAR T cells (effector, E) were co-cultured with cells of the target cells that overexpressed CD19, BCMA, or both antigens (target, T) at E:T ratios of 5:1, 2.5:1, and 1:1, respectively. Target and effector cells were seeded in 96-well plates in a total volume of 100 μL of serum-free RPMI 1640 media (Gibco) and incubated at 37°C for 6 h. After coculture, 50 μL of cell-free supernatant from each well was transferred to a new 96-well plates and mixed with equal volume of lactic acid dehydrogenase substrate mixture for 20 min at room temperature in the dark. The absorbance was recorded at 492 nm using a full wavelength reader Multiskan GO (Thermo Scientific). Tumor (target cell) lysis was calculated with the following formula: % lysis = (experimental LDH release − spontaneous LDH release) / (maximum LDH release − spontaneous LDH release) × 100.
Detection of CD107a
To evaluate CD107a expression on the cell surface as an indirect marker of degranulation, 10 6 CAR T cells were co-cultured with target cells at a 5:1 ratio in 96-well plates with a total of 200 μL of AIM-V (Gibco) medium per well for 6 h. The Golgi inhibitor monensin (Invitrogen, Carlsbad, CA, USA) was added before the incubation. Cocktails (Invitrogen) were added to the positive control group before co-culture. After a 6-h incubation, cells were labeled with anti-CD107a-APC, anti-CD3-FITC, and anti-CD8-PE. All the antibodies were purchased from Becton, Dickinson and Company Co., Ltd. (Franklin Lakes, NJ, USA). Cells were collected, washed twice with PBS, and flow cytometry analysis was performed on an Attune NxT flow cytometer (Thermo Fisher). The results were analyzed by FlowJo V10 software (TreeStar).
Analysis of cytokine release
Cytokine release was evaluated using a Th1/Th2 Cytometric Bead Array (CBA) Kit II (BD Bioscience) according to the manufacturer's instructions. Briefly, CARtransduced T cells were co-cultured with the various K562 cell transfectants at an E:T ratio of 5:1 in a 96-well plate with a total volume of 200 μL of RPMI 1640 medium (Gibco). After 24 h in co-culture, cell-free supernatants were harvested and the levels of various cytokines were evaluated. The capture microspheres for seven specific cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF-a, and IL-17A) were first mixed and then incubated with the sample and fluorescent antibody for 3 h. The mixture was washed and cytokine concentrations were determined by flow cytometry (Thermo Fisher). The concentration of each cytokine was calculated from standard curves.
Mouse xenograft model
Mouse experiments were performed with the approval of the Institutional Animal Care and Use Committee of East China Normal University. Four-to-six-week-old male NOD/scid/γc −/− (NSG) mice were purchased from Biocytogen Co., Ltd. (Beijing, China). Xenograft models were established via injection of K562-CD19-luc and/or K562-BCMA-luc cells. A total of 7 × 10 6 mixed tumor cells at a ratio of 1:1 in 200 μL PBS were injected into mice via the tail vein on day 0. The mice were then randomly divided into four groups that received either (a) 10 7 CD19-CAR T cells (n = 3), (b) 10 7 BCMA-CAR T cells (n = 3), (c) 10 7 tan-CAR T cells (n = 3) or (d) 10 7 un-transduced T cells (n = 3, negative control) via the intravenous route on both days 8 and 10. The mice injected with K562-CD19-luc or K562-BCMA-luc were treated with CD19-CAR T or BCMA-CAR T cells, respectively. Tumor progression was monitored by bioluminescent imaging every 4 days beginning on day 7. The mice were sacrificed when moribund or upon the development of hind-limb paralysis. For in vivo imaging, the mice were injected intraperitoneally with 150 mg/kg D-luciferin (Yeasen, Shanghai, China) and imaged under isoflurane anesthesia using the Xenogen-IVIS system (LI-COR Biosciences, Lincoln, NE, USA). Fluorescence was quantified using Living Image software (IVIS Lumina Series, PerkinElmer, Waltham, MA, USA).
Statistical analysis
Statistical analyses were carried out using GraphPad Prism 8.0. Biological replicates of in vitro (n = 3) and in vivo data (n = 3) are presented as the mean ± SD. Statistical analysis was performed to assess differences between individual treatment groups and the un-transduced control group using one-way ANOVA. Statistically significant findings were defined as *p < 0.05.
Generation and characterization of the tan-CARtransduced T cells
To develop and to evaluate a CAR T-cell strategy that facilitated simultaneous targeting of both CD19 and BCMA, we designed and generated a novel CAR construct with tandem orientation of both CD19-scFv and BCMA-scFv domains. This tandem-CAR T also included the CD28 costimulatory domain and a CD3ζ-mediated activation signaling component. In parallel, we constructed two control CAR Ts with single scFv domains from anti-CD19 (CD19-CAR) and anti-BCMA (BCMA-CAR) with otherwise identical components (Fig. 1a). Transduction efficiency of the lentiviral vectors that contain the tan-CAR, the CD19-CAR, and the BCMA-CAR targeting primary T cells was evaluated. The surface expression of each of these antigens upon T-cell transduction typically yielded 46 to 55% positive cells; this was verified by flow cytometry using L-protein-FITC to detect cell surface expression of variable light chains (Fig. 1b). Similarly, to validate co-expression of both CD19 and BCMA on tan-CAR-transduced T cells, we introduced tan-CAR effector cells to human CD19 protein or human BCMA protein to define specific CAR T cell detection (for details, see Methods). We found that tan-CAR-transduced T cells expressed both scFvs; 59% were BCMA-scFv-positive and 53% were CD19-scFv-positive; these levels are comparable to those of the single scFv CAR T transductants. Similar results were obtained by flow cytometric detection with FITC-conjugated L protein (Fig. 1b). These results suggested that the tandem fusion of two scFv domains could successfully generate CAR T cells with equivalent expression of both receptor antigens. We also examined the impact of the tan-CAR construct on the CD4/CD8 T-cell ratio (Fig. 1c). As shown, tan-CAR T cells generated a comparable CD4 to CD8 transduced T-cell ratio to those that resulted after T-cell transduction with either CD19-CAR or BCMA-CAR constructs. These results suggest that we might anticipate similar anti-tumor efficacy from all three types of CAR T cells.
Tan-CAR-transduced T cells are activated by CD19 and BCMA antigens
CD69 is a standard marker for T-cell activation [20,21]. To demonstrate antigen-specific activation of tan-CARtransduced T cells, cells from the K562 human leukemia line were transfected with lentiviruses encoding CD19 and/or BCMA in order to generate K562-CD19, K562-BCMA, or K562-CD19 + BCMA cells. T-cell activation in response to co-culture with target cells expressed different antigens was analyzed by expression of CD69 on the T-cell surface (Fig. 2a). The tan-CAR T cells responded to both K562-CD19, K562-BCMA, and K562-CD19 + BCMA cells; CD69 expression was detected at levels that were comparable to those observed on activated CD19-CAR and BCMA-CAR T cells. These data indicated that tan-CAR T cells not only express appropriate cell surface antigens but can also be activated by CD19 and BCMA individually or when both are combined. The extent of activation was CAR T-cell-mediated cytotoxicity was evaluated by quantitative assessment of LDH in the supernatants of co-cultured cells as indicated; data are presented as the mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001 vs. un-transduced T cells (NC) from the same donor comparable to that detected among the more conventional single scFv-CAR T cells.
Tumor cell-induce T-cell proliferation in response to CD19 or BCMA
CAR T-cell proliferation upon recognition of the tumor cell antigen is a fundamental principle and crucial factor underlying the augmented anti-tumor efficacy of CAR T cells [22]. Our next step was to determine whether the proliferation of the tan-CAR T cells was dependent on tumor cell-specific expression of CD19 and/or BCMA.
The three different types of CAR T cells were labeled with CFSE and then placed into co-culture with their corresponding target cells; T-cell proliferation was evaluated via detection of decreasing concentrations of the fluorescent dye by flow cytometry. We found that the tan-CAR T cells underwent extensive proliferation in response to activation by target K562 cells overexpressing CD19 and/or BCMA at an effector: target ratio of 1:1. As anticipated, tan-CAR T cells proliferated to an extent that was similar to those of their corresponding single scFv CAR T positive counterparts. In contrast, the NC (control) T cells underwent only limited proliferation after incubation with the target K562 transfected cells (Fig. 2b). Taken together, these results indicated that both single scFv-and tan-CAR T cells exert significant proliferative activities in response to specific antigen stimulation.
Determination of the cytotoxic efficacy and specificity of tan-CAR T cells
To evaluate cytolytic specificity of tan-CAR T cells, we first determined the baseline levels of LDH released from target cells over the time course of the experiment. Effector cells including CD19-CAR T, BCMA-CAR T, tan-CAR T, and untransfected NC T cells were co-cultured with target cells, including K562-CD19, K562-BCMA, K562-CD19 + BCMA and wild-type (un-tranfected) K562 cells as the negative control. After 6 h, significant cytotoxicity was observed in cocultures of tan-CAR T cells with each of the three transfected K562 target cell groups. As anticipated, CD19-CAR and BCMA-CAR T cells were cytotoxic for their corresponding target cells only; the negative control effector T cells (NC-T) had no cytotoxicity for any of the K562 target cells (Fig. 2c).
We also carried out another experiment to compared the cytolytic efficiency of different effector T cells including: a mixture of CD19-CAR T cells and BCMA-CAR T cells with the ratio of 1:1, the T cells transduced with BCMA-CAR followed by CD19-CAR lentivirus, single scFv-CAR T cells and NC T cells. As for target cells, we use the myeloma cell line of 8226, 8226 cells expressing CD19 antigen and a mixture of 8226 and Raji cells at a ratio of 1:1. After 24 h co-culture, as we expected, the tan-CAR showed great cytolytic efficiency towards all kinds of target cells compared with NC T cells (Fig.2d). No significant differences observed among tan CAR T cells, a mixture of two single scFv T cells and T cells transduced with two lentiviruses. Importantly, BCMA-CAR-T showed slightly less cytolytic ability towards the mixture of 8226 cells and Raji cells compared with tan CAR T cells group, indicating BCMA-CAR T cells were cytotoxic for their corresponding target cells only, but not CD19 antigen positive target cells.
Determination of CD107a and cytokines release of Tan-CAR T cells Next, we determined whether cytolytic function was associated with a quantitative increase in the cell surface expression of the lysosomal protein CD107a. After 6-h co-culture with target K562 transfectants, elevated levels of CD107 were detected on tan-CAR T cells compared to levels detected on NC T cells. In parallel, a similar degree of CD107 up-regulation was detected in the CD19-CAR and BCMA-CAR T cells, but only in response to K562 cells expressing their corresponding activating antigen (Fig. 3a).
Finally, proinflammatory cytokine release from CAR T cells was evaluated. We measured cytokine levels in coculture supernatants using the Th1/Th2 CBA Kit II. As anticipated, the tan-CAR T cells released cytokines to an extent that was comparable to that generated by CD19-CAR and BCMA-CAR T cells in response to their respective cognate antigens. Interestingly, NC-T cells also released proinflammatory cytokines, albeit to a more limited extent, when encountering the target K562 cells (Fig.3b). Collectively, these experiments demonstrated that tan-CAR T-cell-mediated cytotoxicity is equivalent to that of conventional CAR T cells.
Tan-CAR-transduced T cells are effective at promoting clearance of tumor cells in vivo
We then performed an in vivo study that was designed to evaluate tumor regression in a mouse xenograft model. In this model, NSG mice were injected with target cells including K562-CD19-luc, K562-BCMA-luc, or the mixture of the two target cells at a 1:1 ratio on day 0. These mice were then treated with tan-CAR T cells, CD19-CAR T cells, BCMA-CAR T cells, or NCs on day 8 and day 10 ( Fig. 4a) and tumor growth was monitored by IVIS imaging; representative images depicting tumor progression or elimination are shown in Fig. 4b. Among the mice that received either K562-CD19-luc or K562-BCMA-luc target cells, those treated with their respective single scFv-CAR T cells experienced a significant decrease in overall tumor burden based on bioluminescence imaging analysis. However, neither of the single scFv-CAR T cells were capable of inhibiting overall tumor growth in mice with the mixed antigen tumor xenograft. By contrast, tan-CAR T cells showed an efficient anti-tumor inhibitory response and were able to promote tumor regression in all three xenograft models. The signal intensity from IVIS imaging as a function of time is shown in Fig. 4c. These results demonstrated that the conventional single scFV-CAR T cells directed against either CD19 or BCMA were capable of targeting tumor cells that express the corresponding antigens but had no impact on other tumor cells. Most notably, the tan-CAR T cells had a remarkable and specific anti-tumor effect in vivo toward tumor cells that were CD19-, BCMA-positive, or both (p < 0.05). Taken together with the results of our in vitro experiments, tan-CAR T cells promote the successful elimination of tumor cells that express either CD19, BCMA, or both; our tan-CAR T cells demonstrate high antigen specificity and in vivo efficacy that is comparable to conventional those of single scFv-CAR T cells.
Discussion
Adoptive transfer of engineered T cells is a promising approach for the treatment of cancer; unfortunately, post-treatment relapse remains a significant challenge, results suggesting that these therapeutic agents might require further optimization [12]. For example, adoptive transfer of anti-BCMA-CAR T cells to patients with refractory or relapsed MM was initially met with a positive therapeutic response; however, relapse can occur very quickly after the completion of CAR T-cell therapy [12,23]. This may be related to the absence of the target antigen from the tumor cell surface that has been observed in response to single scFV-CAR T-cell therapy [12]; Previous data showed CD19 expression on plasma cells with characterized cancer stem cell-like properties is a poor prognostic indicator [24]. Clinical data [15] showed BCMA-CAR combined CD19-CAR T cells resulted in MRD negative completely remission leading us to develop tandem CAR-T targeting both CD19 and BCMA, reducing the cost of manufacturing two kind of CAR-T products, while achieving maintaining the longterm remission of patients.
The linkage of two scFvs into a single tandem-CAR in order to generate a bi-specific chimeric receptor has already been proposed in theory [25], although, to the best of our knowledge, this is the first evidence for its successful execution. The tan-CAR enables T cells to recognize two distinct target antigens and to initiate specific killing of the tumor cells that express one or both of the cognate antigens. Perhaps the most critical component of the tan-CAR design is the need to ascertain cytotoxic efficacy that is at least comparable to that of the conventional single scFv-CAR.
To limit the potential for relapse after completion of therapy with BCMA-CAR T cells, we aligned the scFv of anti-CD19 with that of anti-BCMA in a single targeting domain to generate tandem-CAR (tan-CAR). This tan-CAR construct was used successfully to transduce primary human T cells (tan-CAR T cells). In this study, we carefully evaluated the specificity and efficacy of the tan-CAR T in vitro and with respect to a xenograft disease model. Accordingly, this dual scFv CAR construct was activated by either BCMA or CD19 antigens. Moreover, tan-CAR T cells were highly efficacious against antigenspecific tumor cells in both in vitro and in vivo experiments with responses that were comparable to the single scFv-CAR T cells directed against BCMA or CD19. The intrinsic utility of tan-CAR T-cell therapy was also investigated using an immunodeficient mouse model bearing K562-CD19-luc and K562-BCMA-luc target tumor cells. We determined that tan-CAR T-cell specificity and efficacy was fully comparable to those of its single scFv-CAR T counterparts. Notably, tan-CAR enabled T cells to recognize one or both relevant antigens and significantly limited tumor progression in combined target tumor model in mice; by contrast, the tumor burden in In summary, we present here the first report of an extensive pre-clinical characterization of novel dual tandem CD19/BCMA-scFv-CAR T cells which were developed for the treatment of refractory or relapsing MM. Our study demonstrated that the tan-CAR which included two specific scFvs from anti-CD19 and anti-BCMA promoted cytolysis of target tumor cells via activation via both CD19 and BCMA. Given the known obstacles to effective CAR T-cell therapy, including absence or loss of BCMA expression, the capacity to target two critical antigens with a single cell infusion could be a promising approach toward addressing problems of refractory disease and disease recurrence. This novel approach might also serve to reduce production costs that would be encountered in providing sequential CAR Tcell treatment for myeloma.
Conclusion
We report here the design, generation, and evaluation of tan-CAR T cells that can recognize both CD19 and BCMA B-cell antigens, and that can exert significant (10 7 ) were provided by intravenous injection on days 8 and 10. (b) Bioluminescence radiance was used as a surrogate marker for tumor burden. (c) Time course of tumor growth based on mouse wholebody bioluminescence. The mean signal per mouse ± SD is as shown. Statistical analysis was performed using day 19 data (the last time point at which a sufficient number of mice that did not receive treatments remained viable) using one-way ANOVA followed by Tukey's multiple comparisons test. The data are presented as the mean ± SD (n = 3); ***p < 0.001 in vitro and in vivo cytotoxic effects against tumor cells that express one or both of these antigens. This novel and highly effective tan-CAR construct may ultimately be the basis for a novel and effective option for treatment of refractory and recurrent MM, notably among those who have relapsed after effective BCMA-CAR Tcell treatment. | v3-fos-license |
2018-04-03T00:11:43.775Z | 2018-01-16T00:00:00.000 | 3391501 | {
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} | pes2o/s2orc | Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo
The retinal pigment epithelium (RPE) supports the health and function of retinal photoreceptors and is essential for normal vision. RPE cells are post-mitotic, terminally differentiated, and polarized epithelial cells. In pathological conditions, however, they lose their epithelial integrity, become dysfunctional, even dedifferentiate, and ultimately die. The integrity of epithelial cells is maintained, in part, by adherens junctions, which are composed of cadherin homodimers and p120-, β-, and α-catenins linking to actin filaments. While E-cadherin is the major cadherin for forming the epithelial phenotype in most epithelial cell types, it has been reported that cadherin expression in RPE cells is different from other epithelial cells based on results with cultured RPE cells. In this study, we revisited the expression of cadherins in the RPE to clarify their relative contribution by measuring the absolute quantity of cDNAs produced from mRNAs of three classical cadherins (E-, N-, and P-cadherins) in the RPE in vivo. We found that P-cadherin (CDH3) is highly dominant in both mouse and human RPE in situ. The degree of dominance of P-cadherin is surprisingly large, with mouse Cdh3 and human CDH3 accounting for 82–85% and 92–93% of the total of the three cadherin mRNAs, respectively. We confirmed the expression of P-cadherin protein at the cell-cell border of mouse RPE in situ by immunofluorescence. Furthermore, we found that oxidative stress induces dissociation of P-cadherin and β-catenin from the cell membrane and subsequent translocation of β-catenin into the nucleus, resulting in activation of the canonical Wnt/β-catenin pathway. This is the first report of absolute comparison of the expression of three cadherins in the RPE, and the results suggest that the physiological role of P-cadherin in the RPE needs to be reevaluated.
Introduction
The retinal pigment epithelium (RPE), located between retinal photoreceptor cells and the choroid of the eye, is a single layer of pigmented epithelial cells with cobblestone-like morphology [1]. The RPE is essential for normal vision through multiple activities that support the health and function of retinal photoreceptors. The RPE constantly faces oxidative stress due to its large oxygen consumption and daily phagocytosis of photoreceptor outer segments, leading to accumulation of oxidative damage with age, which is thought to contribute to the loss of epithelial integrity and the development of diseases such as age-related macular degeneration (AMD) [1][2][3].
RPE cells are known to dedifferentiate and lose their fully matured state as a result of a variety of stresses, including oxidative stress and mechanical dissociation of cell-cell junctions [4][5][6][7][8][9][10]. Dissociation of cultured RPE cells leads to dedifferentiation of the cells into fibroblast-like cells through epithelial to mesenchymal transition (EMT) [5,9]. EMT is a process in which cells lose cell-cell junctions and epithelial morphology and become fibroblast-like with increased mesenchymal markers [11][12][13]. RPE cells undergoing EMT contribute to scarring and wound contractions in proliferative vitreoretinopathy (PVR) as well as subretinal fibrosis in advanced AMD [14][15][16].
To maintain the integrity of epithelial cells, adherens junctions are critical by forming cellcell contacts as protein complexes consisting of cadherin homodimers and p120-, β-, and αcatenins that link to actin filaments (F-actin) [17][18][19]. Cadherins are Ca 2+ -dependent cell adhesion molecules that connect neighboring cells through homophilic interaction of two homodimers on the cell surface [18,[20][21][22]. In most epithelial cell types, E-cadherin is the major cadherin responsible for forming and maintaining their epithelial phenotype [18,20,23]. However, it has been reported that RPE cells are different from other epithelial cells in terms of the major cadherin subtype that they express [24,25].
Results of the expression of cadherin subtypes in the RPE have been conflicting. In cultured human RPE cells, N-cadherin rather than E-cadherin was dominantly expressed [25][26][27]. In the center of cultured porcine RPE sheets, where intact RPE cells were located, P-cadherin was abundantly detected, but it was lost at the edge of RPE sheets, where cells were migrating away and showed fibroblastic morphology with the expression of N-cadherin and vimentin [9]. In situ hybridization with mouse embryos showed that the outer layer (RPE) of the optic cup expressed N-cadherin until embryonic day 10.5 (E10.5) but switched to P-cadherin from E12 onward [28]. This study also showed that E-cadherin expression was not detectable in the RPE throughout embryonic and postnatal stages, indicating that each cadherin displays unique spatial and temporal expression patterns [28]. However, drawing general conclusions from these studies is challenging because they differ with regards to species (human, pig, and mouse), RPE source (cultured RPE cells, cultured RPE sheet, and in situ RPE), temporal stage (embryonic, postnatal, and adult), and methodology (Western blot, immunofluorescence, and in situ hybridization). In addition, these methods are not suitable for comparison of the expression levels across different cadherins.
The above-described findings suggest that a dominant cadherin subtype may be different between cultured RPE cells and mature RPE in vivo, and that P-cadherin may be the major cadherin in the latter that is more relevant to RPE physiology, but not in the former. However, this issue has not been clearly addressed so far. Importantly, mutations of human CDH3 (the gene encoding P-cadherin) cause two rare autosomal recessive disorders: Hypotrichosis with Juvenile Macular Dystrophy (HJMD) [29][30][31][32] and Ectodermal Dysplasia, Ectrodactyly, and Macular Dystrophy (EEM syndrome) [33]. HJMD is characterized by early-onset hair loss with severe macular dystrophy, particularly at the RPE. Of note, P-cadherin-deficient mice are viable and fertile with no overt developmental abnormalities including ocular phenotype [34]. Besides the in situ hybridization analyses described above [28], to the best of our knowledge there is only one paper that analyzed P-cadherin in mouse RPE. Using immunofluorescence of P-cadherin, the authors reported that albino RPE cells were irregularly shaped with more loose and wider distribution of adherens junctions at the cell membrane than pigmented RPE cells, suggesting that the lack of melanogenesis may result in impaired RPE cell integrity in albino mice [35].
To address the unsolved issues of the role of cadherin subtypes in the RPE, in this study we revisited the expression of classical cadherins in RPE cells and determined their relative contribution by measuring the absolute cDNA quantity produced from mRNAs of E-, N-, and Pcadherins in human and mouse RPE in situ. Our results show that P-cadherin (CDH3) is the dominant cadherin in mature RPE cells. Using immunofluorescence, we confirmed that Pcadherin protein is localized at the cell-cell border of mouse RPE in situ. In addition, we also show that oxidative stress disrupts the localization of P-cadherin at cell-cell junctions at least temporarily, even when RPE cells look grossly intact, and that this oxidative stress-induced dissociation of adherens junctions leads to the translocation of β-catenin into the nucleus, a sign of activation of the canonical Wnt/β-catenin signaling pathway. This is the first report of absolute comparison of the expression levels of three classical cadherins in the RPE. Based on these results, we suggest that the physiological role of P-cadherin in the RPE needs to be reevaluated.
Mice and sodium iodate (NaIO 3 ) injection
All mice were treated in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Johns Hopkins University Animal Care and Use Committee (Protocol Number: MO15M230). No procedures were performed on live mice, except tail vein injection. Euthanasia was performed with CO 2 from a compressed gas cylinder followed by cervical dislocation, which is consistent with the American Veterinary Medical Association's Guidelines for Euthanasia of Animals. Mice used in this study were 2-10 weeks old male C57BL/6J mice purchased (Jackson Laboratory, Bar Harbor, ME). To induce oxidative stress in vivo, mice (8-10 weeks old) were injected via tail veins with sodium iodate (NaIO 3 ; S4077, MilliporeSigma, St. Louis, MO) in phosphate-buffered saline (PBS) at the dose of 15 mg/kg body weight. Since NaIO 3 is known to cause oxidative damage exclusively in the RPE, with no obvious systemic toxicity or histological damage in other tissues, it does not produce noticeable suffering or distress to mice.
RNA preparation from human RPE
Total RNA of human RPE and retina was previously extracted directly from human donor eyes [36,37]. Total RNAs of human RPE primary cells (named M1 as described below) and various human tissues were previously prepared and purchased, respectively for our prior studies [36,38].
RPE primary cells were cultured following the published protocol [39]. Human donor eyes obtained from Eye Banks were dissected equatorially, and the cornea, lens, and retina were removed gently. The RPE cell layer was incubated with 2.4% dispase (Calbiochem, Millipore-Sigma, Burlington, MA, USA) in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Thermo Fisher Scientific, Grand Island, NY) containing 100 mM sorbitol (MilliporeSigma) at 37˚C for 45 min. The dispase solution was replaced with DMEM with 20% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific), and RPE cells were collected by gentle scraping. The RPE primary cells were maintained in 75 cm 2 flasks coated with laminin (Upstate, Millipore-Sigma) in DMED with 20% FBS and 50 μg/ml of Endothelial Cell Growth Supplement (ECGS; Upstate, MilliporeSigma). The RPE primary cultures became confluent in approximately 3 weeks, and the cells were split and subcultured in DMED with 10% FBS and 10 ng/ml basic FGF (bFGF; Upstate, MilliporeSigma). The medium was changed every 2 days. Among several RPE primary cell preparations, the one named M1 showed the best cobblestone-like morphology. For gene expression analyses, therefore, the third passaged M1 RPE cells were subcultured in DMED with 10% FBS and 10 ng/ml bFGF, and RNA was extracted using Trizol reagent (15596, Invitrogen, Thermo Fisher Scientific) 2 weeks after the cells reached tightly packed confluence (a total of 4 weeks in culture). At the time of RNA extraction, the M1 RPE cells exhibited a uniform cobble-stone-like appearance. We had analyzed the expression of RPE markers, MITF, OTX2, and RPE65, in these M1 cells in our prior studies [36]. The mRNA levels of MITF and OTX2 in M1 RPE cells were similar to those in human RPE in situ; however, the mRNA level of RPE65 was substantially lower (barely detected) in M1 RPE cells compared with that in human RPE in situ [36].
RNA preparation from mouse RPE and choroid individually
To analyze gene expression in mouse RPE and choroid individually, we modified the RNA extraction method reported for only mouse RPE [40]. Briefly, mouse eyes were dissected to remove the cornea and lens, and the retina was peeled off to obtain the eyecup containing the RPE, choroid, and sclera. RPE cells were released by incubating the eyecup in 200 μl of RNAprotect cell reagent (76526, Qiagen, Valencia, CA) in a microcentrifuge tube at room temperature for 10 min followed by gentle tapping of the tube. Then, the choroid/sclera eyecup was transferred to a new tube containing 500 μl of Trizol, the released RPE cells were collected by centrifugation at 650 x g for 5 min at room temperature, and 500 μl of Trizol were added to the RPE cell pellets. The choroid/sclera eyecup and the RPE cell pellets were homogenized separately using a pestle grinder, and RNA was purified from each tissue by the two-step extraction strategy, i.e., first extracting RNA into the aqueous phase with Trizol and then purifying RNA from the aqueous phase with RNeasy Micro Kit (74004, Qiagen) following manufacturers' instructions. To check cross-contamination, the mRNA levels of RPE markers (Sox9, Otx2, and Rpe65) and choroid markers (Vwf and Col6a1) were analyzed in RPE and choroid RNA samples by reverse transcription-quantitative PCR (RT-qPCR) using gene-specific primers (S1 Table).
RT-qPCR
The mRNA levels of three mouse cadherin genes, Cdh1 (the gene encoding E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin), in the RPE and choroid were analyzed by RT-qPCR. Total RNA from mouse RPE and choroid was prepared using the method described above. RT-qPCR was performed as previously described [41] with minor modifications. First-strand cDNA was synthesized from 200 ng of total RNA with random primers using SuperScript III reverse transcriptase (18080044, Invitrogen), and real-time PCR was performed with primers (S1 Table) and SYBR Green master mix using C1000 Thermal Cycler (Bio-Rad, Hercules, CA). Relative gene expression was calculated using the 2 −ΔΔCt method with Gapdh, Hprt, and Actb as reference genes. Each sample was analyzed in triplicate. All gene-specific primers were designed to amplify DNA fragments that encompass the junction of two neighboring exons.
Measurement of absolute cDNA quantity
The absolute quantity of cDNA produced from Cdh1, Cdh2, and Cdh3 mRNAs in mouse RPE was measured as previously described for other genes [36,42] with modifications. To obtain standard curves for quantification, cDNA fragments of Cdh1, Cdh2, and Cdh3 were generated from mouse RPE and choroid RNA by RT-PCR using primers that amplify the region including the segment to be quantified by RT-qPCR (S1 Table). Primer pairs for cDNA quantification were located at each side of exon-exon borders in respective cDNAs, and they were designed in the non-homologous region to avoid cross-reacting with other cadherin cDNAs. The PCR products were fractionated in agarose gels, purified using QIAquick Gel Extraction Kit (28704, Qiagen), and their DNA concentration was measured using NanoDrop Spectrophotometer (Thermo Fisher Scientific). To obtain the molecular complexity similar to the samples, mouse spleen cDNA was chosen to dilute the gel-purified DNA fragments for standard curves because the expression of these three cadherins in the spleen was barely detectable and the lowest among a variety of tissues tested. The purified DNA fragments were diluted into mouse spleen cDNA in a wide range of quantities and tested to determine the best range to generate standard curves that covered threshold cycle (Ct) values of the samples to be analyzed. As a result, the range of DNA fragments from 1 attomole (amole) to 0.1 zeptomole (zmole) was selected for standard curves. Sample cDNAs of mouse RPE were synthesized using 200 ng of total RNA in 20 μl reaction solution, along with mouse spleen cDNA, and all cDNAs were diluted by 20-fold for analyses in triplicate. The final cDNA quantity was calculated for 200 ng of total RNA for each sample by taking into account the process in which 1.5 μl of the 20-fold diluted cDNAs were used for real-time PCR reactions.
The absolute quantity of cDNA generated from CDH1, CDH2, and CDH3 mRNAs in two types of human RPE (RPE in situ and M1 RPE primary cells) was measured in the same manner as described above for mouse RPE with minor modifications. To make standard curves, cDNA fragments of CDH1, CDH2, and CDH3 were amplified from total RNA of small intestine, heart, and M1 cells, respectively by RT-PCR using primers that amplify the region containing the segment to be quantified by RT-qPCR (S1 Table). Primer pairs for cDNA quantification were made with the same design as described above for mouse genes. To dilute the gel-purified DNA fragments for standard curves, human retina, thymus, and retina cDNAs were used for CDH1, CDH2, and CDH3, respectively because these tissues barely express respective genes. The range of DNA fragments from 1 amole to 0.1 zmole was used for standard curves. Sample cDNAs were synthesized from 200 ng of total RNA. Considering the dilution and the volume of cDNA used in real-time PCR, the final cDNA quantity was calculated for 200 ng of total RNA.
Immunofluorescence
For RPE flat-mounts, mouse eyes were dissected at the equator, the cornea and lens were removed, and the retina was carefully peeled off. The remaining eyecups containing the RPE and choroid were immediately fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer for 10 min at room temperature and transferred into PBS. The eyecups were dissected into quarters by four radial cuts from the periphery toward the optic disc, and blocked in 0.25% Triton X-100 in Tris-buffered saline (TBST) with 10% normal horse serum (Z0610, Vector Laboratories, Burlingame, CA) and 1% bovine serum albumin (BSA; A9647, MilliporeSigma) at room temperature for 1 h. The RPE/choroid flat-mounts were incubated with a primary antibody in TBST containing 3% normal horse serum and 1% BSA at 4˚C overnight with gentle shaking. After washing with TBS for 10 min three times at room temperature, the flatmounts were incubated with appropriate secondary antibodies in TBST containing 1% BSA for 30 min at room temperature followed by washing with TBS for 10 min three times. The nuclei were stained with 4',6-diamidino-2'-phenylindole dihydrochloride (DAPI, 10236276001, Roche, Indianapolis, IN) for 10 min at room temperature. The flat-mounts were washed with TBS, mounted in Fluorescent Mounting Medium (S3023, Dako, Carpinteria, CA), and images were acquired using an LSM 510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). Primary antibodies used were anti-ZO-1 (dilution at 1:200; 402200, rabbit polyclonal, Invitrogen), anti-P-cadherin (1:200; AF761, goat polyclonal, R&D Systems, Minneapolis, MN), and anti-β-catenin (1:200; NBP1-32239, rabbit polyclonal, Novus, Littleton, CO). Secondary antibodies were anti-rabbit or anti-goat IgG conjugated with Alexa Fluor 488, 549, or 647 (1:500; Invitrogen). For staining F-actin (filamentous actin), the flat-mounts were incubated with CytoPainter Phalloidin-Fluor 555 Reagent (1:1000; ab176756, Abcam, Cambridge, MA) in PBS with 1% BSA for 30 min at room temperature.
To analyze the localization of P-cadherin and β-catenin, immunofluorescence of retinal sections was performed following the previously published protocols [43]. Briefly, mouse eyes were fixed in ice-cold 4% PFA in 0.1 M phosphate buffer pH 7.4 for 1 h, washed in 0.3% Triton X-100 in PBS (0.3% PBST), cryoprotected in an increasing gradient of sucrose (10%, 20%, and 30%) in PBS at room temperature for 1 h at each concentration, embedded in Tissue-Tek OCT (Ted Pella, Redding, CA), and snap frozen on dry ice in isopentane. Eye sections were cut at 10 μm on a cryostat, and incubated with the same primary and secondary antibodies as used for RPE flat-mounts, and examined in the same manner using an LSM 510 confocal microscope (Carl Zeiss) as described above.
Western blot analysis
Protein lysates of mouse RPE were prepared according to the previously described method [44]. Mouse eyes were dissected in the same manner as described above for RPE flat-mounts. The eyecups were cut into 4 small petals and incubated in RIPA lysis buffer with protease inhibitor cocktail EDTA-free (R0278, MilliporeSigma) for 40 min on ice. After the insoluble fractions were removed by centrifugation at 14,000 rpm for 15 min at 4˚C, the supernatants were collected, and protein concentration was determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific). The same amounts of proteins (25 μg) for each sample were subjected to 4-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were incubated for 1 h at room temperature with a primary antibody in TBS containing 0.05% Tween 20 (pH 7.4) in the presence of 5% nonfat dry milk. After washing the membranes in TBS containing 0.05% Tween 20, secondary antibody reactions were performed with an appropriate source of antibody conjugated with horseradish peroxidase. The signals were detected with an enhanced chemiluminescence (ECL) detection kit (RPN2232, GE Healthcare Life Science, Marlborough, MA) using an ImageQuant LAS 4000 scanner (GE Healthcare Life Science). The intensity of each band was quantified using the ImageJ software (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD). Primary antibodies used were anti-P-cadherin (1:2000; AF761, R&D systems), anti-β-catenin (1:2000; NBP1-32239, Novus), anti-Snail (1:1000; 3895, mouse monoclonal, Cell Signaling, Danvers, MA), and anti-vimentin (1:3000; 5741, rabbit monoclonal, Cell Signaling). For internal control, anti-β-actin antibody (1:100000; A3854, mouse monoclonal, MilliporeSigma) was used.
Statistical analysis
Unpaired Student's t-test was used for statistical analysis. P value less than 0.05 was considered as statistically significant difference (P < 0.05).
RNA extraction from mouse RPE and choroid individually
First, we wanted to establish a method for obtaining RNA separately from mouse RPE and choroid. In most previous studies analyzing mouse RPE gene expression, RNA was extracted from the mixed RPE/choroid due to technical difficulties. To resolve this issue, Wang et al. reported an RNA extraction method for mouse RPE without the choroid [40]. We modified Wang's method to individually purify RNA from both mouse RPE and choroid. RPE cells were released from the RPE/choroid/sclera eyecup in RNAprotect reagent, and the remaining choroid/sclera was transferred to a new tube, which allowed RNA extraction from RPE cell pellets and the choroid/sclera in separate tubes (see the details in the Materials and methods section). Trizol enabled complete lysis of the choroid/sclera, which could not be achieved by the RNeasy lysis buffer, and RNeasy columns enabled to remove melanin pigments that inhibit downstream enzyme reactions.
To test cross-contamination of RNA from mouse RPE and choroid/sclera, we analyzed the expression of RPE markers (Sox9, Otx2, and Rpe65) and choroid markers (Vwf and Col6a1) using RT-qPCR. The mRNA levels of Sox9, Otx2, and Rpe65 in the choroid samples were 27%, 8%, and 4% of those in the RPE samples, respectively (Fig 1A). The result of Sox9 seemed to reflect its expression in choroidal melanocytes. The mRNA levels of Vwf and Col6a1 in the RPE samples were 19% and 2% of those in the choroid samples, respectively (Fig 1B). Vwf encodes von Willebrand factor that is expressed in platelets and vascular endothelial cells [45], which are abundantly present in the choroid, particularly beneath the Bruch's membrane, leading to higher contamination in the RPE samples. Col6a1 encodes collagen type VI alpha 1 chain, an extracellular matrix protein expressed in fibroblasts and adipocytes [46], showing no significant contamination in the RPE samples. The results showed that RPE and choroid/sclera RNAs were slightly contaminated with each other, but not to a substantial degree, suggesting that our method is useful for analyzing gene expression in mouse RPE and choroid individually.
E-, N-, and P-cadherins show distinct preferential expression patterns in the RPE and choroid
Using our RNA extraction method described above, we prepared total RNA from mouse RPE and choroid separately in 3 biological replicates and analyzed the mRNA expression of the three cadherins by RT-qPCR. The mRNA level of Cdh1 was significantly higher in the choroid than in the RPE by 3.4-fold (P = 0.0086) (Fig 1C). In contrast, the mRNA level of Cdh3 was significantly higher in the RPE than in the choroid by 2.9-fold (P = 0.048). The mRNA level of Cdh2 also showed a trend to be higher in the RPE than in the choroid, but the difference was not significant (P = 0.086). These results are suggestive of P-cadherin as a major cadherin in mouse RPE in situ. However, since the specificity and efficiency of gene-specific primers are different for each gene, we wanted to more quantitatively compare the expression levels across different cadherin mRNAs.
P-cadherin is the dominant cadherin in normal mouse RPE in situ
We quantified the absolute amount of cDNA generated from mRNA of the three classical cadherins. Total RNA was prepared from 2 week-old and 2 month-old mouse RPE using our newly established RNA extraction method described above. Based on Ct values of the standard curves, the amount of cDNA produced from 200 ng total RNA was calculated as 57.4, 40.4, and 542 zmole at 2 week-old and 59.7, 22.4, and 365 zmole at 2 month-old for Cdh1, Cdh2, and Cdh3, respectively (Fig 2A). As control for the quantity and quality of RNA, we confirmed that the two RPE samples had the equivalent level of Gapdh expression. Assuming that the efficiency of RT reaction and PCR is the same for all samples and genes, we compared each cadherin mRNA expression directly based on the calculated cDNA quantity. These results show that Cdh3 is the dominant cadherin expressed in mouse RPE in situ at the mRNA level, with Cdh3 accounting for 82~85% of the total of the three cadherin expression.
P-cadherin is the highly dominant cadherin in human RPE in situ, but not in cultured RPE cells
Next, we tested whether P-cadherin was also dominant in RPE cells of different species. Since RNA samples of human RPE, two directly extracted from donor eyes (RPE-1 and RPE-2) and one from RPE primary cells (M1) with cobble-stone-like morphology, were already available The same RNA samples were tested for cross-contamination using choroid markers (Vwf and Col6a1) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for Cdh1 (gene for E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by à (p < 0.05) and Ãà (p < 0.01).
https://doi.org/10.1371/journal.pone.0191279.g001 [36,37], we analyzed these samples using the same quantification procedures as used for mouse RPE. Based on Ct values of the standard curves, the amount of cDNA produced from 200 ng total RNA was calculated as 55.7, 18.2, and 886 zmole in RPE-1, 57.9, 20.7, and 1030 zmole in RPE-2, and 57.0, 81.5, and 161 zmole in M1 for CDH1, CDH2, and CDH3, respectively ( Fig 2B). As control for the quantity and quality of RNA, we confirmed that these RPE samples had the similar level of GAPDH and HPRT expression. As in the case of mouse RPE, these results clearly show that CDH3 is the vastly dominant cadherin expressed in human RPE in situ at the mRNA level, with CDH3 accounting for 92~93% of the total of the three cadherin expression. In contrast, primary RPE cells, even though they had the typical cobblestone-like morphology of well-differentiated RPE, did not show such a dominance of CDH3 expression, and instead expressed CDH2 at the higher level than RPE in situ (P = 0.017). These results confirm the dominance of P-cadherin expression as general characteristics of RPE cells in vivo and suggest the need for caution in interpreting results from cultured RPE cells.
P-cadherin is co-localized with ZO-1, β-catenin, and F-actin at the cell-cell border of mouse RPE in vivo
To assess P-cadherin expression at the protein level, we used immunofluorescence of mouse RPE flat-mounts. We performed double staining for P-cadherin and either of the proteins that are localized at the cell-cell border, ZO-1 (tight junction protein 1, TJP1), β-catenin (a component of adherens junctions), or F-actin (associated with adherens junctions). We obtained a strong and clear signal of P-cadherin at the cell-cell border of mouse RPE in situ, and confirmed that P-cadherin was co-localized with all of the three proteins analyzed (Fig 3). This
Fig 2. P-cadherin is the dominant cadherin in mouse and human RPE in situ.
(A) Absolute quantification of cDNA to assess the mRNA quantity of Cdh1, Cdh2, and Cdh3 in mouse RPE in situ. Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of CDH1, CDH2, and CDH3 in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar). anti-P-cadherin antibody is described as being highly specific to mouse P-cadherin, with less than 0.3% cross-reactivity with recombinant mouse E-cadherin by sandwich immunoassays (AF761, R&D Systems), and has been used to study mouse epidermis and hair follicles [47,48]. We also performed immunofluorescence for both E-and N-cadherins on mouse RPE flatmounts with at least two different antibodies that worked for culture cells, but could not obtain a clear signal. These results confirm that P-cadherin protein is indeed expressed at the cell-cell border, and co-localized with other proteins that are known to compose cell junctions.
Oxidative stress disrupts P-cadherin localization at the cell border
Based on the reported findings that oxidative stress disrupts cell junctions in cultured RPE cells [4,5,7,49], we first tested the effect of oxidative stress on the localization of P-cadherin in mouse RPE in vivo. Sodium iodate (NaIO 3 ), an oxidative stress inducer, was injected into mice via tail vein on Day 0, and immunofluorescence of mouse RPE flat-mounts was performed on Days 1, 2, 3, and 5. After oxidative stress, P-cadherin was no longer detected exclusively at the cell membrane, but instead its diffuse labeling was detected in the cytoplasm, suggesting that oxidative stress may cause P-cadherin dissociation from the adherens junctions. This diffuse cytoplasmic staining was observed most prominently on Days 2 and 3, followed by gradual decrease (S1 Fig). In contrast, the localization of ZO-1 at the cell-cell border was less disturbed by oxidative stress in the experimental conditions used. These results indicate that NaIO 3 -induced oxidative stress, even at a low dose, causes significant changes inside RPE cells at the molecular and structural levels, although RPE morphology looks grossly normal.
Oxidative stress-induced dislocation of adherens junction proteins results in translocation of β-catenin to the nucleus
After observing that oxidative stress resulted in a dramatic change in the distribution of P-cadherin in mouse RPE, we next investigated the localization of β-catenin, another component of adherens junctions as well as a key factor of the Wnt/β-catenin signaling pathway. NaIO 3 was injected into mice in the same manner on Day 0, and double staining of RPE flat-mounts for P-cadherin and β-catenin was performed on Days 1, 3, and 7 ( Fig 4A). Adherens junctions marked by P-cadherin and β-catenin became wider and more diffuse at the cell-cell border, and a portion of these proteins was also detected in the cytoplasm on Day 1 (Fig 4Ad-4Af). The most notable change was observed on Day 3 in that staining of both P-cadherin and βcatenin at the cell membrane was significantly weaker but instead became prominent around or on/in the nucleus (Fig 4Ag-4Ai). By Day 7, however, the localization of these adherens junction proteins mostly returned to the normal state on the cell membrane (Fig 4Aj-4Al). These results raised a critical question where P-cadherin and β-catenin were located on Day 3, outside or inside the nucleus. To answer this question, we performed immunofluorescence of mouse retinal sections on Day 3 after NaIO 3 injection (Fig 4B). The nuclei stained by DAPI were mostly devoid of β-catenin and completely free of P-cadherin on Day 0 (Fig 4Bm-4Bo). In contrast, β-catenin was strongly and clearly detected inside the nuclei on Day 3, with P-cadherin staying negative in the nuclei, indicating that β-catenin was translocated to the nucleus (Fig 4Bp-4Bu). These results suggested activation of the canonical Wnt/β-catenin signaling pathway.
To obtain further evidence for the activation of the Wnt/β-catenin pathway, we analyzed the protein level using Western blotting with mouse RPE protein lysates on Days 1, 3, and 7 following NaIO 3 injection. Indeed, β-catenin was increased by approximately 2-fold on Day 1, suggesting that β-catenin protein was stabilized (Fig 4C). We confirmed this result by repeating the same experiment using Western blotting. These results support that oxidative stress indeed leads to activation of the canonical Wnt/β-catenin pathway through the dissociation of adherens junctions and a subsequent release and stabilization of β-catenin.
Oxidative stress induces EMT-related factors in mouse RPE
The next question was how mouse RPE cells were affected as the consequence of this activation of the Wnt/β-catenin pathway induced by oxidative stress. Based on the literatures reporting that activation of β-catenin can promote EMT in other cell types [12,50,51], we hypothesized that mouse RPE might also acquire EMT-like features by the activated Wnt/β-catenin pathway following dissociation of adherens junctions. Therefore, we analyzed three proteins possibly related to EMT by Western blotting (Fig 4C). The protein level of SNAI1, one of the wellknown EMT transcription factors, increased on Day 1 in a similar manner to that of β-catenin, whereas the level of vimentin, a mesenchymal marker, gradually increased with time. Conversely, the level of P-cadherin, an epithelial marker, gradually decreased with passing days, suggesting that cell-cell contacts by adherens junctions may be weakening, which is consistent with EMT-like change.
Discussion
RPE cells can dedifferentiate and lose their fully matured state through an EMT-like process in response to various stressors, including oxidative stress and Ca 2+ removal, that result in the loss of cell-cell contact [4,5,[7][8][9]. Recently, RPE EMT has drawn increasing interests, not only related to PVR [14,16,52] but also due to its potential relevance to the pathophysiology of dry AMD [10]. To trigger EMT, the loss of cell-cell contact seems critical as an initial event, as suggested by the findings that TGFβ, a known EMT inducer, could not initiate EMT in the central region of cultured porcine RPE sheets where cell-cell junctions were well maintained [9]. Adherens junctions regulate cell-cell contacts with neighboring cells and thereby maintain epithelial cell morphology, structure, and polarity [18][19][20][21][22][23]. Cadherins are key components of adherens junctions, but the cadherin subtype(s) playing a major role in forming and maintaining adherens junctions in RPE cells has been ambiguous. This is because most previous studies Mice were injected with NaIO 3 (15 mg/kg body weight) on Day 0, and the localization of β-catenin (green) and P-cadherin (red) was analyzed along with nuclear stain by DAPI (blue) on Days 0 (a-c), 1 (d-f), 3 (g-i) and 7 (j-l). Double staining: β-catenin (a, d, g, j), Pcadherin (b, e, h, k), and merged images with DAPI (c, f, i, l). The localization of β-catenin and P-cadherin at the cell-cell border was significantly disrupted, and instead prominently detected on/in the nucleus on Day 3. (B) Immunofluorescence of mouse retinal sections with a focus on the RPE nuclei. Mice were injected with NaIO 3 (15 mg/kg body weight) on Day 0, and the localization of β-catenin (green) and P-cadherin (red) was analyzed along with nuclear stain by DAPI (blue) on Days 0 (m-o) and 3 (two representative nuclei; p-r and s-u). Double staining: β-catenin (m, p, s), P-cadherin (n, q, t), and merged images with DAPI (o, r, u). On Day 3, β-catenin was detected in the nuclei of mouse RPE. (C) Western blot analyses of mouse RPE proteins. Mice were injected with NaIO 3 (15 mg/kg body weight) on Day 0, and RPE protein lysates were prepared on Days 0, 1, 3, and 7. The protein levels were analyzed using Western blotting with antibodies against P-cadherin, βcatenin, SNAI1 (Snail), vimentin, and control β-actin. The protein levels of β-catenin and SNAI1 increased similarly on Day 1 following oxidative stress.
have analyzed cultured RPE cells, either ARPE19 cell line or RPE primary cells, with a limited number of in vivo studies, and because the methods used were not suitable for quantitative comparison [4,5,7,8,25,26,28,49,[53][54][55]. In addition, most of these studies have focused on E-and/or N-cadherin, but not P-cadherin. Therefore, we wanted to clarify relative contribution of specific cadherin subtypes to formation of adherens junctions in RPE cells in vivo.
Our results show that P-cadherin mRNA is dominantly expressed in both human and mouse RPE in situ compared with E-and N-cadherins. We designed all cadherin primers for PCR based on the known RefSeq mRNA sequence for a preproprotein that undergoes proteolytic processing to generate a mature protein (National Center for Biotechnology Information, NCBI). These primers produce PCR fragments encompassing an exon-exon border in respective cDNAs in the region that is not homologous across the three cadherins, and are also included in all splice variants currently listed in the GenBank (NCBI). Using RNA from various tissues with different cadherin expression patterns, we confirmed that our primers do not cross-react with other cadherin cDNAs, supporting the validity of our findings.
The degree of dominance of P-cadherin expression in mature RPE in vivo was surprising; however, there are reports, although a limited number, that described P-cadherin expression in RPE cells of various mammalian species. Transcriptome analyses of native human fetal and adult RPE identified both CDH1 and CDH3 among 154 RPE signature genes [56]. However, since microarray data cannot be used to compare the expression of different genes, it was unclear which was the major cadherin in native RPE cells. Studies with human RPE primary cells reported that while N-cadherin was expressed in both early (2-3 days) and late (8 weeks) confluence as well as in both epithelioid and fusiform cells, E-and P-cadherins were detected only in late confluence and epithelioid cells [53]. Using Western blot analyses, these authors showed that both E-and P-cadherins were present in RPE cell extracts prepared from human donor eyes [53]. In mouse eye development, Cdh3 mRNA was not detectable at E10.5 in the outer layer (future RPE) of the optic cup by in situ hybridization, but became highly expressed from E12 onward [28]. For bovine RPE in situ, the presence of P-cadherin protein was confirmed by Western blot analysis [57]. In cultured porcine RPE sheets, P-cadherin protein was abundantly present in RPE cells in the central region, where the cells maintained the cobblestone-like morphology, but lost in migrating cells at the edge of RPE sheets, where such cells began to express the EMT markers vimentin and N-cadherin [9]. Although these studies are not for quantitative comparison, they support our results regarding P-cadherin expression.
Based on these reports and our results, P-cadherin indeed seems to be the major cadherin that forms adherens junctions and maintains the epithelial phenotype of RPE cells in vivo. The functional importance of P-cadherin in the RPE is strongly supported by genetic case studies describing that CDH3 mutations are responsible for two rare autosomal recessive disorders: HJMD [29][30][31][32] and EEM syndrome [33]. HJMD patients show early-onset hair loss and severe retinal dystrophy, particularly at the RPE. EEM syndrome exhibits additional features such as split hand/foot malformation (ectrodactyly) with/without dental malformations in addition to HJMD characteristics. These phenotypic features of CDH3 mutations seem to reflect the expression patterns and function of P-cadherin, which plays a role in epithelial outgrowth and formation during development, including those of hair follicles, limb buds, mammary gland, and RPE [19,34,47,58,59]. The reason why CDH3 mutations exclusively affect RPE cells in the macula is unclear, and answers to this question may also provide mechanistic insights into other macular degeneration including AMD. Based on our results, we speculate that mutated P-cadherin proteins may form weakened adherens junctions, leading to the higher susceptibility to oxidative stress-induced impairment of the RPE integrity. The macular region likely faces stronger oxidative stress due to the higher density of retinal photoreceptors and the densest choroidal vasculature including choriocapillaris in the submacula [60][61][62]. However, further investigation is needed to answer this important question.
In contrast to human HJMD patients, P-cadherin-deficient mice are viable and fertile with no overt developmental abnormalities including ocular phenotype [34]. However, it is unclear how extensively the eyes of P-cadherin knockout mice have been analyzed at the histological and molecular levels. Even with normal mice, surprisingly few reports described P-cadherin in mouse RPE in vivo, and this scarcity of reported analyses of P-cadherin further motivated us to carry out our present studies. Besides the in situ hybridization analyses described above [28], we could find only one other report that analyzed P-cadherin in mouse RPE in vivo by searching in the PubMed (NCBI). Using immunofluorescence, this report described that albino RPE cells are irregularly shaped, and their adherens junctions distribute more widely and loosely at the cell membrane than those of pigmented RPE cells, suggesting that the lack of melanogenesis may impair RPE cell integrity in albino mice [35]. Interestingly, we observed somewhat similar wider and loose distribution of P-cadherin and β-catenin at the cell membrane after oxidative stress.
The surprisingly dominant expression of P-cadherin in RPE in situ at the mRNA level prompted us to test the expression at the protein level. We obtained a clear strong signal for Pcadherin by immunofluorescence of mouse RPE flat-mounts, which showed the co-localization of P-cadherin with β-catenin, F-actin, and ZO-1, proteins known to locate at the cell-cell border. In addition, in response to NaIO 3 -induced oxidative stress, P-cadherin quickly lost its tight localization at the cell-cell border and instead distributed more diffusely in the cytoplasm, suggesting that P-cadherin forms adherens junctions in normal conditions. These results are consistent with the findings that oxidative stress induced by hydrogen peroxide (H 2 O 2 ) disrupts junctional integrity of human ARPE19 cells and porcine RPE primary cells, including the loss of β-catenin localization at the cell border and the leak through tight junctions [4,7]. Light exposure, which induces oxidative stress in the retina, has also been reported to rapidly disrupt the localization of ZO-1, β-catenin, and N-cadherin at the cell-cell border of mouse RPE, leading to their redistribution to the cytoplasm [8]. In these studies, however, β-catenin and N-cadherin, but not P-cadherin, were analyzed as junctional proteins as in other previous studies using cultured RPE cells.
Following the observation of oxidative stress-induced dissociation of adherens junctions, an important question was whether dislocation of β-catenin resulted in activation of the canonical Wnt/β-catenin signaling pathway. Indeed, we found both an increase of β-catenin protein and its translocation to the nucleus, hallmarks of activation of the canonical Wnt/βcatenin pathway. Our results seem consistent with the findings previously reported in other cell systems in several aspects as summarized below. 1) Effect of oxidative stress on the canonical Wnt/β-catenin pathway. NIH3T3 or HEK293 cells treated with H 2 O 2 showed an increase of β-catenin and activation of T-cell factor (TCF), a transcriptional activator with which βcatenin forms a complex to regulate its target genes [63]. This β-catenin activation by oxidative stress was independent of Wnt ligands and triggered by the dissociation of dishevelled (DVL), an intermediate component of Wnt/β-catenin signaling, from nucleoredoxin, a member of the thioredoxin family that interacts with DVL and keeps it in an inactive state [63,64]. It has also been reported that 4-hydroxynonenal, a lipid peroxidation product, activated the Wnt/β-catenin pathway in a rat model of diabetic retinopathy [65]. 2) Intersection of two β-catenin pools. β-catenin is an essential component of both cadherin-based adherens junctions and the canonical Wnt/β-catenin signaling pathway, and a connection of these two β-catenin pools was suggested and later demonstrated [66][67][68]. In A431 epidermoid carcinoma cells exposed to lysophosphatidic acid that forces rapid dissociation of adherens junctions, cadherin-bound βcatenin was internalized together with E-cadherin, accumulated at the perinuclear endocytic recycling compartment, and translocated into the nucleus, suggesting that dissociation of adherens junctions can affect β-catenin levels available for the Wnt/β-catenin pathway [67]. 3) The Wnt/β-catenin pathway and EMT. β-catenin in the nucleus binds to members of the TCF/ LEF family of transcription factors to promote EMT [50,51]. During mouse embryonic development, a stabilized form of β-catenin in epiblasts led to activation of Wnt/β-catenin target genes, and cells of the embryonic ectoderm exhibited a premature EMT as a consequence [51]. Snail, one of the key EMT transcription factors, has multiple intersections with the Wnt/βcatenin pathway. Activation of this pathway results in upregulation of Snail, and Snail protein stability and subcellular localization are regulated through phosphorylation by glycogen synthase kinase-3β (GSK-3β), an intermediate component of the Wnt/β-catenin pathway [69,70]. In addition, Snail interacts with β-catenin and increases its transcriptional activity, indicating a positive feedback stimulation of the Wnt/β-catenin pathway by Snail [71]. Accordingly, although multiple pathways are involved in EMT, oxidative stress-induced EMT through activation of the Wnt/β-catenin pathway needs to be considered as one of the consequences of oxidative stress in RPE cells.
The findings reported so far suggest that cadherin subtype profiles are different between cultured RPE cells and mature RPE in vivo, and that P-cadherin be the major cadherin in the latter, but not in the former. However, this issue has not been clearly addressed thus far. In most epithelial cell types, E-cadherin is the major cadherin that is responsible for forming and maintaining their epithelial phenotype [18,20,23]. In contrast, it has been reported that RPE cells dominantly express and use N-cadherin to form adherens junctions [24][25][26]. However, it should be noted that these results were obtained from cultured RPE cells, such as ARPE19 human RPE cells and human RPE primary cells. Retrospectively, these results are not surprising because our quantification data show that M1 human RPE primary cells with the cobblestone-like appearance lost the dominant expression of CDH3 and instead acquired the expression of CDH2 at the level significantly higher than that in RPE in situ. In our previous studies, we also found that while M1 cells expressed RPE markers such as MITF and OTX2, they were quite different from in situ RPE cells with regard to the expression profile of MITF isoforms and the level of RPE65 expression [36]. Therefore, this difference in cadherin expression profiles is another example to add to the accumulating evidence of the difference between cultured RPE cells and RPE in situ.
The role of typically epithelial cadherins, E-and P-cadherins, and mesenchymal cadherin, N-cadherin, has become recognized as being more complex than originally defined, with both overlapping and distinct functions [17,19,72]. Interestingly, studies of epidermal sheet formation and maintenance showed that the level of cadherin is more critical than the subtype [48]. This finding seems consistent with the observation that the outcomes of P-cadherin overexpression in breast cancer depend on the cellular context of E-cadherin. In cells in which E-cadherin is highly expressed and maintained at the cell-cell border, P-cadherin disrupts Ecadherin function and promotes invasion, whereas in cells without E-cadherin, P-cadherin promotes adhesion and suppresses invasion [19,59,73]. In the case of in situ RPE with the barely detectable level of E-cadherin, P-cadherin likely promotes adhesion by forming adherens junctions. However, the situation in cultured RPE cells is quite different. Without the dominant abundant expression of typically epithelial cadherin, E-or P-cadherin, it is likely that N-cadherin plays a role in forming adherens junctions in cultured RPE cells, although Ncadherin is usually associated with EMT in most of other epithelial cell types [12,72].
Conclusions
We have established the RNA extraction method to purify RNA from mouse RPE and choroid individually. Using this method, we identified P-cadherin as the highly dominant form of cadherin in mouse RPE in situ. P-cadherin was also dominantly expressed in human RPE in situ, but not in cultured RPE cells. In addition, we found that oxidative stress led to dislocation of adherens junction proteins, P-cadherin and β-catenin, from the cell membrane to cytoplasm, resulting in nuclear translocation of β-catenin, a sign of activation of the canonical Wnt/β-catenin signaling pathway, and ultimately EMT-like molecular changes as a consequence. Based on this study, we would like to suggest that the expression and function of cadherins, especially P-cadherin, in the RPE should be reevaluated.
Supporting information S1 Table. Primer sequences. Sequences of all primers used in this study for expression analyses and generation of cDNA fragments for standard curves. | v3-fos-license |
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} | pes2o/s2orc | Cyclophospholipids Increase Protocellular Stability to Metal Ions
the vesicle size was reduced to below 500 nm with centrifugal spin column filters (Ultrafree–MC–Durapore 0.45 µ m with polyvinylidene difluoride membrane, Millipore) more than five times. Samples were diluted for each measurement to stay within the manufacturer’s recommended particle rate range (100–3000 particles min − 1 ) to avoid overestimation of the concentration. For the effect of dodecanol experiments, the final total lipid concentrations in the flow cell were as following, with respect to dodecanol: 0 mol% = 25 × 10 − 3 m , 9 mol% = 5 × 10 − 3 m , 16 mol% = 2.5 × 10 − 3 m , and 33 mol% = 1 × 10 − 3 m . For the analysis of dextran-containing vesicles, the final total lipid concentration was 1 × 10 − 3 m . The absolute concentration was then determined by taking into account the dilution factor of each sample.
Introduction
A major problem in our understanding of how protocells could have emerged is the lack of identified prebiotically plausible lipids that form robust vesicles capable of surviving the environments of the early Earth. Typically, mixtures of fatty acids, fatty alcohols, and the glycerol monoesters of fatty acids, i.e., monoglycerides, have been used to construct model compositions of protocellular membranes. [1,2] This is, in part, because RNA molecules retain their activity within fatty acid-based model protocells, [3,4] and such protocells can grow, divide, [5][6][7] Model protocells have long been constructed with fatty acids, because these lipids are prebiotically plausible and can, at least theoretically, support a protocell life cycle. However, fatty acid protocells are stable only within a narrow range of pH and metal ion concentration. This instability is particularly problematic as the early Earth would have had a range of conditions, and extant life is completely reliant on metal ions for catalysis and the folding and activity of biological polymers. Here, prebiotically plausible monoacyl cyclophospholipids are shown to form robust vesicles that survive a broad range of pH and high concentrations of Mg 2+ , Ca 2+ , and Na + . Importantly, stability to Mg 2+ and Ca 2+ is improved by the presence of environmental concentrations of Na + . These results suggest that cyclophospholipids, or lipids with similar characteristics, may have played a central role during the emergence of Darwinian evolution. and acquire nutrients from the environment. [8,9] Fatty acids are found in carbonaceous meteorites [10] and are abiotically synthesized by Fischer-Tropsch chemistry. [11] However, fatty acid vesicles are unstable; they only form over a narrow range of pH, [12,13] and rapidly disassemble in the presence of low concentrations of divalent cations, [4,14] and precipitate at the concentration of monovalent cations typically found in the environment. [14][15][16] Admixtures with fatty alcohol or glycerol monoesters of fatty acid increase stability to more alkaline conditions but do not sufficiently increase stability to cations. [4,12,14] Such characteristics appear at odds with the conditions of the early Earth and greatly limit the regions where the Earth's first cells could have emerged. Therefore, the identification of prebiotically plausible membrane compositions that can withstand a wide variety of chemical conditions, including the concentrations of metal ions necessary for the folding and activity of biomolecules, would be advantageous.
Recently, a prebiotically plausible small molecule, diamidophosphate (1, DAP), was found to phosphorylate nucleosides, amino acids, and glycerides. [17,18] Specifically, a mixture of glycerol and nonanoic acid was phosphorylated by DAP 1 to produce cyclophospholipid 4 (Figure 1a), which formed vesicles in the same reaction milieu. [18] Phospholipid 4 belongs to a family of naturally occurring lipids with a cyclic phosphate headgroup. [19] We reasoned that the decreased affinity of Mg 2+ for the cyclic phosphate headgroup, in comparison to a carboxylate or a phosphatidic acid headgroup, would render the vesicles more resistant to hard divalent metal ions. We, therefore, sought to determine the stability of model protocells built with prebiotically plausible, short-chain, saturated lipids with a cyclic phosphate headgroup. We find that vesicles built from cyclophospholipids are stable over a broad range of pH and salinity, suggesting that such vesicles could have harbored early chemical systems on the path toward life.
Fatty Alcohols Stabilize Cyclophospholipid Vesicles
To start with, we observed that fatty alcohols enhanced the formation of cyclophospholipid vesicles, consistent with previous work with fatty acid vesicles. [12] It was previously suggested that such stabilization resulted from decreased charge repulsion between polar headgroups and the strengthening of interacyl chain packing. [12,20] By epifluorescence microscopy, we found a dramatic increase in the number of vesicles formed with CPC9 4 as the mole fraction of the fatty alcohol nonanol 6 increased (Figure 2a). While only a few vesicles were detected in the absence of nonanol 6, many well-defined vesicles were observed with 2:1 CPC9 4:nonanol 6 at the same total lipid concentration of 40 × 10 −3 m.
Cyclophospholipid Vesicles are Stable to Changes in pH
To determine if vesicles composed of cyclophospholipids were more stable to changes in pH than fatty acid vesicles, 40 × 10 −3 m 2:1 CPC9 4:nonanol 6 and 2:1 CPC10 10:dodecanol 11 were dispersed in solutions at different pH with a constant Na + concentration of 200 × 10 −3 m and evaluated by epifluorescence microscopy. Vesicles clearly formed between pH 4 and pH 10 (Figures S16 and S17, Supporting Information). Conversely, 2:1 decanoic acid 5:dodecanol 11 formed vesicles only between pH 7 and 10. Since monoglycerides are frequently employed to increase the stability of fatty acid vesicles, a ternary mixture of fatty acid, fatty alcohol, and monoglyceride was tested. A 4:1:1 decanoic acid 5:dodecanol 11:decanoyl monoglyceride 9 mixture also formed vesicles, but only between pH 7 and pH 10.
To confirm the increased stability of vesicles composed of cyclophospholipid to pH in comparison to fatty acid, the retention of fluorescently labeled 10 kDa dextran was assessed. Remarkably, 2:1 CPC10 10:dodecanol 11 retained greater than 75% of the dextran at all pH values tested after 24 h of incubation (Figure 3a). In contrast, 2:1 decanoic acid 5:dodecanol 11 vesicles did not leak dextran at pH 7 and pH 8 (Figure 3a), and 4:1:1 decanoic acid 5:dodecanol 11:decanoyl monoglyceride 9 vesicles were comparatively unstable ( Figure 3a). 2:1 CPC9 4:dodecanol 11 were stable between pH 7 and pH 10 ( Figure S18, Supporting Information). Control experiments demonstrated that there was no hydrolysis of the fluorophore from the dextran at low pH ( Figure S19, Supporting Information). Additionally, the cyclic phosphate headgroup of CPC10 10 was not hydrolyzed at low (pH ≈ 3.9) or high (pH = 10.5) pH at 24 h ( Figure 3b) at room temperature. At the extreme basic pH of 10.5, the 31 P NMR showed two peaks (≈18 ppm) that developed over 24-96 h ( Figure S20a, Supporting Information). We initially ascribed these two peaks to the cyclic phosphate moiety existing in different environments (e.g., bilayers, micelles, or monomers). However, later work suggested that the newer second peak could belong to the carboxyl-ester hydrolyzed product, glycerol-1,2-cyclophosphate. This interpretation was supported by electrospray ionization-mass spectrometry (ESI-MS). Apart from the molecular ion peak corresponding to the CPC10 10, a relatively small molecular ion peak for glycerol-cyclophosphate was also observed ( Figure S21, Supporting Information), and confirmed by comparison with the authentic spectra of glycerol-1,2-cyclophosphate ( Figure S20b, Supporting Information). The relative intensities of the two peaks corresponding to the cyclophosphate species in 31 P NMR spectra at pH 10.5 (Figure 3b) have to be reconciled with the fact that glycerol-cyclophosphate is more soluble than CPC10 10 in water and may not be representative of the actual ratio of the mixture. This hypothesis is supported by a) the presence of the molecular ion mass of CPC10 10 in the sample at pH 10.5 after 96 h ( Figure S21, Supporting Information); b) the changes in the relative intensities of the two peaks upon dilution of the NMR sample ( Figure S22, Supporting Information); and c) by acquiring 31 P NMR on the same 72 h sample after lyophilization and resuspension in methanol, which showed a single peak ( Figure S23, Supporting Information) ascribed to CPC10 10. Nevertheless, despite the carboxyl-ester bond cleavage, our functional data indicate that protocells consisting of cyclophospholipid species can still retain the inner contents much more efficiently than fatty acid counterparts. Hydrolysis of the cyclophosphate moiety could be achieved by incubation at pH 2.3 within 24 h, as observed by 31 P NMR and ESI-MS ( Figure S24, Supporting Information).
A likely explanation for the formation and stability of cyclophospholipid vesicles over a broader range of pH than for fatty acid vesicles could be attributed to the lower pK a of the cyclic phosphate headgroup. For example, pure fatty acid vesicles form at the pK a of the headgroup of the bilayer-associated acid (≈7-9) [21,[23][24][25] because of the stabilizing interactions between the protonated and deprotonated forms of the carboxylate. [26] Similarly, pure dodecyl phosphate forms vesicles at the first pK a of the phosphate headgroup near pH 2. [21] Although vesicles are not formed when fatty acids or alkyl phosphates are fully protonated, fully deprotonated, or possess more than a single negative charge, the incorporation of a nontitratable hydrogen-bond donor, such as a fatty alcohol, in the membrane allows for the formation of vesicles under more alkaline conditions. [25] Since cyclic phosphates typically have a lower pK a than carboxylates, a mixture of cyclophospholipids with fatty alcohol would be expected to form vesicles at all pH values where deprotonated lipids exist. In bulk aqueous solution, the pK a of the cyclic phosphate headgroup was measured to be 2.3 ( Figure S25, Supporting Information), which was similar to the pK a1 of dodecyl phosphate. [21] However, large changes in pK a are common between free and membrane localized lipids, [27,28] and so the pK a may be different when embedded in a membrane.
Cyclophospholipid Vesicles are Stable to the Presence of Monovalent and Divalent Metal Ions
Having demonstrated that the vesicles containing cyclophospholipid were more stable over a broad range of pH compared to fatty acid vesicles, we next asked whether this increased stability extended to high concentrations of Na + and Mg 2+ . Epifluorescence microscopy showed that 2:1 CPC9 4:nonanol 3 and 2:1 CPC10 10:dodecanol 11 vesicles were stable between 0.2 and 2.2 m Na + (Figures S26-S28, Supporting Information), although nonvesicular aggregates were also observed at high concentrations of Na + . For comparison, the concentration of Na + in modern day seawater is between 0.4 and 0.5 m. [29] In contrast to cyclophospholipid vesicles, all of the fatty acid vesicles tested began to aggregate beyond 0.6 m Na + , consistent with previous studies. [14][15][16][30][31][32] The incorporation of monoglyceride did not improve the stability of fatty acid vesicles to Na + ( Figures S26 and S27, Supporting Information).
The retention of fluorescently labeled 10 kDa dextran after 24 h was more sensitive to the stability of the vesicles than could be observed by microscopy. Vesicles formed by a 4:1:1 decanoic acid 5:dodecanol 11:decanoyl monoglyceride 9 mixture never retained more than 26% of the dextran from 0.2 m NaCl and above (Figure 4). Cyclophospholipid vesicles were much more stable but did show a linear dependence on the concentration of Na + . Although greater than 85% of the dextran was retained by 2:1 CPC10 10:dodecanol 11 vesicles at 0.2 m Na + , approximately 10% more dextran was lost per 0.2 m increase of Na + between 0.2 and 1 m Na + . High concentrations of Na + promoted the formation of nonvesicular aggregates, as noted above, which may have led to the co-elution of lipid aggregates with dextran. Similar behavior was observed by others with fatty acid vesicles. [33] However, high concentrations of K + did not noticeably induce the formation of nonvesicular aggregates and thus showed a linear dependence of vesicle stability between 0.4 and 1.5 m K + ( Figure S29 The effect of Mg 2+ on vesicle stability mirrored the results above with cyclophospholipid vesicles outperforming their fatty acid counterparts. For example, epifluorescence microscopy showed Mg 2+ -induced aggregation of fatty acid vesicles composed of 2:1 decanoic acid 5:dodecanol 11 at 5 × 10 −3 m of Mg 2+ . Similarly, 4:1:1 decanoic acid 5:dodecanol 11:decanoyl monoglyceride 9 vesicles were aggregated at Mg 2+ concentrations above 10 × 10 −3 m (Figures S30 and S31, Supporting Information). In contrast, the cyclophospholipid vesicles (i.e., 2:1 CPC10 10:dodecanol 11) were observed in solutions with up to 25 × 10 −3 m Mg 2+ (Figure 5a and Figures S31 and S32, Supporting Information). For comparison, the concentration of Mg 2+ in modern day seawater is ≈50 × 10 −3 m. [29] The retention of dextran data confirmed the large difference in stability between fatty acid and cyclophospholipid vesicles. While fatty acid vesicles completely lost the encapsulated dextran by 5 × 10 −3 m Mg 2+ , the cyclophospholipid vesicles retained more than 50% of the entrapped dextran at 25 × 10 −3 m Mg 2+ (Figure 5b).
In addition to magnesium, the prebiotic Earth was rich in calcium. Since phospholipid membranes can interact with and be disrupted by calcium ions, [34] we next asked if cyclophospholipid membranes could withstand the presence of Ca 2+ . After 24 h, 2:1 CPC10 10:dodecanol 11 vesicles were morphologically unchanged in the presence of 5 × 10 −3 m Ca 2+ and began to crystallize at 10 × 10 −3 m Ca 2+ ( Figure S33, Supporting Information). The stability to Ca 2+ was assessed by measuring the retention of fluorescently labeled dextran. Approximately 50% of the entrapped dextran was retained after 24 h in the presence of 10 × 10 −3 m Ca 2+ (Figure 6a). The concentration of Ca 2+ in the contemporary ocean is ≈10 × 10 −3 m. [35] The observed stability to pH and metal ions is in contrast to that observed with alkyl phosphates [36] and fatty acids. [37,38] Although mixtures of alkyl phosphate and fatty alcohol form vesicles over a broad range of pH, experiments with alkyl phosphate vesicles were always performed either in deionized water [25] or in the presence of chelators of metal ions. [21] The prebiotic environment likely contained a complex mixture of molecules that cannot be well represented by solutions containing a single type of metal ion. To better assess the plausibility of cyclophospholipid vesicles surviving environments of complex composition, the effect of mixtures of cations on the retention of entrapped dextran was evaluated after 24 h. The presence of an additional 0.2 m Na + increased the stability of 2:1 CPC10 10:dodecanol 11 vesicles to 25 × 10 −3 m Mg 2+ and 10 × 10 −3 m Ca 2+ by more than 20% and 10%, respectively. That is, 71% ± 4% of the dextran remained entrapped within the cyclophospholipid vesicles in solutions containing 25 × 10 −3 m Mg 2+ and 0.4 m Na + , and 61% ± 4% of the dextran was retained with 10 × 10 −3 m Ca 2+ and 0.4 m Na + (Figures 5b and 6a). Protection by Na + was possible because the disruptive effects per Na + were 20-fold less than per Mg 2+ ( Figure S34, Supporting Information). The data were confirmed with entrapped, fluorescently labeled DNA in place of the dextran (Figure 6b), demonstrating the ability of cyclophospholipid vesicles to hold genetic material. Next, a ternary mixture of metal ions was added to the cyclophospholipid vesicles. 2:1 CPC10 10:dodecanol 11 vesicles retained ≈50% of entrapped dextran and 75% ± 3% of fluorescently labeled DNA after 24 h in a solution containing 5 × 10 −3 m Ca 2+ , 20 × 10 −3 m Mg 2+ , and 0.4 m Na + (Figure 6b).
Taken together, cyclophospholipid vesicles can withstand high concentrations of metal ions in the absence of chelators, such as citrate (Figure 7 and Figure S35, Supporting Information). [9] Although prebiotically plausible short-chain cyclophospholipid vesicles may have been incapable of surviving long periods of time in seawater, such vesicles would have been capable of surviving conditions of higher salinity than found in many present day hot springs. [31,32] That is, the increased stability to salinity expands the regions where protocells could have survived, thus increasing the overall likelihood of their emergence.
Conclusion
Stability to metal ions has long been considered a problem in our understanding of protocell chemistry. The Earth is rich in metal ions, so biology has evolved in a way that exploits this abundant resource. [39,40] For example, Mg 2+ plays structural and catalytic roles necessary for the activity of extant nucleic acids, proteins, and small molecules. Although there are places with low concentrations of metal ions, [31,32] even bodies of fresh water would experience transiently high concentrations of salt during the types of wet-dry cycles frequently invoked to aid the dehydration reactions needed for the synthesis of biological polymers. [41,42] Previous reports demonstrated that vesicle stability can be improved with admixtures of prebiotically plausible short chain fatty acids with alkyl amines, [30] and that some single chain lipids can assemble into vesicles that withstand high salinity. [43] However, such lipids are either prebiotically unlikely or cannot be clearly assigned a transitional role between prebiotic chemistry and extant lipids.
The esterification of a glycerol to a fatty acid renders the resulting vesicles more stable to Mg 2+ and Ca 2+ , [14] in part, because the binding site of the metal ion is weakened. However, vesicles containing mixtures of fatty acid and monoglyceride are still susceptible to changes of pH, because of the loss of polarity that results from the protonation of the fatty acid component of the membrane. Cyclic phosphate headgroups improve both of these features at once. The cyclophospholipid likely has decreased affinity for Mg 2+ and Ca 2+ and a lower pK a that allows for vesicle formation over a broader range of pH in the presence of suitable hydrogen-bond donors.
Past attempts to determine environmental conditions compatible with the existence of protocells have tended to focus on either identifying regions of low salinity, the presence of prebiotic chelators of metal ions, or lipid additives that can withstand the effects of Mg 2+ alone. What has been less considered is the effect of Na + , which is important because high concentrations of Na + can interfere with the binding of other cations. [44] Here, we show that vesicles composed of cyclophospholipid and fatty alcohol are more resistant to Na + , Mg 2+ , and Ca 2+ individually than fatty acid and alkyl phosphate vesicles. [21,25] More significantly, the increased stability to Na + gives rise to an increased tolerance to Mg 2+ and Ca 2+ . Therefore, not only could such cyclophospholipid systems survive a wider variety of chemical conditions more compatible with what is known about the prebiotic Earth, [45] but also cyclophospholipid protocells would be able to persist in conditions ideal for the nonenzymatic polymerization of nucleotides [46,47] and the evolution of extant-like nucleic acid and protein folds. It should be noted that a recent report showed a stabilizing effect on fatty acid vesicles by prebiotic amino acids. [33] Whether similar stabilization of cyclophospholipid vesicles is possible has yet to be tested. Contemporary diacyl phospholipids can be broken down into component parts that may chart a historical path starting from fatty acids. The coupling of glycerol to the fatty acid gives a lipid that forms vesicles that are more stable to pH and the presence of metal ions. Similarly, phosphorylation of this monoglyceride to give a cyclophospholipid increases the stability to pH and metal ions even further. Such incremental improvements with each discrete chemical step, presumably by energy-dissipative cycling, [48] suggest a path in which environmental selective pressures could lead to modern day lipids. The remaining step needed to convert the monoacyl cyclophospholipid to a contemporary diacyl phospholipid would greatly improve stability at the expense of the ability to acquire nutrients and grow and divide without protein machinery. [49] It may be that cyclophospholipids played a critical role in the space between proteinindependent and protein-dependent (proto)cells.
Experimental Section
General Experimental: Thin layer chromatography was performed with a silica gel 60 ÅF254 from Angela Technologies and visualized by UV lamp and/or a stain solution of phosphomolybdic acid in ethanol. Flash chromatography was performed on a biotage isolera. NMR was recorded at 298 K with a Bruker DRX-600 or AV-600 (600 MHz for 1 H and 150 MHz for 13 C). 31 P NMR spectra were acquired using a Bruker DPX-400; chemical shifts (δ) in parts per million (ppm), spin multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constants (J) in Hertz (Hz), number of protons. Mass spectra were collected with an Agilent ESI-TOF or a ThermoElectron Finnigan LTQ ion trap mass spectrometer. The pH electrode was from Hanna instrument from Spectrum Chemicals and Laboratory products.
Cyclophospholipid Synthesis: Reagents and solvents were from Sigma-Aldrich, VWR International, Fisher Scientific, and Acros. Cyclophospholipids CPC9 4 and CPC10 10 were synthesized and purified by employing previously reported protocols. [18] Spectroscopic and spectrometric data are presented in the Supporting Information (Figures S1-S8, Supporting Information).
Preparation of Vesicles: Fatty acid vesicles were prepared by oildispersion. [8] More specifically, fatty acids and their nonphosphorylated glycerol monoesters were heated to 55 °C. Subsequently, aliquots of the lipid oils (total: ≈10 µmol) were quickly mixed with 100 µL aqueous solution and vortexed for ≈30 s. The resulting solution was then repeatedly heated to 55 °C and vortexed for ≈30 s between five and eight times. The solution was allowed to reach room temperature (23 °C) before use. Cyclophospholipids were handled similarly, except that the powder was added directly to the aqueous solution. Buffer compositions are reported in Table S1 in the Supporting Information.
Vesicle Stability Assay: Unless otherwise noted, all vesicles were extruded through 100 nm track-etched polycarbonate membranes using an Avanti Polar mini extrusion system (11 passes). Vesicles were prepared in the presence of 25 × 10 −6 m anionic (not poly-anionic nor dextran-sulfate) 10 kDa dextran conjugated with Alexa Fluor 488 (ThermoFisher Scientific) and purified within 2 h of extrusion. For the encapsulation of oligonucleotide, vesicles were extruded to 200 nm to increase encapsulation efficiency. 10 × 10 −6 m Alexa Fluor 555 DNA oligonucleotide (5′-GGCTCGACTGATGAGGCGCG-Alexa Fluor 555-3′) was hybridized to 10 × 10 −6 m Alexa Fluor 488 labeled oligonucleotide (5′-Alexa Fluor 488-CGCGCCGAAACACCGTGTCTCGAGC-3′) by heating to 95 °C and cooled to 4 °C at a rate of 0.5 °C s −1 in the presence of 1 × 10 −3 m MgCl 2 . Size exclusion chromatography with sepharose 4b was used to separate vesicles from unencapsulated dye to afford purity of > 95%. Columns were pre-equilibrated and run with buffer containing lipid above the critical aggregate concentration (20 × 10 −3 m for cyclophospholipid vesicles and 40 × 10 −3 m for fatty acid vesicles). Fractions were collected with either a FC203B or FC204 Gilson fraction collector. Optical density at 600 nm and fluorescence (λ ex = 485 nm and λ em = 515 nm) measurements were taken with a Tecan Infinite M200 plate reader. Vesicles were then incubated in the dark at 23 °C. For Na + , K + , Mg 2+ , Ca 2+ stability, vesicles were diluted twofold into solutions containing empty vesicles (with a final total lipid concentration of ≈10 × 10 −3 m) and salts. The percent solute retained inside of the vesicles was determined by comparing the vesicle and free dye fractions after a second round of size exclusion chromatography.
Determination of the Critical Aggregate Concentration (CAC): The CAC was determined by following previously published procedures. [13] Briefly, 100 µL of 200 × 10 −6 m merocyanine 540, 0.2 m HEPES, pH 8.0 was dispensed in the wells of a 96-well plate (Costar 3603, black clear bottom, Corning). Solutions were then diluted twofold with vesicles of varying concentrations prepared in 0.2 m HEPES, pH 8.0 and incubated at room temperature (23 °C) for 10 min. Absorbance was read with a Tecan Infinite M200 plate reader at 570 and 530 nm. The A 570 nm /A 530 nm ratio indicated the aggregation state of the lipid, because the absorbance of merocyanine 540 at 570 nm reflects ordered lipid structures, whereas absorbance at 530 nm is correlated to free molecules in solution. [50] No lipid negative controls gave A 570 nm /A 530 nm values between 0.5 and 0.65. Measured values above the corresponding negative control were interpreted as points where the lipids formed aggregates.
TRPS Measurements: Data were collected with a qNano Gold (Izon Science) instrument. The influence of fatty alcohol content made use of Nanopore NP400 (pore size range = 200-800 nm), and the effect of dextran was assessed with Nanopore NP200 (pore size range = 100-400 nm). For consistency, an identical instrumental setup was used throughout the same-day analyses with current amplitudes of 120 ± 10 nA (>100%, as recommended by manufacturer). The parameters were as follows: voltage = 0.34 ± 0.06 V, stretch = 46.75 ± 0.25 mm, pressure = 7 ± 3 mbar. For statistical significance, 500-1000 particles per run were collected with a particle rate maximum of 4000 particles min −1 . Multiple dilutions (of different samples) and readings (of the same sample) were analyzed and reported as technical replicates (±SD). The instrument and nanopore were calibrated with standard calibration particles from the manufacturer (Izon Science) with mean a diameter of either 210 nm for NP200 or 340 nm for NP400. For dextran analysis, vesicles were prepared as indicated above. For the analysis of the effect of fatty alcohol content, vesicles were prepared from a single stock of 50 × 10 −3 m CPC10 10 solution at pH 8.0 with 0.2 m HEPES and left tumbling for 24 h. This stock of CPC10 10 was then aliquoted into new glass vials containing varying amounts of dodecanol. The solution was then adjusted to a final total lipid concentration of 25 × 10 −3 m in a final volume of 200 µL. Prior to TRPS measurements, the vesicle size was reduced to below 500 nm with centrifugal spin column filters (Ultrafree-MC-Durapore 0.45 µm with polyvinylidene difluoride membrane, Millipore) more than five times. Samples were diluted for each measurement to stay within the manufacturer's recommended particle rate range (100-3000 particles min −1 ) to avoid overestimation of the concentration. For the effect of dodecanol experiments, the final total lipid concentrations in the flow cell were as following, with respect to dodecanol: 0 mol% = 25 × 10 −3 m, 9 mol% = 5 × 10 −3 m, 16 mol% = 2.5 × 10 −3 m, and 33 mol% = 1 × 10 −3 m. For the analysis of dextran-containing vesicles, the final total lipid concentration was 1 × 10 −3 m. The absolute concentration was then determined by taking into account the dilution factor of each sample.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author. | v3-fos-license |
2019-04-03T13:03:25.566Z | 2019-03-27T00:00:00.000 | 91187757 | {
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} | pes2o/s2orc | Bioactivity of Bioceramic Materials Used in the Dentin-Pulp Complex Therapy: A Systematic Review
Dentistry-applied bioceramic materials are ceramic materials that are categorized as bioinert, bioactive and biodegradable. They share a common characteristic of being specifically designed to fulfil their function; they are able to act as root canal sealers, cements, root repair or filling materials. Bioactivity is only attributed to those materials which are capable of inducing a desired tissue response from the host. The aim of this study is to present a systematic review of available literature investigating bioactivity of dentistry-applied bioceramic materials towards dental pulp stem cells, including a bibliometric analysis of such a group of studies and a presentation of the parameters used to assess bioactivity, materials studied and a summary of results. The research question, based on the PICO model, aimed to assess the current knowledge on dentistry-based bioceramic materials by exploring to what extent they express bioactive properties in in vitro assays and animal studies when exposed to dental pulp stem cells, as opposed to a control or compared to different bioceramic material compositions, for their use in the dentin-pulp complex therapy. A systematic search of the literature was performed in six databases, followed by article selection, data extraction, and quality assessment. Studies assessing bioactivity of one or more bioceramic materials (both commercially available or novel/experimental) towards dental pulp stem cells (DPSCs) were included in our review. A total of 37 articles were included in our qualitative review. Quantification of osteogenic, odontogenic and angiogenic markers using reverse transcriptase polymerase chain reaction (RT-PCR) is the prevailing method used to evaluate bioceramic material bioactivity towards DPSCs in the current investigative state, followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining (ARS) to assess mineralization potential. Mineral trioxide aggregate and Biodentine are the prevalent reference materials used to compare with newly introduced bioceramic materials. Available literature compares a wide range of bioceramic materials for bioactivity, consisting mostly of in vitro assays. The desirability of this property added to the rapid introduction of new material compositions makes this subject a clear candidate for future research.
The aim of this study is to present a systematic review of available literature investigating bioactivity of dentistry-applied bioceramic materials towards dental pulp stem cells; including a bibliometric analysis of such group of studies and a presentation of the parameters used to assess bioactivity, materials studied and summary of results.
Materials and Methods
This systematic review was conducted in accordance with the PRISMA guidelines or preferred reporting items for systematic reviews and meta-analyses [18]. Our review was not eligible for registration with PROSPERO, as it did not involve health studies in which participants were people nor animal research studies exclusively.
In terms of the research question, based on the PICO model, our review aimed to assess the current knowledge on dentistry-based bioceramic materials by exploring to what extent they express bioactive properties in in vitro assays and animal studies when exposed to dental pulp stem cells, as opposed to a control or compared to different bioceramic material compositions, for their use in the dentin-pulp complex therapy.
Inclusion and Exclusion Criteria
Studies assessing bioactivity of one or more bioceramic materials (both commercially available or novel/experimental) towards DPSCs were included in our review. We established bioactivity assessment as any test or measurement for odontogenic, osteogenic, angiogenic and/or mineralization potential of DPSCs exposed both directly or indirectly to bioceramic materials. Studies assessing cytotoxicity and/or biocompatibility alone i.e., cell viability or proliferation were excluded. Studies assessing any other type of stem cell apart from DPSCs were also excluded.
The series of inclusion and exclusion criteria were established by a consensus reached from all authors after discussion, considering the research question and the objectives of the study while aiming for an ample range of results to be provided from the search.
Sources of Information
To identify potentially relevant studies, a thorough electronic search was made in PubMed, Web of Science, Scopus, Embase, Cochrane, and Lilacs databases. Study search was performed during October, November and December 2018. In particular cases, the authors of the articles were contacted by email to request missing information. The structured search strategy and data extraction were conducted by an individual examiner.
Study selection
Articles identified using the search terms were exported to RefWorks (ProQuest, MI, USA) to check for duplicates. Once duplicates were discarded, a first screening of record titles and abstracts
Study Selection
Articles identified using the search terms were exported to RefWorks (ProQuest, MI, USA) to check for duplicates. Once duplicates were discarded, a first screening of record titles and abstracts was carried out according to the previously described inclusion and exclusion criteria. Remaining studies were assessed for eligibility and qualitative synthesis by full-text screening.
Study Data
For the bibliometric analysis, the following variables were recorded for each article: author and year of publication, journal, country, and institution. For the synthesis of study methodology, a summary of the materials and methods of included studies was transcribed by listing the following variables: study type, bioceramic materials used, bioactivity analysis and duration of the analysis. For the synthesis of results, studies were categorized in terms of the significant results found, the duration in which these significant results were found, and their significance level.
Quality Assessment
The quality of the studies was assessed using a modified CONSORT checklist of items for reporting in vitro studies of dental materials [19] and the ARRIVE guidelines for reporting animal research [20].
Study Selection and Flow Diagram
The search identified 1023 preliminary references related to the bioactivity of bioceramic materials towards dental stem cells, of which 355 were found in PubMed, 473 in Web of Science, 179 in Embase, 15 in Scopus, and 1 in Cochrane databases. Search made in LILACS produced no results. After excluding 203 duplicates, the remaining 820 were screened. Of these, 783 were excluded on reading the title and abstract as they did not fulfil our inclusion criteria. The resulting 37 articles were examined at full-text level, and all of them resulted to be eligible for our review (Figure 2).
Bibliometric Analysis
All corresponding authors of the included studies were associated with an academic institution or university. The distribution of included studies by year of publication, country, and journal is presented in Figure 3.
Bibliometric analysis
All corresponding authors of the included studies were associated with an academic institution or university. The distribution of included studies by year of publication, country, and journal is presented in Figure 3.
Bioactivity Analysis
A wide range of analyses of bioactivity were presented from the included studies. The most common analysis was the quantification of the expression of odontogenic, osteogenic and/or angiogenic markers or genes using reverse transcription polymerase reaction (RT-PCR), followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining (ARS) to assess mineralization potential.
Other analyses include western blot, micro-computed tomography (micro-CT), scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), transmission electron microscopy (TEM), histological analysis, immuno-fluorescence, and immuno-histochemical assays. Bioactivity analyses alongside with their duration and a description of the study associated with them are presented in Table 1.
Bibliometric Analysis
All corresponding authors of the included studies were associated with an academic institution or university. The distribution of included studies by year of publication, country, and journal is presented in Figure 3.
Study Type
Articles included fell into two main categories in terms of type of study: in vitro, or animal study. In some cases, articles presented both an in vitro and an animal study [26,27,37]. There were two studies which analyzed bioactivity of bioceramic materials towards hDPSCs ex vivo [33,42].
Cell Variant
All studies included used dental pulp stem cells (DPSCs) as their cell variant to assess bioceramic material bioactivity.
Bioceramic Materials Used
Bioceramic materials studied ranged from commercially available ( Table 2) to novel or experimental materials (Table 3). A separate category was presented for bioceramic materials which were combined with an additive for their analysis (Table 4).
Quality Assessment
All in vitro studies analyzed using the modified CONSORT checklist [19] (Table 5) presented a structured abstract (item 1) and an introduction which provided information about the background of the bioceramic material and/or bioactivity analysis studied (item 2a). Within the introduction, the majority of studies presented clear objectives and hypotheses (item 2b). Description of methodology as well as of the variables studied was sufficiently clear to allow for replication in all studies (items 3 and 4), but none of them presented a detailed report of the calculation of sample size or random allocation sequence (items 5-9). All studies indicated the statistical method used (item 10), but presented significance level as p values, and not confidence intervals (item 11). Discussions generally included a brief synopsis of the key findings and comparisons with relevant findings from other published studies, but often failed to address the limitations of the studies, which we considered as a reason for non-fulfillment (item 12). Sources of funding (if any) were indicated in the majority of studies (item 13), and indications for access to full trial protocols were obviated in all studies (item 14). Table 5. Results of the assessment of in vitro studies by the use of the modified CONSORT checklist [19]. Cells marked with an asterisk "*" represent study fulfilment for the given quality assessment parameter. Cells left blank represent non-fulfilment.
Studies
Modified Only three out of the five animal studies analyzed using the ARRIVE guidelines [20] (Table 6) were headed with a sufficiently descriptive title (item 1), but all of them provided a detailed abstract (item 2). All studies provided sufficient scientific background (item 3a) and established clear objectives (item 4) in the introduction, but failed to justify the use of the animal species studied to address the scientific objectives (item 3b). Ethical statements were clear in all studies (item 5), and study design, experimental procedures were detailed enough in all except one (items 6 and 7). Details about the experimental animals and how they were distributed in the study design were included in every study (items 8-11 and 14), but housing and husbandry information was obviated in all cases (item 9). Both experimental outcomes and statistical methods were described in all studies (items 12 and 13). All studies reported the results for each analysis carried out with a measure of precision (item 15), but all of them failed to report baseline data about health status of the animals studied and any adverse effects they could have suffered after the experiment (items 14 and 17). Lastly, items referring to the discussion were fulfilled by all studies (items 18-20). Table 6. Results of the assessment of animal studies by the use of the ARRIVE guidelines [20]. Kyung-Jung et al. [26] * * * * * * * * * * * * * * * * Wongsupa et al. [27] * * * * * * * * * * * * * * * * * * Atalayin et al. [29] * * * * * * * * * * * * * * * Zhu et al. [37] * * * * * * * * * * * * * * * Daltoé et al. [50] * * * * * * * * * * * *
Study Tesults
Significant results from included in vitro studies are presented in Tables 7-17, and significant results from included animal research studies are presented in Table 18.
Studies comparing a bioceramic material and a non-bioceramic material did not show positive significant results for the bioceramic materials studied. One of the studies showed that DDM produced a greater bioactivity-related gene expression than HA-CPC [26]; and the other one showed mixed results for Ca 3 SiO 3 , which produced a greater expression of some markers but not others compared to Ca(OH) 2 [57] (Table 10).
Both studies comparing a bioceramic material and an additive with the bioceramic material itself showed positive significant results for the bioceramic material in combination with the additive (γION-CPC and αION-CPC, [24]; GNP-CPC, [25]) (Table 12).
Results for ALP Activity
There was only one study comparing a bioceramic material with MTA in terms of ALP activity, and it produced negative results for the bioceramic material studied (Quick-Set2, [31]). The rest of the studies compared two different biomaterials or different concentrations of the same bioceramic material (Table 14).
Results for Other Bioactivity-Related Analyses
Western blot analyses showed mixed results for Zn0/1/2/3 compared to a control [28], and a higher expression of bioactivity-related markers by PR-MTA compared to Quick-Set2, and by both of them compared to a control [31]. ATR-FTIR showed positive results for PR-MTA compared to Quick-Set2, and for both of them compared to a control [31]. ELISA showed mixed results for MTA and CEM [39]. Assessment of the level of grey in mineralization nodules using Gene Tool showed positive significant results for PLGA/TCP compared to PLGA/HA and PLGA/CDHA [42]. Lastly, both the TRACP & ALP assay kit (Takahara, Shiga, Japan) and the OC and DSP emzyme-linked immunosorbent assay kit (Thermo Fisher Scientific, Waltham, MA, USA) showed that the addition of polydopamine to PR-MTA produced better results than PR-MTA itself. Table 11. Summary of the results of included studies showing significant differences between various bioceramic materials or different concentrations of the same bioceramic material for ARS staining.
Discussion
The attractiveness of bioceramic materials for their desirable properties added to their constant development, the demand for new advances and the ampliation of treatment indications results in an overflow of related literature over time. Therefore, it seems convenient to establish an updated and organized vision of the commercially available and experimental dentistry-applied bioceramic materials' characteristics. With this in mind, the aim of this study was to present a systematic review of available literature investigating bioactivity of these materials towards dental pulp stem cells.
In terms of results, it can be highlighted that the most common method used to assess bioactivity in the included studies was the expression of bioactivity-related markers using reverse transcriptase polymerase chain reaction or RT-PCR. A recent systematic review illustrates this tendency by assessing gene expression of dental pulp cells in response to tricalcium silicate cements [58]. Studies also tended to compare new bioceramic materials with the established mineral trioxide aggregate or the more recently introduced Biodentine, as shown in Table 2, in which they appear as the most studied materials.
The use of additives in combination with bioceramic materials looks promising, in some cases enhancing or positively influencing the material's results in bioactivity assays in comparison with the bioceramic material itself. For example, positive significant results have been shown for iron oxide [24], gold [25], and bioactive glass [32] nanoparticles in combination with calcium phosphate. However, we need to interpret these results with caution, being able to extrapolate them to clinical practice only when a clear dosage or ratio for the additive and bioceramic material has been established in controlled clinical trials.
New material compositions being studied also need to be taken into consideration for future investigations, as some of them have shown positive significant results in bioactivity assays. Novel materials like Exp. PPL [22], Gelatin-HA-TCP [23] and Zinc Bioglass (Zn0/1/2/3) [28] have all shown positive significant results for ARS staining and ALP activity assay compared to a control, and more specifically, Exp. PPL has shown a greater expression of DSPP and OCN compared to MTA and a control; Gelatin-HA-TCP has shown a greater expression of RUNX2, OSX and BSP compared to a control; and Zinc Bioglass (Zn0/1/2/3) has shown a greater expression of RUNX2, ON, CON, MEPE, BSP, and BMP-2 compared to a control. So again, in order to extrapolate these results to clinical practice, it would be interesting to carry out further studies investigating these biomaterials in different conditions. When assessing quality and risk of bias, included studies referred a similar structural pattern. They reported essential data like a sufficient abstract, a clear objective or objectives, a detailed description of methodology, a mention of the statistical tests used and relevant conclusions; but often failed to justify the sample size used, to describe the randomization process used (if any), and most importantly to address the study's limitations in the discussion. It may be worth noticing for future reviews that a checklist for reporting in vitro studies or "CRIS" guideline is under development [59] to address the need for uniform methodology in the assessment of this type of studies.
The introduction of new bioceramic materials and the use of additives in combination with them calls for updated research in the field. At the current state, bioactivity assessment of these materials towards dental pulp stem cells centers on in vitro assays or animal research at most. For future studies, it could be interesting to explore the mechanisms with which this bioactivity is achieved and move on towards in vivo trials.
Conclusions
Quantification of osteogenic, odontogenic and angiogenic markers using reverse transcriptase polymerase chain reaction or RT-PCR is the prevailing method used to evaluate bioceramic material bioactivity towards DPSCs in the current investigative state, followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining (ARS) to assess mineralization potential. Mineral trioxide aggregate and Biodentine are the prevalent reference materials used to compare with newly introduced bioceramic materials. Available literature compares a wide range of bioceramic materials for bioactivity, consisting majorly of in vitro assays. The desirability of this property added to the rapid introduction of new material compositions makes this subject a clear candidate for future research. | v3-fos-license |
2020-12-24T09:11:34.625Z | 2020-01-01T00:00:00.000 | 235094961 | {
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} | pes2o/s2orc | Studying the biochemical transformations in sherry wines subjected to biological aging
. The fundamental role in the process of biological aging is given to yeast which enrich the wine with substances that contribute to the formation of the characteristic organoleptic properties of sherry. We studied 55 samples of wine in the dynamics of biological aging. The purpose of this research was to study the transformation of wine components during biological aging and to determine their technological value. Generalization of experimental data showed that biological aging determines changes in the composition of wines and physical-chemical characteristics: reduction of the volume fraction of ethanol, the mass concentration of the components of the acid complex (malic, lactic and acetic acids), amine nitrogen and glycerin as sources of energy and carbon nutrition for yeast. Decrease in the content of phenolic substances, the value of the redox-potential of wine and optical density D 420 confirms the protective function of the sherry film that prevents non-enzymatic oxidation of wine components. The biological aging is also characterized by an increase in the mass concentration of aroma-forming substances: acetaldehyde, diacetyl, acetoin, dioxanes, dioxolans, lactones. Based on the data obtained, the role of the main components of wines in the process of biological aging and the ranges of their variation were determined.
Introduction
The fundamental role in the process of biological aging is given to yeast which, as a result of its development, as well as autolysis, enrich the wine with substances that contribute to the formation of the characteristic organoleptic properties of sherry [1][2][3][4][5][6]. The physiological state and metabolism of the yeast cell is influenced by the content of nutrient components, process inhibitors (sulfurous acid, lactic and acetic acid bacteria), as well as technological factors [1,5,7]. The oxidative metabolism of yeast results in the produced substances that provide the typical aroma and flavor properties of sherry wines [2,[8][9][10][11][12][13][14][15].
Presently, control over the biological aging is carried out by the accumulation of the mass concentration of acetaldehyde, as well as by the transformation of the organoleptic characteristics of the product. Studying the qualitative and quantitative changes in the component composition under the action of flor yeast to substantiate additional criteria for assessing the quality of Sherry-type white wine called "Fino'" is relevant.
The purpose of this research was to study the transformation of wine components in the process of biological aging, as well as to determine their technological value.
Study objects and methods
The objects of our research were base wines from grapes of the 'Aligoteʼ and 'Rkatsiteliʼ varieties, enriched with ethyl alcohol to provide the conditions of 16.0% vol. Biological aging of the samples was carried out by a film method using the sherry yeast race Jerez 20 C/96. The formation of a continuous sherry film on the surface of the wine was observed after 9-13 days. We analyzed 55 samples of wines in dynamics (before laying for biological aging, 1, 3, 5 months after sowing sherry film). Microbiological control and monitoring of the state of the yeast was performed every 2 weeks.
In wines, optical and potentiometric characteristics were determined [16,17]. The organic acid profile and glycerol content were determined by the HPLC method on a Shimadzu LC20 Prominance chromatograph (Japan). The component composition of the aromatic complex of wines was carried out using high performance liquid chromatography by means of the Agilent Technologies system.
Results and Discussion
Generalization of the literature data and research results shows that the metabolism of sherry yeast which can adapt to conditions of a limited amount of nutrients is the basis of complex biochemical transformations of the components of wine, as well as the formation of specific organoleptic characteristics of sherry wine. The main sources of carbon and energy nutrition of sherry yeast include ethyl alcohol, the consumption of which in the process of biological aging is 1-3% vol., and glycerin, the content of which is reduced to trace levels. Yeast also assimilates organic (malic, lactic, acetic) and amino acids as substrates. Analysis of the literature data [2,5,8] and the results of studies of the dynamics of biological aging made it possible to establish the role of the main components of wine under the action of flor yeast, as well as to determine the ranges of their change (Table 1). As a result of oxidative metabolism of sherry yeast, the medium is enriched with new components that determine the specific organoleptic properties of sherry (Table 2). Due to the oxidation of ethanol, acetaldehyde is formed under the action of the alcohol dehydrogenase enzyme the amount of which can reach 1000 mg/l and higher. As a result of its high reactivity, acetaldehyde reacts with the components of wine forming specific products for sherry-type wine: 1,1-diethoxyethane with ethanol, dioxanes and dioxolanes with glycerin, and sotolon with α-ketobutyric acid. In addition, acetoin and diacetyl are formed enzymatically [1,4,8,14]. In addition to biochemical transformations, the optical and potentiometric characteristics of wine undergo changes under the action of flor yeast (Table 3): the optical density of D420 and the oxidation reduction potential decrease which confirms the protective function of the sherry film which, by consuming dissolved oxygen, prevents non-enzymatic oxidation of wine components [5,11]. As a result of coagulation with yeast cells and subsequent sedimentation, the content of phenolic complex components decreases.
Conclusion
Thus, as a result of the studies carried out, it has been shown that the process of biological aging of wines under a sherry film causes a change in the component composition of the wine and its physicochemical characteristics: -reduction of the volume fraction of ethyl alcohol, the mass concentration of the components of the acid complex (malic, lactic and acetic acids), amine nitrogen and glycerol as sources of energy and carbon nutrition for yeast; -decrease in the content of phenolic substances, the value of the oxidation reduction potential of wine and optical density D420; -increase in the mass concentration of aromatic substances formed during the oxidative metabolism of yeast: acetaldehyde, diacetyl, acetoin, dioxanes, dioxolans, lactones.
Based on the data obtained, the role of the main components of wines in the process of biological aging was established, and the ranges of their variation were determined. The important role of acetaldehyde and products of its interaction with wine components in the formation of specific organoleptic properties of Sherry is shown. | v3-fos-license |
2019-04-29T13:07:22.895Z | 2013-05-31T00:00:00.000 | 137713342 | {
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} | pes2o/s2orc | Application of Eclipta Leaves, Achras Zapota Leaves and Nyctanthes Arbortristis Flowers on Organic Cotton Fabric with Bio Wash
Scouring was done using Sapindus seeds without using of chemicals. Sapindus seeds (Figures 4a and 4b) were collected from Coimbatore Agricultural University. The sapindus seeds were dried under shade and ground to fine powder. The scouring of organic cotton yarn was done in the bath with liquor ratio 1:20 with 5 g/l of sapindus seeds solution (soap nut) using laboratory winch machine. The yarn was loaded into the bath at 400°C to 500°C for 3hours.The yarn was then thoroughly washed with cold water and dried.
Introduction
Environmental issues are becoming more crucial all over world. Textile processing industry is characterized not only by the large volume of water required for various unit operations but also by the variety of chemicals used for various processes [1].
Organic cotton fabrics are generally understood as cotton that is grown from plants without chemical fertilizers or pesticides which are not genetically modified, though organic cotton has less environmental impact than conventional cotton and it costs more for its production [2,3].
The three herbs Eclipta Leaves, Achras Zapota Leaves and Nyctanthes Arbortristis Flowers were selected since Eclipta leaves are anti toxicity in nature, Zapota leaves demonstrated character of antioxidant activity and Nyctanthes Arbortristis flowers has antiviral and antifungal activities in vitro [9][10][11].
Today enzymes have become an integral part of textile processing. Enzyme application is inevitable tool in modern industry where environmental aspect plays critical role to sustain in the competitive market. Enzyme (Cellulase) treatment gives the fabric a smoother and glossier appearance [4][5][6][7][8].
Selection of yarn
Organic cotton was purchased in yarn stage from the yarn dealer, Erode, Tamil Nadu which is of 80s count since the end use is going to be used for baby wear. Fabric Specifications are shown in Table 1a.
Scouring
Scouring was done using Sapindus seeds without using of chemicals. Sapindus seeds (Figures 4a and 4b) were collected from Coimbatore Agricultural University. The sapindus seeds were dried under shade and ground to fine powder.
The scouring of organic cotton yarn was done in the bath with liquor ratio 1:20 with 5 g/l of sapindus seeds solution (soap nut) using laboratory winch machine. The yarn was loaded into the bath at 400°C to 500°C for 3hours.The yarn was then thoroughly washed with cold water and dried.
Bleaching
Catalase Enzyme is the bleaching agent which is chosen for the study to bleach the organic cotton yarn.
The bleaching of organic cotton yarn was done in the bath with liquor ratio 1:20 with catalase enzyme treated with 3% using laboratory winch machine. The bath was heated to 900°C and the yarns were bleached for 60 mins. The yarn was washed twice pin hot water and cold water.
Abstract
To rescue from the harmful effects of those chemical wastes the research work was focused on eco friendly natural dyes. The best in eco-friendly fabrics, "Organic cotton" was selected for the study. Extracted natural dyes from the selected natural resource (Eclipta Leaves, Achras Zapota Leaves and Nyctanthes Arbortristis Flowers) were applied onto organic cotton yarn with no using of chemicals & mordents. Enzymatic Bio wash has been further done to the naturally dyed organic cotton fabric. The colorfastness properties of natural dyed organic cotton fabric were observed and concluded. flowers have been collected from Coimbatore area and it is used as natural eco friendly dyeing agent. The specified three (Eclipta leaves, Achras Zapota leaves and Nyctanthes Arbortristis flowers) herbs were selected (Figures 5a-5c).
These three herbs were selected because of the following aspects as per the literature review, Eclipta leaves are anti toxicity in nature, Zapota leaves demonstrated character of antioxidant activity and Nyctanthes Arbortristis flowers has antiviral and antifungal activities in vitro.
Main active principle constituents of Eclipta leaves are coumestans such as wedelolactone and demethylwedelolactone, polypeptides, polyacetylenes, thiophene derivatives, steroids, triterpenes and flavonoids, Structure of wedelolactone is shown in Figure 1 and the structure of Zapota leaves in Figure 2.
Extraction of natural dye solution from Eclipta
250 gms of Eclipta leaves are shadow dried and ground well to fine powder. 1 liter of boiling water was added to the finely powdered dried eclipta leaves for about 2-3 hours. Then it is filtered using nylon cloth.
Extraction of natural dye solution from Achras Zapota
Achras Zapota fresh leaves were boiled in 1 litre of water for about 2-3 hours. Then it is filtered using nylon cloth.
Extraction natural dye solution from Nyctanthes Arbortristis
Nyctanthes Arbortristis fresh flowers were boiled in 1 litre of water for about 2-3 hours. Then the extracted dye solution is filtered using nylon cloth.
Application of Natural Dyes on Organic Cotton Yarn
The dyeing of organic cotton yarn was done in the bath with liquor ratio 1:20 with 10 gpl of dye solution using Laboratory winch dyeing machine. The yarn was loaded into bath at 900°C to 1200°C for 2-3
Hand loom weaving
The yarn is then woven into fabric using pit loom.
Bio wash
Cellulase Enzyme is the washing agent which is chosen for the study for the bio wash process of dyed organic cotton fabrics.
The bio washing of organic cotton fabrics was done in the bath with the liquor ratio 1:15 with 3 gpl of Cellulase Enzyme using laboratory washing machine. The fabric was loaded into the bath at 450°C to 550°C for 10 minutes. The sample washing was repeated for 5 times and then, the samples were rinsed three times in de-ionized water. Finally, the samples were left to dry at room temperature for 24 hours.
Evaluation of color fastness
Colorfastness to washing: Wash fastness of all dyed samples was measured by the ISO 105-C03 testing method. Dyed samples were taken, stitched with one of the shorter side of the adjacent bleached fabric and was put to the bath containing 3 gpl of soap, 2 gpl of sodium carbonate and 1:30 MLR ratio at 60°C for 30 minutes. Then the specimen was washed with hot water, cold water and then it was dried. Then the dried fabrics were evaluated for color change and staining using grey scale.
Colorfastness to rubbing: Rubbing fastness of all dyed samples was measured by dry and wet rubbing method. The dyed sample was fastened to the flat base of the crock meter and the bleached 100% cotton measuring 5 cm×5 cm was mounted on the rubbing finger. After mounting the samples, the handle was rotated to ten complete turns at the rate of one turn per second to slide the covered finger back and forth twenty times. Then both the dyed and bleached fabric was evaluated with the grey scale for color change and staining.
Colorfastness to sunlight: Sunlight fastnesses of all dyed samples were exposed to sun for a period of time and then compared with an unexposed sample. A sample size of 35 cm×12 cm was cut from dyed organic cotton fabrics. The sample was divided into nine equal parts and marking was made on it. The strip was covered with a black chart papers, marked with equal number of divisions. First division was cut and exposed on sun light. The second division was cut and exposed on sun light and so on. Finally the first division after exposure for seven days was assessed for colour change in comparison with the original using a grey scale rating.
Colour fastness to washing
Colour fastness to washing of Eclipta, Achras Zapota and Nyctanthes Arbortristis dyed organic cotton fabric is shown in the Table 1b. It is found that the dyed sample shows good colour fastness.
Colour fastness to rubbing
Colour fastness to rubbing of Eclipta, Achras Zapota and Nyctanthes Arbortristis dyed organic cotton fabric is shown in Table 2. It is found that the dyed sample shows good colour fastness.
Colour fastness to sun light
Colour fastness to Sunlight of Eclipta, Achras Zapota and Nyctanthes Arbortristis dyed Organic cotton fabric is shown in Table 3. It is found that the colour fastness to sunlight of dyed Eclipta, Achras Zapota and Nyctanthes Arbortristis performed to be best.
Conclusion
Organic cotton is an eco-friendly fiber that suits for children wear. The natural dyeing is carried out in yarn stage to enhance the durability of dyeing. Then bio wash is given to soften the fabric. | v3-fos-license |
2019-04-02T13:11:48.133Z | 2017-09-25T00:00:00.000 | 90380347 | {
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} | pes2o/s2orc | Influence of Hypoxia and Hypercapnia on Fatty Acid Composition of Lipids in White Muscles of Common Carp Cyprinus carpio
Study of the involvement of fish tissue fatty acids into the organism reactivity caused by an influence of exogenous factors is crucial for investigation of cell mechanisms underlying hypobiotic effect. Gas chromatography was used to estimate the composition and quantities of fatty acids of total lipids in white muscles of the Ukrainian scaly carp species. Twenty eight fatty acids were identified and their quantitative redistribution was revealed under hypoxic-hypercapnic action following a decrease in the environmental temperature (artificial hibernation). The decrease in total content of saturated and an increase in the one of unsaturated fatty acids was mainly due to the ω-3 and ω-6 polyunsaturated acids family. The optimum ω-3/ω-6 ratio under the studied conditions was supposedly maintained due to acyl-lipid ω-3 and ω-6-desaturase activity. Modified content of total lipids fatty acids in carp white muscles was likely a part of the cell mechanism of hypobiosis factors action in the fish organism.
Taking into account the habitat conditions in fish, the lipids play a significant role in biochemical adaptation [14][15][16][17], in particular, this concerns fatty acids (FAs), the most labile component of lipid molecules rapidly responding to exogenous environmental influences [16]. It should be noted that the involvement of fatty acids into adaptation, occurring in fish organism due to the effect of lowered temperature (hypothermia) [20,14,1], leads notably to an increased content of polyene fatty acids (especially ω-3 and ω-6 acids), providing thereby the required viscosity of biological cell membranes and maintaining the thermal homeostasis of the body [8,16]. However, it is necessary to consider the peculiarities of hypoxic-hypercapnic influence jointly with hypothermia on the lipid component of cells of various organs of animals, including fish [10].
Since fatty acids as structural components of biological membranes and energy substrates [7] are involved into the organism reactivity due to the effects of environmental factors [5,11], the study of the fatty acid profile of fish tissues being in hypobiosis, is perspective and relevant.
The research aim was to study the qualitative and quantitative composition of fatty acids of total lipids of Ukrainian scaly white carp under hypoxic-hypercapnic influences when the environmental temperature was reduced.
Materials and methods
Experiments were carried out in the Ukrainian scaly carps (Cyprinus caprio L.) weighing 250-270 g, which were obtained from Ivanivka Fish Processing Plant of the Kyiv region. The fish were caught in autumn and were kept in a 2,000 dm 3 pool for three days to adapt. The animals were divided into two groups (n = 5 each): 1 (control) -active fish; 2 (experimental) -fish under an artificial hypobiotic state (the influence of hypoxia, hypercapnia, with the decrease of environmental temperature) [12]. For the experiment the fish were placed into a closed glass aquarium with a water temperature of 8…10°C, supplied with the gas mixture of carbon dioxide and oxygen in 1:1 ratio for half an hour at a blowing rate of 150-200 cm 3 /min (at 50-100 dm 3 water). Under conditions of lowered temperature and increasing hypoxia-hypercapnia fish gradually switched to a hypobiotic state (suspended animation).
Fish of groups 1 (control) and 2 (at the 6 th and 24 th hour of exposure to artificial hypobiosis) were dissected and the white muscle tissues were extracted. Homogenization and extraction of lipids were performed поступово переходили у гіпобіотичний стан (знижена життєдіяльність).
with chloroform methanol mixture according to the Folch method [4]. The fatty acid methyl esters were prepared according W.W. Christi [2] and analyzed by means of Trace GC Ultra gas chromatograph (Thermo Scientific, USA). The separation was carried out with a high-polar capillary chromatographic column SPTM-2560 (Supelco, USA). Acids were identified using the standard mixture of methyl esters of fatty acids 37 Compone FAME Mix (Supelco) [3]. For quantification of individual FAs, the method of area normalization was used and the relative content of FAs was represented as the percentage to their total amount.
The obtained results were processed by the method of variation statistics using Student's t-criterion and Excel software (Microsoft, USA). Changes were considered as statistically significant at p < 0.05.
Investigation of the FA spectrum of total lipids of carp white muscles under hypoxic-hypercapnic effect found no qualitative changes in respect of the control of a pool of FA lipids, however, there was a redistribution of their content versus the control (Table 1). There was a decrease in total amount of UFAs, in particular due to a reduced content of С 14:0 , С 15:0 , С 16:0 , С 17:0 , С 18:0 , C 20:0 , C 21:0 , C 22:0 , C 23:0 , C 24:0 acids to the 6 th and 24 th hour of hypoxic-hypercapnic effect in average by 41.7 and 65.9%, respectively, relative to the control (Fig. 1). This may be due to their expenditure as an energy substrate [18,5].
Total content of UFAs was increased due to a rise in the level of MUFAs and PUFAs if compared with the control (Fig. 1). Under hypoxic-hypercapnic effect the unsaturation ratio (FAs/UFAs ratio) decreased to the 6 th and 24 th hour if compared to the control and was 0.23 and 0.12, respectively.
It is known that PUFAs are precursors of biologically active substances [8,1]. Arachidone ω-6 PUFA derivatives are a series of thromboxanes and leukotrienes that enhance the permeability of the membrane and cause inflammation, and the ω-3 PUFA metabolites, which are anti-platelets and anti-inflammatory agents, contribute to the stabilization of membranes. Therefore it is important to maintain the physiological ratio of ω3/ω6 PUFAs.
The results of our studies indicate that at the 6 th and 24 th hours of hypoxic-hypercapnic exposure on fish organism the ratio of (ω-3/ω-6) in case of white muscle lipid PUFAs decreased if compared with the control by 11.5 and 14.0%, respectively, along with a more significant rise in the content of ω-3 and ω-6 fatty acids. This dynamics may be related to desaturation and fatty acid elongation [20].
It is known that a change in the unsaturation degree of fatty acids (especially at the expense of PUFAs of ω-3 and ω-6 families) can occur due to the participation of acyl-lipid ω-3 and ω-6-desaturases, which perform the desaturation of fatty acids at positions 3 and 6, respectively [19]. Their activity is evidenced by the change in the values of desaturation indices (C 22:6ω3 / C 18:3ω3 and C 20:4ω6 /C 18:2ω6 ratio). The ratio C2 0:4ω6 / C 18:2ω6 , which shows the conversion rate of linoleic acid to arachidone one, for carp white muscles increased, and to the 6 th and 24 th hours of exposure was 0.36 and 0.42 respectively, whereas in the control group this index was 0.28. The ratio C 22:6ω3 /C 18: 3ω3 , which reflects the level of metabolism of the family ω-3 acids, decreased, and at the 6 th and 24 th hours of exposure was 3.77 and 2.88 respectively, while in the control group that was 4.76.
Consequently, the changes in activity of acyl-lipid ω-3 and ω-6-desaturase are observed in lipids of white carp muscles after hypoxic-hypercapnic effects. Similar changes in the activity of desaturases under artificial hypobiosis are found in other organs of fish [20,13,10]. The activity of these highly specific enzymes under the influence of external factors is likely aimed at maintaining an optimal (ω-3 / ω-6) ratio by control of PUFAs content.
Thus, since PUFAs are directly involved into regulation of the majority of cellular processes, the observed changes in the ω-3 and ω-6 families PUFA spectra under hypoxic-hypercapnic effect (artificial hypobiosis) can be considered as mobilization of body adaptive responses.
Conclusions
The performed studies of the FA spectrum of carp white muscles total lipids indicated a redistribution of the content of fatty acids under hypoxic-hypercapnic effect following a decrease in temperature (artificial hypobiosis), which led to a reduction of the saturated and an increase of unsaturated fatty acids content. The ω-3 and ω-6 PUFAs underwent the most prominent changes, in particular docosahexaenoic, eicosapentaenoic and arachidonic acids, characterized by a high metabolic activity. It is assumed that the optimal ω-3/ω-6 ratio could be maintained by acyl-lipid ω-3 and ω-6-desaturases, and this is a manifestation of biochemical adaptation. The revealed modifications of the content of FA lipids of white carp could be explained by the involvement of FAs into systems of reactivity of an organism under the effect of hypobiosis factors, that provided an optimal performance of all metabolic processes. модифікації вмісту ЖК ліпідів білих м'язів коропа можна пояснити залученням ЖК до систем реактивності організму за дії гіпобіотичних чинників, що забезпечує оптимальну роботу всіх метаболічних процесів. | v3-fos-license |
2020-01-09T09:10:42.588Z | 2020-01-01T00:00:00.000 | 210702351 | {
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} | pes2o/s2orc | Impact of Type and Enzymatic/High Pressure Treatment of Milk on the Quality and Bio-Functional Profile of Yoghurt
The objective of the present study was to investigate the effect of the high pressure (HP) processing and transglutaminase (TGase) treatment of bovine (cow) or ovine (sheep) milk, when applied individually or sequentially, on the quality parameters and anti-hypertensive and immunomodulatory properties of yoghurt. Low-fat (2% w/w) bovine or ovine milk samples were used. Results showed that HP treatment of milk led to acid gels with equivalent quality attributes to thermal treatment, with the more representative attributes being whey separation and firmness, which ranged from 47.5% to 49.8% and 23.8% to 32.2% for bovine and ovine yoghurt, respectively, and 74.3–89.0 g and 219–220 g for bovine and ovine yoghurt, respectively. On the other hand, TGase treatment of milk, solely or more effectively following HP processing, resulted in the improvement of the textural attributes of yoghurt and reduced whey separation, regardless of milk type, exhibiting values of 32.9% and 8.7% for the whey separation of bovine and ovine yoghurt, respectively, and 333 g and 548 g for the firmness of bovine and ovine yoghurt, respectively. The HP processing and TGase treatment of milk led to the preservation or improvement of the anti-hypertensive activity of the samples, especially in the case in which ovine milk was used, with Inhibitory activity of Angiotensin Converting Enzyme (IACE) values of 76.9% and 88.5% for bovine and ovine yoghurt, respectively. The expression of pro-inflammatory genes decreased and that of anti-inflammatory genes increased in the case of samples from HP-processed and/or TGase-treated milk as compared to the corresponding expressions for samples from thermally treated milk. Thus, it can be stated that, apart from the quality improvement, HP processing and TGase treatment of milk may lead to the enhancement of the bio-functional properties of low-fat yoghurt made from either bovine or ovine milk.
Introduction
Over the last decades, in order for consumers' demands for health-promoting food products to be satisfied, a large amount of research has been conducted in functional foods. These products include
•
Milk homogenization was performed in a single stage process at 150 bar; • Thermal treatment of milk was conducted at 95 • C for 5 min; • TGase (ACTIVA YG, Ajinomoto, DE) was inoculated at an enzyme concentration of 2.0 U·g −1 protein (reference activity: 100 U·g −1 ); • The commercial starter culture used was Yo-Mix (Danisco, DK) prepared as a 1:4 (w/w) dilution in commercial UHT (Ultra-High-Temperature) skim milk.
Fermentation Kinetics
A high precision pH meter (AMEL 338, AMEL Instruments, Milano, Italy) was used for pH measurements during the fermentation procedure. The pH of yoghurt samples during the fermentation process was continuously monitored using a 12 mm glass electrode (HI1131B, Hanna Instruments, Winsocket, RI, USA). The pH was recorded every 20 min at the beginning and every 10 min after 2.5 h of fermentation, until the end point of fermentation (pH 4.75). The pH values were plotted against time, and the parameters describing the fermentation kinetics were determined by fitting the data to the modified Gompertz equation (Equation (1)), as previously proposed by de Brabandere and de Baerdemaeker [26]: where pH∞ and pH 0 show the final (end point) and initial pH values respectively, µ shows the maximal acidification (pH drop) rate expressed in pH·min −1 , and λ is the duration of the lag phase (min).
Study of the Quality Characteristics of Yoghurt
The microbiological quality of the prepared samples was tested in weekly intervals with respect to total viable counts (TVC), yeasts and molds, and starter culture growth, as described in Tsevdou et al. [2]. Briefly, 10-fold serial dilutions of yoghurt samples were either spread or pour-plated in the appropriate growth media in Petri dishes for the enumeration of different microorganisms. Total viable counts were enumerated in Plate Count Agar (Merck, DE) after incubation at 25 • C for 72 h under aerobic conditions. Viable yeast and molds were enumerated on Rose Bengal Chloramphenicol (RBC) Foods 2020, 9, The acidity of yoghurt samples was measured using a pH meter (AMEL 338, AMEL Instruments, IT) and by the titration of a 1:1 mix of yoghurt/deaerated-deionized water with 0.1 N NaOH using phenolphthalein as an indicator, and expressed as % lactic acid [27].
The susceptibility of yoghurt to whey separation was determined using a drainage method and was expressed as the grams of separated whey from 100 g of sample after incubation at 4 • C for 3 h. Briefly, 100 g of yoghurt was transferred to a funnel with Whatman paper #1 placed on a conical flask. The flask was stored at 4 • C and the amount of eliminated serum was weighted after 3 h of storage.
Texture analysis was performed using a TA-XT Plus texture analyzer (Stable Micro Systems, Surrey, UK) and the microstructure of the prepared acid gels was examined with scanning electron microscopy (SEM), as previously described in Tsevdou et al. [2]. Briefly, for texture analysis, samples were tempered at 10 • C before testing, and then they were subjected to a double compression test using a clear acrylic cylinder probe TA3/1000 of 25.4 mm in diameter and 35 mm in length (Brookfield Viscometers Ltd., Harlow Essex, UK). For SEM analysis, samples were freeze-dried using a laboratory scale freeze-drying unit (Alpha 1-4LDplus, CHRIST, Germany) and then gold-palladium-coated in vacuum using a sputtering device (Polaron 5100). The microstructure was examined with a FEI Quanta 200 (FEI Company, Hillsborough, OR, USA) scanning electron microscope using a large-field detector (LFD) operating at 25 kV.
Preparation of Water-Soluble Extracts (WSEs)
Water-soluble extracts (WSEs) were obtained from all samples after 3 and 42 days of storage using the method proposed by Kuchroo & Fox [28]. Briefly, a mixture of 1:2 yoghurt/deionized water was prepared and homogenized in a Bag Stomacher (BagMixer Interscience, FR) for 10 min, followed by incubation in a water-bath of 45 • C for 1 h. The incubated samples were then centrifuged (Heraeus Megafuge 16R, Thermo Fischer Scientific, OR, USA) at 3000× g and 20 • C for 30 min. The supernatant was collected, vacuum-filtered and stored in a freezer until the analysis. According to the above procedure, one third (ca. 34%) of the total water-soluble nitrogen was extracted.
Determination of ACE-Inhibitory Activity
ACE-inhibitory activity was determined using the method proposed by Nakamura et al. [29], as modified by Donkor et al. [20]. Each assay mixture (250 µL) contained the following components at the indicated final concentrations: (a) 180 µL of a hippuryl-L-histidyl-L-leucine solution (5 mM) in sodium borate (100 mM) (pH 8.3), buffer-treated with 300 nM NaCl; (b) 50 µL of WSE or peak material or water as a blank; (c) 20 µL of a 2 mU ACE (from rabbit lung, Sigma-Aldrich, St. Louis, MO, USA) aqueous solution. The mixture was incubated at 37 • C for 90 min, and the reaction was stopped with 250 µL of 1 N HCl. The produced hippuric acid was extracted with 1.7 mL of ethyl acetate. In order to separate the organic phase, samples were centrifuged at 1000 rpm and 15 • C for 10 min. Samples were then heat-evaporated at 100 • C for 15 min, re-dissolved in 1 mL of distilled water and measured spectrophotometrically at 228 nm (SPECTROStar Nano, BMGLabtech, DE). The ACE-inhibitory (IACE) activity was expressed as the percentage according to the following equation: Foods 2020, 9, 49 5 of 16 where A is the absorbance of the ACE-buffer solution, B is the absorbance of the buffer solution, C is the absorbance of ACE in WSE, and D is the absorbance of the WSE-buffer solution.
Determination of Immunomodulatory Activity
The immunomodulatory activity of WSEs was evaluated by measuring their ability to modulate the expression of the pro-inflammatory genes IL1B, IL12B, NOS2 and PTGS2, and of the anti-inflammatory genes IL10 and TGFB1 by ovine monocytes. Ovine monocyte isolation, treatment with WSEs, RNA isolation and subsequent enzymatic reactions were performed as described by Theodorou and Politis [30].
Statistical Analysis
The data represent the means with their standard deviation of two independent experiments. Within each experiment, each treatment was performed in triplicate. A factorial ANOVA was applied for the determination of the main effects of the investigated factors (milk type, milk treatment, storage time) and their interactions on the experimental data. Duncan's multiple range test was used to separate the means of data when significant differences (p < 0.05) were observed. All statistical analyses were performed using Statistica ® release 7 software (StatSoft Inc., Tulsa, OK, USA).
Effect of Milk Type and Applied Treatment on the Fermentation Kinetics of Milk
The fermentation kinetic parameters (lag phase duration, λ, and maximal acidification rate, µ) were estimated with a modified Gompertz model (Equation (1)) and are presented in Table 2. The enzymatic treatment with TGase of bovine milk led to a significant (p < 0.001) reduction in the lag phase duration, whereas the HP treatment of both bovine and ovine milk led to a slight-yet significant (p < 0.01)-increase in the lag phase duration. However, the sequential application of the HP and TGase treatment of milk led to a pronounced reduction of the duration of the lag phase compared to samples from either thermally or HP-treated milk. Additionally, the milk type, which is directly related to compositional differences, had a significant (p < 0.001) effect on lag phase duration, as previously reported [31,32]. In particular, ovine yoghurt samples exhibited a decreased lag phase as compared to bovine samples, which might be related to the increased protein content of ovine milk and thus to the higher availability of free amino acids, which is related to the proteolytic activity of the starter culture [31]. In both bovine and ovine milk, TGase treatment led to a significant (p < 0.05) increase in the maximal acidification rate when HP treatment was previously applied. This behavior might be correlated with extended cross-linking among milk proteins and the increased availability of free amino acids for the proteolytic action of the starter culture microorganisms [33]. The HP treatment of milk also led to a significant (p < 0.01) increase in the maximal acidification rate, as compared to the corresponding value of thermally treated milk samples, regardless of the milk type, as a result of the protein unfolding and refolding induced by HP treatment [9,33].
Effect of Milk Type, Applied Treatment and Storage Time on the Quality Attributes of Yoghurt
According to the microbiological analyses, all samples exhibited growth below the detection limit (<2 logCFU/g) for yeasts and molds, whereas the total counts of the starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) remained above the acceptable limit of 7 logCFU/g, during the 42 days of storage, as also reported in previous studies [2,8].
With regard to acidity, it was found to be significantly (p < 0.01) dependent on milk type, TGase treatment of milk and storage time for all tested samples. In particular, in the case of bovine yoghurt samples, the initial pH ranged from 4.18-4.51 for samples from alternatively treated milk compared to 4.48 for samples from thermally treated milk (Table 3), and these values decreased to 4.18-4.21 and 4.12, respectively, after 42 days (data not shown). As has been also reported in previous studies [34,35], this is probably related to the slow acidity development induced by the delayed multiplication of the starter culture microorganisms. Similar results were observed in the case of ovine yoghurt samples, with the difference that, in general, ovine samples exhibited higher pH values than bovine samples, indicating that the type of milk, and consequently the milk composition, plays an important role in the development of quality attributes-e.g., post-acidification-in fermented products [31].
The pre-treatment of milk (thermally or HP), TGase treatment, milk type and storage time all had a significant (p < 0.001) effect on whey separation percentages. The HP treatment of milk led to either a similar or slight-yet significant (p < 0.05)-increase in whey separation as compared to samples from thermally treated milk for either bovine or ovine yoghurts ( Table 3). The subsequent thermal treatment and enzymatic cross-linking of milk proteins resulted in a significant (p < 0.001) decrease in whey percentages after 3 days of production as compared to samples from either thermally or HP-treated milk, regardless of the milk type. The decrease in syneresis, which is associated with an improvement in the protein network of the studied yoghurt samples [34], was even more pronounced in the case in which TGase was combined with a prior step of HP treatment. As a result, yoghurt samples from HP-TGase-treated milk exhibited the lowest percentages of whey separation (with statistical mean percentages of 32.9% and 8.76% for bovine and ovine samples, respectively). Similar results have been also previously reported by Anema et al. [36] and Tsevdou et al. [2], who identified that both simultaneous TGase treatment under HP conditions and/or the subsequent HP processing and TGase treatment of bovine milk are capable of causing the extensive denaturation of whey proteins and dissociation of micelles, reforming new intra-and inter-molecular cross-links and leading to strengthened networks with contiguous protein molecules and thus with reduced water permeability. With regard to the effect of milk type on the whey separation, previous studies in goat, sheep and cow milk showed that yoghurt gels prepared from ovine milk exhibit the lowest percentages of whey separation compared to yoghurts from either goat or bovine milk due to the different chemical composition of these milks regarding their proportion of the four major caseins [31]. The major textural attributes (firmness and adhesiveness) of both bovine and ovine yoghurt samples were found to be significantly (p < 0.001) dependent on all designed parameters-milk pre-treatment, TGase treatment, storage time, as well as milk type. The TGase treatment of milk, when applied individually-or to a greater extent subsequently to HP treatment-resulted in the highest values of firmness for all tested samples (Table 3). However, the HP treatment of milk led to a slight, but not significant, decrease in the firmness values of bovine samples as compared to those of samples prepared from thermally treated milk. Both the treatment of milk and milk type had effects in the same manner as in the case of whey separation on the firmness of the prepared yoghurt samples, which is related to both the protein denaturation and further cross-linking of the milk proteins and the different protein content of bovine and ovine milk. As expected, storage time had a significant (p < 0.001) effect on firmness for all tested samples-regardless of milk composition, milk type or the applied treatment in milk-as a result of the continuous starter culture multiplication during storage and thus the strengthening of the coagulum and an increase in firmness values.
Concerning the parameter of adhesiveness, this was found to be significantly (p < 0.001) dependent on all experimental designing factors, with the exception of storage time ( Table 3). The application of the HP treatment of bovine milk led to samples with high absolute values of adhesiveness (with a statistical mean value of −44.6 g·s), especially when combined with a subsequent step of TGase treatment (with a statistical mean value of −121 g·s). This observation had been previously correlated with a smoother and creamier sensorial perception of those products as compared to products prepared from thermally treated milk [37]. Yoghurt samples prepared from ovine TGase-treated milk exhibited the lowest absolute value of adhesiveness, which was previously correlated with a more gel-like sensorial perception of these products [2]. However, samples prepared from HP-TGase-treated milk exhibited lower value of adhesiveness (with a statistical mean value of −94.3 g·s) compared to samples from either thermally (with a statistical mean value of −152 g·s) or HP-treated (with a statistical mean value of −96.1 g·s) milk, but these values were higher than those of samples from TGase milk (with a statistical mean value of −80.1 g·s), suggesting that the HP treatment of milk maintains the smooth and creamy structure of the coagulum, while the combination with the TGase treatment of milk may also lead to the improvement of the protein network of acid gels.
In order to confirm our observations concerning the improvement of the protein network in our yoghurt samples prepared from alternatively treated milk, samples prepared from bovine milk were analyzed by scanning electron microscopy (Figure 1a-d). As illustrated in Figure 1a-b, the observed protein network of TGase-treated samples was more compact than that of thermally treated samples, which is related to the smaller porosity and strengthening of the protein network [34,38]. Similar improvements were observed in the case in which HP treatment was applied (Figure 1c). In the case of yoghurt prepared from HP-TGase treated milk, the samples exhibited an even tighter protein network as compared to all other samples (Figure 1d), revealing more continuous cross-links between protein molecules and fewer and smaller lacunae (depicted as black areas on SEM images) in comparison with samples prepared from HP or TGase-treated milk, thus supporting the hypothesis regarding the synergistic effect of the HP and subsequent TGase treatment of milk on its yoghurt-making properties.
improvements were observed in the case in which HP treatment was applied (Figure 1c). In the case of yoghurt prepared from HP-TGase treated milk, the samples exhibited an even tighter protein network as compared to all other samples (Figure 1d), revealing more continuous cross-links between protein molecules and fewer and smaller lacunae (depicted as black areas on SEM images) in comparison with samples prepared from HP or TGase-treated milk, thus supporting the hypothesis regarding the synergistic effect of the HP and subsequent TGase treatment of milk on its yoghurtmaking properties.
Effect of Milk Type, Applied Treatment and Storage Time on the Anti-Hypertensive Activity of Yoghurt
The anti-hypertensive activity of the prepared yoghurt samples (as expressed through % IACE) was found to be significantly (p < 0.001) dependent on all designed parameters-milk pre-treatment, TGase treatment, storage time, as well as milk type ( Figure 2).
Effect of Milk Type, Applied Treatment and Storage Time on the Anti-Hypertensive Activity of Yoghurt
The anti-hypertensive activity of the prepared yoghurt samples (as expressed through % IACE) was found to be significantly (p < 0.001) dependent on all designed parameters-milk pre-treatment, TGase treatment, storage time, as well as milk type ( Figure 2).
Ovine WSEs samples exhibited significantly (p < 0.001) higher %IACE percentages than the bovine WSEs samples (with statistical mean percentages of 76.9% and 88.5% for bovine and ovine WSEs, respectively), which is in agreement with previous observations on commercially available fermented products made from thermally treated bovine or ovine milk, suggesting that the proteolysis of ovine milk probably leads to the release of peptides which are more capable of inhibiting the ACE-system [21,39]. Regarding the HP treatment of milk, it led to a significant (p < 0.001) increase in %IACE as compared to the corresponding percentage of samples from thermally treated milk, especially when combined with a subsequent enzyme treatment (with statistical mean percentages of 78.4%, 83.9% and 87.9% for samples from thermally, HP and HP-TGase-treated milk, respectively). Concerning the effect of storage period on the anti-hypertensive activity, it was observed that there is a noteworthy synergistic effect of both storage time and milk type, as samples prepared from different milk samples behaved differently, with samples from HP and HP-TGase-treated milk showing the best preservation or even enhancement of anti-hypertensive activity of yoghurt samples prepared from ovine and bovine milk, respectively. To date, there have been no studies on the effect of either the HP or TGase treatment of milk on the anti-hypertensive activity of fermented milk products, and therefore, a more detailed analysis of specific peptidic fractions with ACE-inhibitory activity may reveal important information regarding the mechanism of these effects on the release of bioactive peptides. Foods 2020, 9, x FOR PEER REVIEW 10 of 16 Ovine WSEs samples exhibited significantly (p < 0.001) higher %IACE percentages than the bovine WSEs samples (with statistical mean percentages of 76.9% and 88.5% for bovine and ovine WSEs, respectively), which is in agreement with previous observations on commercially available fermented products made from thermally treated bovine or ovine milk, suggesting that the proteolysis of ovine milk probably leads to the release of peptides which are more capable of inhibiting the ACE-system [21,39]. Regarding the HP treatment of milk, it led to a significant (p < 0.001) increase in %IACE as compared to the corresponding percentage of samples from thermally treated milk, especially when combined with a subsequent enzyme treatment (with statistical mean percentages of 78.4%, 83.9% and 87.9% for samples from thermally, HP and HP-TGase-treated milk, respectively). Concerning the effect of storage period on the anti-hypertensive activity, it was observed that there is a noteworthy synergistic effect of both storage time and milk type, as samples prepared from different milk samples behaved differently, with samples from HP and HP-TGasetreated milk showing the best preservation or even enhancement of anti-hypertensive activity of yoghurt samples prepared from ovine and bovine milk, respectively. To date, there have been no studies on the effect of either the HP or TGase treatment of milk on the anti-hypertensive activity of fermented milk products, and therefore, a more detailed analysis of specific peptidic fractions with ACE-inhibitory activity may reveal important information regarding the mechanism of these effects on the release of bioactive peptides.
Effect of Milk Type, Applied Treatment and Storage Time on the Immunomodulatory Properties of Yoghurt
The immunomodulatory properties of WSEs were found to be significantly (p < 0.001) dependent on milk type and milk pre-treatment, whereas storage time did not affect the relative expression of the tested genes by ovine monocytes, with the exception of the pro-inflammatory IL12B (Table 4). For all tested genes, their expression by monocytes was significantly (p < 0.001) higher when cells were treated with ovine WSEs than when treated with bovine WSEs (Table 4). Table 4. Immunomodulatory properties of bovine and ovine yoghurt WSEs as evaluated though the expression of pro-inflammatory and anti-inflammatory genes by ovine monocytes, and analyzed after 3 (D+3) and 42 (D+42) days of yoghurt sample production. Milk Type (MT) *** *** *** *** *** *** Milk Treatment (MT) *** *** *** *** *** *** Storage Time (ST) n.s. n.s. *** n.s. n.s. n.s. MO × MT × ST *** *** *** *** *** *** For all tested genes, their expressions were found not to be affected by the applied processing of the milk, with the exception of TGFB1, where HP-applied individually or in combination with a subsequent step of enzymatic treatment-led to a significant (p < 0.001) decrease in expression (Table 4). Additionally, the fact that the expression of the pro-inflammatory NOS2 was not affected by the applied treatment in milk is important, as this gene is associated with the defense mechanism in humans (e.g., attack by parasites, bacterial infections, tumors growth, etc.) and also plays a critical role in many diseases with an autoimmune etiology [40]. With regard to IL10 gene expression, although no differences were observed among samples after 3 days of production, at the end of the storage period (42 days), its relative expression was significantly (p < 0.001) higher as compared to its initial expression only in the case in which ovine milk was subjected to both HP and TGase treatment.
IL1B
Concerning the effect of ovine WSEs on the relative expression of pro-inflammatory genes by ovine monocytes, it was observed that the treatment of monocytes with WSEs from yoghurts produced with TGase-treated milk led to a significant (p < 0.001) decrease in both IL12B and IL1B expressions (Table 4), whereas treatment with WSEs from yoghurts produced with HP treatment of milk led to a significant (p < 0.05) decrease in NOS2. In the case of PTGS2, no differences were detected among samples. Similar results have been previously reported by Cermẽno et al. [41], who found that the addition of TGase prior to and after the hydrolysis of sodium caseinate showed a significant decrease in the release of the pro-inflammatory gene IL-6.
Considering that the levels of IL12B and IL1B are associated with auto-inflammatory syndromes [23] and diseases of the nervous system [22], respectively, their expression is required to be maintained at low levels. Regarding the relative expression of the tested anti-inflammatory genes by ovine monocytes, it was observed that each gene was influenced differently by the applied treatment in milk. The HP processing of milk led to a significant (p < 0.05) increase in TGFB1 expression (Table 4, with a statistical mean value of 3.058), whereas it led to a significant (p < 0.05) decrease in IL10 expression (Table 4, with a statistical mean value of 2.491), as compared to the corresponding values of monocytes treated with WSEs from thermally treated milk samples (with statistical mean values of 2.448 and 3.052 for TGFB1 and IL10, respectively). The enhancement of TGFB1 expression is desirable as it plays an important role in controlling the immune system and has a key role in the resolution of inflammation [42]. It is also worth noting that both bovine and ovine WSE, affected the expression of pro-and anti-inflammatory cytokines in a way that indicates that yoghurts from both ovine and bovine milk exhibit mostly anti-inflammatory properties.
Principal Component Analysis
In order to further evaluate the effect of the milk type and milk treatment on the quality and bio-functional properties of yoghurt samples, the data obtained from the physicochemical (pH, serum, protein), textural (firmness, adhesiveness) and bio-functional (%IACE, IL1B, IL12B, PTGS2, IL10, TGFB1, NOS2) attributes of the prepared yoghurt samples at D+3 were submitted to principal component analysis (PCA). According to the PCA biplot (Figure 3), principal components (PC) 1 and 2 accounted together for 72.7% of the total explained variance.
The capability of coagulum to retain water (serum), adhesiveness and TGFB1 gene expression was positively correlated with PC1, whereas firmness, anti-hypertensive activity (%IACE) and IL1B gene expression were negatively correlated with the same principle component. Moreover, the gene expression of PTGS2 was negatively correlated with PC2, whereas protein content, and IL12B and IL10 gene expression were positively correlated with PC2. With regard to NOS2 gene expression, according to the estimated table of factor coordinates of variables, it was found to be strongly and negatively correlated with principal component 4 (representing 8.3% of total variance). Moreover, it was observed that milk type was correlated to PC2, whereas milk treatment was strongly correlated to PC1 (as shown by vectors). expression of PTGS2 was negatively correlated with PC2, whereas protein content, and IL12B and IL10 gene expression were positively correlated with PC2. With regard to NOS2 gene expression, according to the estimated table of factor coordinates of variables, it was found to be strongly and negatively correlated with principal component 4 (representing 8.3% of total variance). Moreover, it was observed that milk type was correlated to PC2, whereas milk treatment was strongly correlated to PC1 (as shown by vectors). From the PCA biplot, it is obvious that milk type and the kind of applied treatment in milk affect the quality profile of yoghurt, resulting in a clear classification of bovine and ovine yoghurt samples into two main groups, with the use of ovine milk promoting the production of samples with improved firmness, enhanced anti-hypertensive activity and immunomodulatory properties. Additionally, according to the sample distribution, it is evident that differentiations in milk treatment led to the production of yoghurt samples with completely different characteristics. Consequently, the implementation of the TGase and/or HP-TGase treatment of milk led to the production of acid gels with reduced whey separation, improved firmness and enhanced bio-functional characteristics, From the PCA biplot, it is obvious that milk type and the kind of applied treatment in milk affect the quality profile of yoghurt, resulting in a clear classification of bovine and ovine yoghurt samples into two main groups, with the use of ovine milk promoting the production of samples with improved firmness, enhanced anti-hypertensive activity and immunomodulatory properties. Additionally, according to the sample distribution, it is evident that differentiations in milk treatment led to the production of yoghurt samples with completely different characteristics. Consequently, the implementation of the TGase and/or HP-TGase treatment of milk led to the production of acid gels with reduced whey separation, improved firmness and enhanced bio-functional characteristics, whereas the individual application of HP in milk led to products with similar technological characteristics and slightly enhanced bio-functional characteristics compared to those of samples from thermally treated milk.
Conclusions
Modern consumer demands are related not only to high nutritional value foods but also foods with functional properties. These foods include yoghurt produced with alternative technologies, such as the high pressure and transglutaminase treatment of milk and milk proteins, respectively. Moreover, yoghurt made from both bovine and-especially-ovine milk is known for its functional properties. Previous studies have shown that the application of HP and/or TGase treatment of milk during yoghurt manufacturing could be more inexpensive compared to the addition of external protein sources in the pursuit of the formation of a stable and compact acid coagulum and to overcome the common problems of non-fat or low-fat dairy products (e.g., weak texture, phase separation, sensorial defects, etc.). This study revealed that, apart from the improvement of the quality characteristics of yoghurt, these technologies led to the maintenance or even improvement of the bio-functional profile of either bovine or ovine yoghurt. More specifically, yoghurt prepared from bovine HP-TGase-treated and ovine HP-treated milk exhibited the greatest ACE-inhibitory activity, even after 42 days of storage. In addition, yoghurt prepared from TGase-treated milk exhibited the best results in eliciting the same levels of expression of important anti-inflammatory genes, such as TGFB1, while at the same time leading to reduced levels of the expression of pro-inflammatory genes, such as IL1B and IL12B from monocytes. With regard to milk type, it was observed that yoghurt produced from ovine milk had higher anti-hypertensive and immunomodulatory properties than yoghurt from bovine milk, regardless of the applied treatment in milk. Nevertheless, further research needs to be performed in specific peptidic fractions with anti-hypertensive activity and in simple systems of different types of milk in order to identify the mechanisms of the effect of HP and TGase treatment of milk on the IACE and immunomodulatory activity of fermented dairy products. | v3-fos-license |
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} | pes2o/s2orc | The C-Terminal O-S Acyl Shift Pathway under Acidic Condition to Propose Peptide-Thioesters
Peptide-thioester is a pivotal intermediate for peptide ligation and N-, C-terminal cyclization. In this study, desired pathway and the side products of two C-terminal handles, hydroxyethylthiol (HET) and hydroxypropylthiol (HPT) are described in different conditions as well as kinetic studies. In addition, a new mechanism of C-terminal residue racemization is proposed on the basis of differentiation of products derived from the two C-terminal handles in preparing peptide thioesters through an acid-catalyzed tandem thiol switch, first by an intramolecular O-S acyl shift, and then by an intermolecular S-S exchange.
Results and Discussion
In the study for the effect of acidity in the reaction medium, a very important factor for O-S acyl shift is monitoring the TFA conditions without TfOH, such as 5% Thiocresol-TFA (TC-TFA) or TFA alone, by TR6-HET disappearance ( Figure 1A). The conditions gave very slow disappearance rates, even though the 5% TC-TFA condition accelerated a little after 30 min. Furthermore, the main conversion product within a short period of time in both conditions (Figures S1 and S2) was the racemized TIGGIR-OEtSH (TR6-HET), which was confirmed through direct synthesis ( Figure S5), while the 5% TC-TFA condition led to CF3CO-acetylated products after a long period ( Figure S2). These results were consistent with previous findings [43] that the HET handle of peptide ester 1 requires an increase in acidity to protonate the ester carbonyl for the thioester conversion. However, a systematic approach for O-S acyl shift has not been fully established because the core structures for O-S acyl shift were susceptible to side reactions such as hydrolysis and C-terminal racemization [31,32,37,40], which have been found in both intermolecular [31,32,37] and intramolecular [33][34][35][36][38][39][40] methods using various metal-mediated [31,32], acidic [33,37,43], neutral [35], and basic [34,36,[38][39][40][41][42] conditions. Similar with other studies [31][32][33]37,40] the two critical problems from TIGGIR-OEtSH (TR6-HET) 3 which were encountered, are the alkylation of 8, the guanidino side chain of Arginine (Arg) by ethylene sulfide 5 (Scheme 2) and the C-terminal racemization (Scheme 3). Due to lack of reasonable approaches, it had been difficult to explain the side products pathway from an application-oriented design with only the HET handle structure. In the present study, the product pathway is demonstrated using HET and hydroxypropylthiol (HPT) handles as well as the kinetic studies, and a new mechanism of C-terminal racemization which was deduced from the results of the universal C-terminal handles in the preparation of TR6-thioester 6 is proposed.
Results and Discussion
In the study for the effect of acidity in the reaction medium, a very important factor for O-S acyl shift is monitoring the TFA conditions without TfOH, such as 5% Thiocresol-TFA (TC-TFA) or TFA alone, by TR6-HET disappearance ( Figure 1A). The conditions gave very slow disappearance rates, even though the 5% TC-TFA condition accelerated a little after 30 min. Furthermore, the main conversion product within a short period of time in both conditions (Figures S1 and S2) was the racemized TIGGIR-OEtSH (TR6-HET), which was confirmed through direct synthesis ( Figure S5), while the 5% TC-TFA condition led to CF 3 CO-acetylated products after a long period ( Figure S2). These results were consistent with previous findings [43] that the HET handle of peptide ester 1 requires an increase in acidity to protonate the ester carbonyl for the thioester conversion. To examine the effect of further increase in acidity, the first-order rates of TR6-HET in TFA and 5% TC-TFA conditions were compared with 0.1%, 0.25%, and 0.5% TfOH (v/v) additions. The experiment gave rise to distinct rates of 1,2-elimination and O-S acyl shift, while the main products detected were peptide-TCs in TC-TfOH-TFA (Scheme 2A,B), peptide-TFA adducts in TfOH-TFA ( Figure S2), and peptide-OHs in TFA only ( Figure S1). The 1,2-elimination progressed relatively slowly, compared to To examine the effect of further increase in acidity, the first-order rates of TR6-HET in TFA and 5% TC-TFA conditions were compared with 0.1%, 0.25%, and 0.5% TfOH (v/v) additions. The experiment gave rise to distinct rates of 1,2-elimination and O-S acyl shift, while the main products detected were peptide-TCs in TC-TfOH-TFA (Scheme 2A,B), peptide-TFA adducts in TfOH-TFA ( Figure S2), and peptide-OHs in TFA only ( Figure S1). The 1,2-elimination progressed relatively slowly, compared to To examine the effect of further increase in acidity, the first-order rates of TR6-HET in TFA and 5% TC-TFA conditions were compared with 0.1%, 0.25%, and 0.5% TfOH (v/v) additions. The experiment gave rise to distinct rates of 1,2-elimination and O-S acyl shift, while the main products detected were peptide-TCs in TC-TfOH-TFA (Scheme 2A,B), peptide-TFA adducts in TfOH-TFA ( Figure S2), and peptide-OHs in TFA only ( Figure S1). The 1,2-elimination progressed relatively slowly, compared to the five-membered O-S acyl shift ( Figure 1B). Furthermore, the S-S exchange seems to be a crucial step to isolate the stable thioester peptides; since otherwise the 1,2-elimination which produces the acid form, becomes more dominant than the unstable 5-membered O-S acyl shift. The catalytic TfOH additions into peptide-HET allowed a similar product profile with subtle differences in a broad range (0.1%-0.5%) instead of the expected narrow optimal point (Table S1).
The kinetic behavior of five-and six-membered O-S acyl shifts was comparatively examined on the basis of disappearance rates of the starting materials. The model peptide handles, TR6-HET for five-membered acyl shift and TR6-HPT for six-membered acyl shift, were constructed, using the same peptide sequence (TIGGIR), to perceive only structural effect of C-terminal handle. It showed that TR6-HET was approximately twice as fast as TR6-HPT ( Figure 1B), even though the product profiles from the two handles were only minimally different (Figure 2). Though it means that the five-membered acyl shift of HET handle is faster, it is more feasible to the production of side products than the six-membered acyl shift of HPT handle. Furthermore, a minute structural difference at the C-terminal residue between two peptides TR(r)6-HET with land d-Arg, showed that l-Arg was faster than d-Arg ( Figure 1B). The C-terminal difference, however, only slightly affected the disappearance of the initial compound without changing the product pathway ( Figure S5). Figure 1B). Furthermore, the S-S exchange seems to be a crucial step to isolate the stable thioester peptides; since otherwise the 1,2-elimination which produces the acid form, becomes more dominant than the unstable 5-membered O-S acyl shift. The catalytic TfOH additions into peptide-HET allowed a similar product profile with subtle differences in a broad range (0.1%-0.5%) instead of the expected narrow optimal point (Table S1). The kinetic behavior of five-and six-membered O-S acyl shifts was comparatively examined on the basis of disappearance rates of the starting materials. The model peptide handles, TR6-HET for fivemembered acyl shift and TR6-HPT for six-membered acyl shift, were constructed, using the same peptide sequence (TIGGIR), to perceive only structural effect of C-terminal handle. It showed that TR6-HET was approximately twice as fast as TR6-HPT ( Figure 1B), even though the product profiles from the two handles were only minimally different (Figure 2). Though it means that the fivemembered acyl shift of HET handle is faster, it is more feasible to the production of side products than the six-membered acyl shift of HPT handle. Furthermore, a minute structural difference at the C-terminal residue between two peptides TR(r)6-HET with l-and d-Arg, showed that l-Arg was faster than d-Arg ( Figure 1B). The C-terminal difference, however, only slightly affected the disappearance of the initial compound without changing the product pathway ( Figure S5). From product differentiation and kinetic studies of the two handles, two main problems could be explained: alkylation at the Arg site and racemization at the C-terminal in the acidic conditions as mentioned in a previous study [43]. The alkylation gave a reversible side product of the guanidino side chain of Arg from TR6-HET and Tr6-HET by ethylene sulfide, forming Tr(EtSH)6-TC and TR(EtSH)6-TC (peaks 6, 7 in Figure 2A) while ca. 60% of TR(r)6-TC products was obtained (peaks 4 and 5 in Figure 2A). However, only 11% of TR6-HPT side products (peaks 9 and 10 in Figure 2B) were obtained from TR(r)6-TC products (peaks 6 and 7 in Figure 2B). It might be a reasonable explanation that the formation of propylene sulfide through the four-member ring intermediate of the HPT handle is substantially slower than that of the ethylene sulfide, through the three-member ring intermediate or, the released mercaptoethanol from the 1,2-elimination (4 and 10→5 in Scheme 2).
Racemization of O-S acyl shift has been one of the major concerns in the conversion steps of the oxazolone pathway and peptide ligation [31][32][33]37,43]. Normally, the pathway (Scheme 3A) was established in a manner that the activated peptide esters are prone to racemization at the C-terminal residue via an oxazolone intermediate [37,[44][45][46] even though this is unclear under some conditions [31,32]. However, a possible oxazolone pathway involving intermolecular interactions was not suitable to explain the racemization, since the conversion rate order was: racemization > O-S acyl shift > S-S exchange for TR6-HET and O-S acyl shift ≥ S-S exchange > racemization for TR6-HPT ( Figures S3 and S4). Thus, it could not support the fact that no effect from other conditions except the different handle structure was there. The other rational argument against the oxazolone pathway was that Tr6-HET was detected at an early reaction time (peaks 3 and 5 in Figure S3, 5 min). This observation was not applicable to the oxazolone pathway because the released 2-mercaptoethanol has not to act as a nucleophile instead of thiocresol, which is in excess and appears as a better nucleophile in acid. Therefore, a plausible mechanism can be proposed for the C-terminal racemization on the basis of differentiation of the two handles that could play a dual role as both a nucleophile and base in the acidic conditions.
The proposed racemization mechanism is 7-and 8-membered ring interactions between the α-proton of C-terminal Arg, and thiol handle groups as a base (Scheme 3B). The hypothesis was that the α-proton should become more susceptible in an approaching motion of the thiol handles, which are proximal to the C-terminal carbonyl group and in cis configuration for O-S acyl shift. The situation would cause a fast enol formation in TR6-HET, but a much slower one in TR6-HPT, which is a more flexible handle. The proposed mechanism was well matched with Gellman's study [47] where a restricted beta-peptide with Z configuration presented a strong NOE interaction in 6-and 7-membered rings. Further evidence of the 7-membered ring interaction has been established to give rise to a folding evidence, by the internal hydrogen bond in a small peptide-like structure [48].
Synthesis of Handles
All reagents and solvents were obtained from commercial suppliers and were used without further purification. Side-chain protected Fmoc amino acids used in the experiment were d-Arginine (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) to make the coupling complete. A triple coupling method was used, with a longer reaction time at each coupling to load the first amino acid on the resin. After chain assembly was completed, the peptides were deprotected and cleaved from the resins (250 mg, each) by treatment with reagent K (TFA/thioanisole/PhOH/H 2 O/EDT/TIS: 81.5/5/5/5/2.5/1) for 2 h for TR6-HET and TR6-HPT at room temperature. The crude peptides were precipitated with diethyl ether, dissolved in aqueous acetonitrile, and purified by preparative RP-HPLC on a Waters 600 HPLC module, using a C18 Phenomenex preparative column (250 mm × 10 mm, 10 µ). The yield of two handles was 70%-80%. The molecular weight of peptides was confirmed by 4800 MALDI TOF/TOF Analyzer from Applied Biosystems.
Tandem Switch Experiment for O-S Acyl Shift
The conversion of tandem switch for 5-and 6-membered O-S acyl shift was conducted in 2-3 mg peptides within 1 mL total volume including TFA, 0.05%-1.5% TfOH as a freshly pre-made solution of 10% TfOH/TFA by volume, and 5% thiocresol (50 µL) at room temperature for 2 h, 4 h, and more. The remaining thiocresol was removed by ether precipitation twice. The yield of all peptide-TC products was obtained by analytical RP-HPLC purification on a Shimadzu 10 series module with Diode Array UV detection, using a Vydac C18 column, with a linear gradient of 2% buffer B to 100% buffer B for 40 min and retained for 10 min in 100% buffer B after 40 min (Buffer A = 0.05% TFA in water; buffer B = 0.045% TFA in 60% acetonitrile in water). However, the area percentage of HPLC for other side products of small amounts such as "before 22 min" and "after 34 min" was employed (Supplementary Materials Table S1). The molecular weight of all products was confirmed by 4800 MALDI TOF/TOF Analyzer from Applied Biosystems.
Conclusions
In summary, the O-S acyl shift of peptide-HET and peptide-HPT was dependent on acidity and the protonation state of the ester carbonyl. Addition of a catalytic TfOH accelerated the O-S acyl shift to afford a thioester. The addition of an external thiol into the reaction mixture permitted the second "thiol switch" reaction and favored the formation of stable thioester 6 (peak 5 in Figure 2) by terminating the equilibration of the acyl migrations. The design of the handle difference provides a new finding that the thiol groups can be used as a base in the acidic conditions. The C-terminal HPT handle for O-S acyl shift could be a more promising structure to reduce alkylation and racemization even though both handles were manipulated from convenient starting materials which are simple and feasible to provide thioester peptides compatible with Fmoc chemistry. However, the acidic effect on structures of other C-terminal amino acids and longer peptide sequences will be further studied to establish a relationship between O-S acyl shift and side products. | v3-fos-license |
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} | pes2o/s2orc | S-nitrosylation of ARH is required for LDL uptake by the LDL receptor.
The LDL receptor (LDLR) relies upon endocytic adaptor proteins for internalization of lipoproteins. The results of this study show that the LDLR adaptor autosomal recessive hypercholesterolemia protein (ARH) requires nitric oxide to support LDL uptake. Nitric oxide nitrosylates ARH at C199 and C286, and these posttranslational modifications are necessary for association of ARH with the adaptor protein 2 (AP-2) component of clathrin-coated pits. In the absence of nitrosylation, ARH is unable to target LDL-LDLR complexes to coated pits, resulting in poor LDL uptake. The role of nitric oxide on LDLR function is specific for ARH because inhibition of nitric oxide synthase activity impairs ARH-supported LDL uptake but has no effect on other LDLR-dependent lipoprotein uptake processes, including VLDL remnant uptake and dab2-supported LDL uptake. These findings suggest that cells that depend upon ARH for LDL uptake can control which lipoproteins are internalized by their LDLRs through changes in nitric oxide.
DTT reduction assay
Cell lysate (20 µg) was incubated with DTT (0 to 10 mM) at room temperature for 30 min. Lysates were then mixed with protein loading buffer, separated on SDS-PAGE, and immunoblotted for ARH using the 4034 antibody.
Generation of mouse embryonic fi broblasts lacking both ARH and dab2 expression
Fibroblasts from Arh Ϫ / Ϫ ;Dab2 fl ox/fl ox ;hLDLR +/+ mice were kindly provided by Dr. Helen Hobbs. These cells were infected with adenovirus encoding Cre-recombinase at an MOI of 500. Cells were then clonally selected in 96-well plates for cells that lack expression of dab2 (Arh Ϫ / Ϫ ;Dab2 Ϫ / Ϫ ;hLDLR +/+ fi broblasts). An outline of the procedure is shown in Fig. 2 .
Generation of expression plasmids encoding ARH variants and transfection into 293 cells
ARH variants were generated using Quickchange II XL sitedirected mutagenesis kit (Stratagene) and cloned into pCDNA3 mammalian expression vector. Then 293 cells were transfected with 1 g of plasmid per well in 6-well dishes. Cells were cultured for two days and then lysed with lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Triton X-100]. Lysates were run on SDS-PAGE and immunoblotted using either the V5 mAb (Invitrogen) or the ARH mAb (Santa Cruz Biotechnology).
Generation of ARH retroviruses and ARH-expressing cell lines
Human ARH variants were generated using Quickchange II XL site-directed mutagenesis kit (Stratagene), and cloned into the pMX bicistronic retroviral vector ( 21 ). These vectors use the 5 ′ UTR of the virus to drive mRNA expression. The multicloning site is 5 ′ to the internal ribosome entry site (IRES), while GFP is encoded 3 ′ to the IRES element. Retroviral vectors were cotransfected with the pAmpho vector (Clontech) into 293T cells to produce infectious, replication-defective retroviruses. These viruses were used to infect Arh Ϫ / Ϫ ;Dab2 Ϫ / Ϫ ;hLDLR +/+ mouse fi broblasts. Because both ARH and GFP are encoded on the same viral mRNA, GFP expression correlates with ARH expression, allowing ARH-expressing cells to be isolated by fl uorescence-activated cell sorting (FACS). MOI for ARH virus infection was kept low (<5% cell infection) to enrich for cells with only single genomic integration events. Single integration yields populations of cells with similar mRNA production and hence similar protein expression ( 21 ).
LDL initial rate endocytosis assay
Endocytic rates of lipoprotein internalization were determined as previously described ( 10,22 ). Briefl y, cells were incubated with 10 g/ml 125 I-LDL for 1 h at 4°C in MEM medium [MEM supplemented with 10% fetal lipoprotein-poor serum (FLPPS)]. Media was changed for the times indicated with warm DMEM medium (high-glucose D-MEM supplemented with 10% FLPPS) ( 19,20 ), indicating that dab2 could support LDL uptake in these cells were it expressed.
Here we show that the activity of ARH is regulated by nitric oxide. Nitric oxide nitrosylates ARH at two cysteines, and nitrosylation is necessary for ARH to support LDL uptake by the LDLR. Nitric oxide is not required for either VLDL remnant uptake or dab2-supported LDL uptake. We suggest that the ability of ARH to be regulated is the reason why hepatocytes and leukocytes normally use ARH and not dab2 for LDL uptake.
Materials
Human LDL and rabbit  -VLDL were provided by Drs. Michael Brown and Joseph Goldstein (UT Southwestern). Rabbit polyclonal Identifi cation of a posttranslational modifi cation in the C-terminal half of ARH. HEK293 cells were transfected with C-terminal V5-tagged human ARH constructs encompassing the entire ARH protein (WT-V5), residues 1-187 (NT-V5), or residues 188-308 (CT-V5), and then lysed. Lysates were run on SDS-PAGE and immunoblotted for V5. (C) Posttranslational modifi cation resides between residues 284 and 292. HEK293 cells were transfected with untagged human ARH variants bearing premature stop codons at the indicated residue positions and lysed. Lysates were run on SDS-PAGE and immunoblotted for ARH. (D) C286 is posttranslationally modifi ed. HEK293 cells were transfected with human ARH variants bearing alanine mutations at the indicated positions and lysed. Lysates were run on SDS-PAGE and immunoblotted for ARH. (E) C286 participates in an intramolecular disulfi de bond. Lysates of normal human fi broblasts were treated with the indicated concentrations of DTT for 30 min at room temperature, run on SDS-PAGE, and immunoblotted for ARH. (F) C286 forms a disulfi de bridge with C199, and both C199 and C286 are nitrosylated. The C-terminal 121 residues of human ARH have two cysteines, C199 and C286. HEK293 cells were transfected with the indicated ARH variants and lysed. Lysates were divided into two portions. The fi rst portion was processed by biotin switch to label nitrosylated cysteines with biotin. Biotinylated proteins were precipitated with neutravidin agarose and run with the second, untreated portion of the lysate on SDS-PAGE and immunoblotted for ARH. CC>AA indicates the ARH variant with both the C199A and C286A mutations.
were incubated with the cells for 1-4 h. Cells were harvested hourly, washed with PBS, fi xed with 3% paraformaldehyde, and held on ice for fl ow cytometry. Mean cellular fl uorescence from 10,000 cells per time point was determined using a BD FACScalibur. As a negative control, all assays included cells without FLPPS treatment. Uptake of both LDL and  -VLDL by cells expressing wild-type (WT) ARH increased ف 20-fold following LPPS treatment and was consistent with the fold induction of LDLR expression. In all reported data, the uptake by cells without FLPPS treatment was subtracted from FLPPS-treated cells at each time point. Relative rates of uptake were determined by linear regression analysis using Prism 4.0 software.
LDL-binding assay
LDL was labeled with 125 I using the Bolton-Hunter protocol ( 24 ). Binding assays were performed as previously described ( 10,25 ).
Surface LDLR expression assay
Surface expression was measured by fl ow cytometry using the C7 monoclonal antibody to the LDLR as previously described ( 23 ). Briefl y, cells were treated with LPPS medium overnight, fi xed with 3% paraformaldehyde, and blocked with PBS containing 0.1% BSA. Cells were then incubated with 10 g/ml C7 antibody for 1 h at room temperature, washed, and incubated for 1 h at room temperature with a secondary antibody coupled to allophycocyanin. Cells were lifted from the dishes, and cellular fl uorescence determined by fl ow cytometry.
Biotin switch assay for protein nitrosylation
Nitrosylated proteins were identifi ed by replacing S-nitrosyl groups with biotin using the S-nitrosylated protein detection assay kit (Cayman Chemical Co., Cat. No. 10006518), which is based upon the protocol developed by Jaffrey and Snyder ( 26 ). Biotinylated proteins were then purifi ed using neutravidin-agarose, separated on SDS-PAGE, and immunoblotted for ARH.
Electronic microscopy
Colloidal gold-conjugated LDL (LDL-gold) was produced as previously described ( 27,28 ). Surface labeling with LDL-gold was performed by incubating cells with 10 g/ml LDL-gold in minimal essential media supplemented with 10% LPPS at 4°C for 2 h. The cells were washed three times with PBS and fi xed with 3% paraformaldehyde followed by 0.8% glutaraldehyde. The cells were then embedded, sectioned, counter-stained, and visualized using an FEI Tecnai electron microscope operating at 120 kV as previously described ( 28 ). Micrographs of each cell type were coded, and the length of the noncoated pit membranes, the length of the coated pit membranes, and the number of gold particles associated with each class of membrane were determined using ImageJ software.
RT-PCR
RNA was isolated from white adipose tissue of a C57BL/6 mouse or from WT cells using the RNA STAT-60 kit (TEL-TEST also containing 10 g/ml 125 I-LDL. Cells were extensively washed with ice-cold PBS and incubated with 1 mg/ml protease K in protease buffer [PBS + 1 mM EDTA (pH 8.0] for 2 h at 4°C. The cell suspension was then centrifuged at 5000 g for 10 min over a cushion of 10% sucrose in PBS. The tubes were frozen in liquid nitrogen, cut to separate the cells (internal) from the solution (surface-bound material released by protease K), and counted on a ␥ counter. Nonspecifi c activity was assessed in parallel experiments in the presence of 250 g/ml unlabeled lipoprotein. Nonspecifi c activities were subtracted from mean values for each data point. Data are means ± SEM of four replicate trials from four experiments (n = 16).
Lipoprotein uptake assay
LDL and  -VLDL uptake assays used previously published protocols ( 23 ). Briefl y, cells were fi rst treated with FLPPS medium (D-MEM supplemented with 10% fetal lipoprotein-poor serum) overnight to induce LDLR expression. Alexa546-labeled LDL (10 g/ml) or Alexa546-labeled  -VLDL (5 g/ml) in LPPS medium ARH, C199, was required for the faster mobility form of ARH. As with the C286A mutation, the C199A mutation eliminated the fast mobility form of ARH ( Fig. 1F ). Formation of disulfi de bonds in cytosolic proteins is rare because the cytosol is normally a reducing environment; however, nitric oxide is an endogenously produced oxidant that has been shown to promote disulfi de bond formation ( 29 ). Nitric oxide reacts with the sulfhydryl of cysteine to form S-nitroso cysteine, which can then react with other cysteines and nitrosylated cysteines to form disulfi de bonds. To determine whether ARH is nitrosylated, we introduced into HEK293 cells WT ARH, ARH-C199A, ARH-C286A, or ARH-C199A/C286A and tested for nitrosylation using biotin switch assays. These assays showed that WT ARH, ARH-C199A, and ARH-C286A, but not ARH-C199A/C286A, are nitrosylated ( Fig. 1F ), indicating that ARH is nitrosylated at both C199 and C286 and that these cysteines can form a disulfi de bond.
Nitrosylation of ARH is required for LDL uptake
To test whether the nitrosylation status of ARH infl uences lipoprotein uptake, we used fi broblasts from Arh Ϫ / Ϫ ; Dab2 fl ox/fl ox ;hLDLR +/+ mice to make stable fi broblast cell lines that i ) express Dab2 but not ARH (Dab2); ii ) neither ARH nor Dab2 (Vector); iii ) WT human ARH but not Dab2 (WT); iv ) human ARH-C199A but not Dab2 (C199A); v ) human ARH-C286A but not Dab2 (C286A); or vi ) human ARH-C199A/C286A but not Dab2 (CC>AA) ( Fig. 2A, B ). These cells were then assayed for their ability to support lipoprotein uptake using a FACS-based assay that measures steady-state rates of lipoprotein accumulation. We observed that C199A and C286A supported rates of LDL accumulation that were similar to that of WT cells, while CC>AA cells supported a rate of LDL accumulation that was similar to that of Vector cells ( , consistent with the prior observation that  -VLDL uptake does not require ARH, dab2, or the FDNPVY sequence ( 10 ). These observations show that ARH-supported LDL uptake requires either of the two nitrosylated cysteines.
To confi rm the role of nitric oxide, we tested whether inhibition of nitric oxide synthase activity reduced LDL uptake in our fi broblast cell lines. Mammalian genomes encode three nitric oxide synthases (NOS1-3), and our fi broblasts express primarily NOS2 (iNOS) with lower levels of both NOS1 (nNOS) and NOS3 (eNOS) (supplementary Fig. II). This expression pattern is consistent with prior reports of NOS expression in fi broblasts ( 30 ). To reduce nitric oxide synthase activity, we used l-N G -nitroarginine methyl ester (L-NAME), a cell-permeable arginine analog that inhibits all three NOS enzymes ( 31 ). Pretreatment of WT, C199A, or C286A cells with 4 mM L-NAME sharply reduced their rates of LDL uptake ( Fig. 3A and supplementary Fig. I), indicating that nitrosylation of ARH by nitric oxide is necessary for ARH to support LDL uptake. Importantly, L-NAME had no effect on dab2-supported LDL uptake or  -VLDL uptake ( Fig. 3A and supplementary Inc.) according to the manufacturer's instructions. RNA was then treated with DNA-free kit (Ambion) and reverse-transcribed into cDNA using the Taqman Reverse Transcription kit (Applied Biosystems). PCR was performed using the following primers: NOS1: 5 ′ -GACCAAGCCCTGGTGGAGATTAAC, 5 ′ -GCCTCTGCCAATT-TCTTGAAGCCA; NOS2: 5 ′ -CTGCTGGTGGTGACAAGCACAT-TTG, 5 ′ -CGTTCTTTGCATGGATGCTGCTGAG; NOS3: 5 ′ -CAG-TTCCCGGAAAGAGGGATTGTG; 5 ′ -GCATATGAAGAGGGCAG-CAGGATG. All reactions used SpeedSTAR HS polymerase (Takara) with 50 ng of cDNA over 30 cycles. Expected product sizes are 332 bp for NOS1 (nNOS), 439 bp for NOS2 (iNOS), and 214 bp for NOS3 (eNOS).
Statistics
P -values were calculated by one-way ANOVA using PRISM 4.0.
ARH is nitrosylated
The reason to suspect that ARH might be subject to posttranslational modifi cation originated from the fi rst immunoblots of ARH, which showed that ARH exists in two forms of differing electrophoretic mobility ( 17 ). To ensure that the difference in mobility did not result from proteolysis, we introduced into HEK293 cells C-terminally V5-tagged human ARH variants encoding full-length ARH, an N-terminal fragment of ARH (residues 1-187), or a C-terminal fragment of ARH (residues 188-308). V5 immunoblots showed that cells expressing full-length and the C-terminal half of ARH produced two V5 immunoreactive bands, while the N-terminal fragment produced only a single band ( Fig. 1B ). Thus, proteolysis is not responsible for two ARH bands because hydrolysis at the N-terminal end of ARH would have produced a second band for the N-terminal fragment, while hydrolysis at the C-terminal end of ARH would have removed the V5 tag. To identify what part of the C-terminal half was responsible for the second ARH band, we introduced a series of untagged human ARH variants with C-terminal deletions into HEK293 cells. Deletions removing the last 17 amino acids had little effect on the presence of two ARH bands, but deletions of 25 or more residues eliminated the faster migrating band ( Fig. 1C ). We identifi ed which residues were required for the fast mobility species by introducing ARH variants bearing single alanine substitutions. Cells expressing ARH variants with alanine mutation at T277, H282, Y283, or S288 produced both slow and fast mobility ARH forms, but cells expressing ARH-C286A produced only the slow mobility form ( Fig. 1D ). These observations indicate that C286 is required for posttranslational modifi cation of ARH.
Most posttranslational modifi cations slow electrophoretic mobility; however, intramolecular disulfi de bonds are an exception, because this modifi cation reduces the hydrodynamic radius of SDS-denatured proteins. We tested whether C286 was involved in an intramolecular disulfi de bond by treating lysates of normal human fi broblasts with increasing concentrations of DTT. Concentrations above 0.3 mM eliminated the faster mobility form of ARH ( Fig. 1E ). Disulfi de bonds require two cysteines, and we tested whether the other cysteine in the C-terminal half of Quantifi cation of the LDL-gold distribution showed that Vector cells had little enrichment of LDL-gold in coated pits, while WT cells had a 10-fold enrichment. C199A, C286A, and Dab2 cells had coated pit enrichments that were equal to or better than that of WT cells; however, CC>AA cells had enrichment that was nearly as poor as Vector cells ( Fig. 5B ). These observations show that ARH requires the ability to be nitrosylated for coated pit targeting of LDL.
The ability of ARH to be nitrosylated also correlated with the ability of ARH to support LDL internalization. Assays of LDL internalization were performed by loading surface LDLR with 125 I-LDL at 4°C, shifting to 37°C for various periods of time, and measuring surface and internal pools of LDL. Fibroblasts expressing endogenous levels of both ARH and dab2 show uptake rates of 0.065-0.085 min Ϫ 1 ( 10,32 ). WT and Dab2 cells were both in this range with LDL internalization rates of 0.081 and 0.067 min Ϫ 1 , respectively ( Fig. 5C ). Vector cells, which express neither an ARH nor dab2, only slowly internalized LDL with a rate of 0.0059 min Ϫ 1 . CC>AA cells were similar to Vector cells with a rate of 0.0099 min Ϫ 1 . C199A and C286A cells were intermediate with rates of 0.030 and 0.029 min Ϫ 1 , respectively. These results indicate that loss of both cysteines prevents ARH from supporting LDL internalization, while loss of either cysteine slows LDL internalization. During LDL uptake, the LDLR cycles between the cell surface where it binds LDL and endosomes where it releases LDL. In fi broblasts at steady state, half of the total LDLR pool is exposed on the cell surface, while the remainder is in transit through the endosomal and recycling systems ( 4 ). In fi broblasts, which lack both ARH and dab2 or which express only LDLRs that lack a functional FDN-PVY sequence, the internal pool of LDLRs is lost and all receptors are present on the cell surface ( 10,13,18,28 ). Consistent with these prior fi ndings, our Vector cells, which lack both adaptors, had twice as many surface LDLRs and bound twice as much LDL as WT cells ( Fig. 4 ), despite having a similar total LDLR content as WT cells ( Fig. 2 ). C199A and C286A cells had LDLR surface expression and LDL binding that was similar to WT cells; however, CC>AA cells had levels of surface LDLR and LDL binding that were similar to Vector cells ( Fig. 4 ), despite WT levels of total LDLR ( Fig. 2 ). These fi ndings suggest that nitrosylation is required for ARH to engage the endocytic machinery of coated pits.
To test whether nitrosylation is necessary for ARH-dependent targeting of LDL-LDLR complexes to coated pits, we loaded surface LDLRs of the fi broblast cell lines with LDL-gold and then visualized the location of LDL-LDLR complexes by thin section electron microscopy ( Fig. 5A ). Fig. 3. ARH nitrosylation is necessary for LDL uptake but not  -VLDL uptake. Vector, WT, C199A, C286A, CC>AA, and Dab2 cells were pretreated or not with 4 mM L-NAME for 30 min and then assayed by FACS for lipoprotein uptake at 1, 2, 3, and 4 h using 10 g/ml Alexa546-labeled LDL or 5 g/ml Alexa546labeled  -VLDL. Data for each time point are shown in supplementary Fig. I. Linear regression of uptake data was used to determine rate constants for LDL uptake and  -VLDL uptake. Rate data are plotted as mean rates ± SD (n = 3 trials; 10,000 cells per time point per trial). Signifi cance determined by one-way ANOVA. ‡ P < 0.05, ‡ ‡ P < 0.005 for untreated cells versus L-NAMEtreated cells; * P < 0.05, ** P < 0.005 for untreated cells compared with untreated WT cells. ARH-C286A than with ARH-CC>AA. Pretreatment of cells with L-NAME reduced the amount of AP-2 and clathrin coprecipitating with ARH in WT, C199A, and C286A cells to the level observed with CC>AA cells. We confi rmed the preferential interaction of AP-2 with nitrosylated ARH by immunoprecipitating AP-2 and comparing coprecipitation of ARH. Immunoprecipitation of AP-2 coprecipitated more ARH from lysates of WT, C199A, and C286 cells than from CC>AA cells or from cells treated with L-NAME. AP-2 has its own binding sites for clathrin heavy chain, and coprecipitation of clathrin with AP-2 was unaffected by L-NAME treatment or mutation of ARH. No differences were observed among WT ARH, ARH-C199A, and ARH-C286A
Nitrosylation is necessary for normal interaction of ARH with AP-2
Published experiments have shown that  -arrestin2, an endocytic adaptor that promotes G-protein coupled receptor (GPCR) uptake, is nitrosylated and that this modifi cation promotes association of  -arrestin2 with AP-2 ( 33 ). Like  -arrestin2, ARH binds to the  2-adaptin component of AP-2 ( 5 ), suggesting that nitrosylation may promote binding of ARH to AP-2. We tested this possibility using immunoprecipitation and found that ARH effi ciently coprecipitated AP-2 from lysates of WT, C199A, and C286 cells, but not from lysates of CC>AA cells ( Fig. 6 ). Clathrin also coprecipitated better with WT ARH, ARH-C199A, and ability to target LDL-LDLR complexes to coated pits. Coated pit enrichment is reported using the summation of 10 random micrographs ± SEM of enrichments calculated from each micrograph separately. The P -value relative to WT is indicated above the error bar. (C) CC>AA cells have poor ability to support LDL internalization. Surface receptors were saturated with 10 g/ml 125 I-LDL at 4°C and then shifted to 37°C in the presence of 10 g/ml 125 I-LDL. At the indicated times, internalization was stopped and surface-bound and internalized pools of LDL were assayed as described in Materials and Methods. Data is shown as the mean ratio of internal/surface ± SEM, n = 16.
analogous mechanism to control AP-2-binding activity. While  -arrestins and ARH have no sequence conservation outside their AP-2-binding sites, ARH may hold its AP-2-binding sequence in an inactive, nonhelical state. S-nitrosylation at C199 and C286 may release the AP-2binding sequence, allowing it to adopt the active, helical state.
In addition to activating AP-2 binding, S-nitrosylation at C199 and C286 may play a role in LDL internalization. Mutation of either cysteine slowed LDL internalization more than 2-fold ( Fig. 5C ). The contribution of C199 and C286 to LDL uptake was not at the level of coated-pit targeting because both C199A and C286A cells displayed normal LDL-gold enrichment in coated pits and because binding of AP-2 and clathrin to ARH-C199A and ARH-C286A were normal ( Figs. 5 and 6 ). The contribution of these cysteines does not appear to infl uence the internalization of nonlipoprotein-loaded LDLRs because the surface LDLR levels on C199A and C286A cells were normal ( Fig. 4 ). Only a fraction of LDL-LDLR complexes that reach coated pits are internalized through the budding portion of coated pits ( 40 ), and nitrosylation of both cysteines may help to anchor LDL-LDLR complexes in budding portion of coated pits. Importantly, the decreases in LDL internalization rate caused by the C199A and C286A mutations had only a small effect on overall LDL accumulation rates ( Fig. 3 ). LDL accumulation is a function of LDL binding, LDL-LDLR traffi cking to coated pits, internalization, LDL release, receptor recycling, and LDL resecretion. The differences between LDL accumulation and LDL internalization by WT, C199A, and C286A cells suggest that internalization is not rate limiting for LDL accumulation in WT cells.
Of the two cysteines that are nitrosylated in human ARH, only C286 is conserved across vertebrate species (supplementary Fig. III). The strong conservation of C286 from fi sh to primates suggests that nitrosylation at C286 plays a conserved role in ARH-supported LDL uptake; however, the lack of a cysteine near residue position 199 in nonprimate ARH proteins suggests that the function of with regards to association with AP-2 in either set of immunoprecipitation. These observations indicate that nitrosylation of either C199 or C286 is suffi cient to facilitate the binding of ARH to AP-2.
DISCUSSION
The central fi nding of this study is that nitric oxide regulates the ability of ARH to support LDL uptake. LDL uptake by the LDLR requires either ARH or dab2, which binds to AP-2, clathrin, and the FDNPVY sequence of the LDLR cytoplasmic domain. The combination of these three interactions is necessary for effi cient targeting LDL-LDLR complexes to coated pits for endocytosis ( 10,13,34 ). The ability of ARH to associate strongly with AP-2 requires that ARH be nitrosylated at either C199 or C286 ( Figs. 1F and 6 ). Failure to nitrosylate ARH cripples the ability of ARH to cluster LDL-LDLR complexes in coated pits ( Fig. 5A, B ) and impairs LDL uptake ( Figs. 3 and 5 ). C199 and C286 can form a disulfi de bond in cells ( Fig. 1 ), and nitrosylation may catalyze this disulfi de bond formation. Together, these fi ndings suggest that ARH cycles between an inactive unnitrosylated state, an active nitrosylated state, and a disulfi de-bonded state ( Fig. 7 ). Interchange between these states may depend upon NOS activity, denitrosylation activities, and the cellular redox state.
ARH binds to the  2-adaptin component of AP-2 through a sequence that has homology with the AP-2binding sequence of  -arrestins. Structural studies have shown that the AP-2-binding sequences of both ARH and the  -arrestins adopt an ␣ -helical conformation when bound to  2-adaptin (35)(36)(37). In the case of  -arrestins, the AP-2-binding activity is regulated by GPCRs. When  -arrestins are in the inactive/basal state, their AP-2-binding sequences are stretched into a  -strand conformation ( 38,39 ). Interaction of  -arrestins with active, phosphorylated GPCRs induces a conformational change in  -arrestins that allows the AP-2-binding sequence to coil into the active, ␣ -helical conformation. In  -arrestin2, the C-terminal cysteine is nitrosylated in response to GPCR activation, and this nitrosylation event facilitates the conformational change that activates AP-2 binding ( 33 ). ARH may use an Fig. 6. Nitrosylation is required for normal interaction of ARH with AP-2. Cells were treated or not with 4 mM L-NAME for 30 min, and then lysed and immunoprecipitated with antibodies to ARH or AP-2. Immunoprecipitants were run on SDS-PAGE and immunoblotted for the presence of ARH, AP-2, and clathrin heavy chain (CHC). Fig. 7. Model for regulation of ARH activity. We propose that ARH exists in three states: a free sulfhydryl state, a nitrosylated state, and a disulfi de-bonded state. The free sulfhydryl form can react with nitric oxide produced by NOS enzymes to generate the active, nitrosylated form of ARH. Nitrosylation catalyzes disulfi de bond formation, leading to inactivation of ARH. Cytosolic reductases (e Ϫ ) reduce disulfi de-bonded ARH to the free sulfhydryl form. Active ARH is presented as a hexagon, and inactive ARH is presented as a square. falls sharply following the consumption of a large meal ( 59,60 ) and this drop may reduce ARH nitrosylation, thereby focusing the endocytic activity of hepatic LDLRs on uptake of chylomicron and VLDL remnants, which fl ood the circulation in the postprandial state. Remnant particles are more atherogenic than LDL ( 61 ) and focusing the endocytic activity of the LDLR on remnants may help prevent atherosclerosis both by more rapidly reducing the circulating number of remnants and by preventing the conversion of VLDL remnants into LDL. Peripheral blood leukocytes also express ARH, but not dab2 ( 14 ), and the absence of dab2 may protect leukocytes from excessive LDL uptake in atherosclerotic lesions. Atherosclerotic lesions are infl ammatory responses to lipoprotein accumulation in the intima of arteries ( 62 ). As part of the infl ammatory response, peripheral blood leukocytes extravasate into the lesion ( 63 ). Once in a lesion, leukocytes shut down LDLR function via transcriptional pathways that are triggered by the elevated cholesterol and oxysterols in the lesion. Both cholesterol and oxysterols suppress LDLR mRNA expression through inactivation of sterol response-element binding proteins (SREBP), which are transcription factors required for LDLR mRNA transcription ( 64 ). Elevation of oxysterols also promote LDLR turnover by activating liver X receptors (LXR), which are transcription factors that drive production of Idol/MYLIP, an E3-ubiquitin ligase that promotes lysosomal degradation of the LDLR ( 65 ). Because both processes are dependent upon changes in transcription, they require time for new protein synthesis, and changes in ARH function may complement transcriptional inactivation of LDLR function. Atherosclerotic lesions have abundant reactive oxygen species (ROS), which react with nitric oxide and reduce nitric oxide availability ( 66 ). Loss of nitric oxide inactivates ARH ( Figs. 2-7 ) and may halt LDLR-dependent LDL uptake while LDLR mRNA and protein are being destroyed.
In summary, the data presented here show that LDL uptake by the LDLR requires S-nitrosylation of ARH. Nitrosylation promotes interaction of ARH with AP-2 and is required for ARH to target LDL-LDLR complexes to coated pits for internalization.  -VLDL uptake and dab2supported LDL uptake are not dependent upon nitric oxide, indicating that the infl uence of nitric oxide on LDLR function is specifi c to ARH-supported LDL uptake. Thus, cells that express ARH, but not dab2, can reduce LDL uptake in response to reductions in nitric oxide without affecting either LDLR expression or LDLR-dependent uptake of VLDL remnants. C199 is primate-specifi c. Other species may use different cysteines to form a disulfi de bond with C286, because mouse ARH has a fast-migrating form ( 17 ), despite the absence of a cysteine near position 199. The most likely role for disulfi de bond formation is denitrosylation. Most protein denitrosylation involves transnitrosylation events that transfer the nitric oxide moiety from a nitrosylated protein to either glutathione or thioredoxin. The nitric oxide moiety of glutathione and thioredoxin is then removed through a process that involves disulfi de bond formation followed by disulfi de bond cleavage ( 41 ). The ability of ARH to form an intramolecular disulfi de bond may provide an intrinsic mechanism that accelerates denitrosylation of ARH.
Importantly, the involvement of nitric oxide is specifi c to ARH-supported LDL uptake. Neither dab2-supported LDL uptake nor VLDL remnant uptake are impaired by inhibitors of nitric oxide synthase activity ( Fig. 3 ). The ability of dab2 to support LDL uptake in the absence of NOS activity may allow cells that express dab2 to support LDLR-dependent uptake of LDL when nitric oxide levels are low. For example, the adrenal glands and ovaries of humans use LDL-derived cholesterol to produce steroid hormones (42)(43)(44). Nitric oxide inhibits steroid production ( 45 ), and the high levels of dab2 expressed in steroidogenic tissues ( 46,47 ) may facilitate LDL uptake when nitric oxide levels are low and cholesterol demand for steroid production is high. More generally, activation of ERK induces LDLR expression in dividing cells to supply the cholesterol needed for membrane biogenesis ( 48 ). The moderate levels of dab2 that are expressed in most tissues may allow dividing cells to use the LDLR to supply LDL-derived cholesterol irrespective of nitric oxide levels.
Liver hepatocytes express ARH but not dab2, suggesting that changes in nitric oxide production may dictate whether hepatic LDLRs internalize LDL. In humans, reductions in the rate of whole-body nitric oxide production correlate strongly with increased circulating LDL-cholesterol in age-matched individuals ( 49 ). Whole-body nitric oxide production also decreases with age ( 50,51 ), coincident with a decrease in LDL clearance rates ( 52,53 ). The ability of lipoproteins to inhibit eNOS activity ( 54,55 ) likely explains part of the correlation between LDL-cholesterol and nitric oxide production; however, the correlation is much stronger in individuals with two normal LDLR alleles than in individuals with a defective LDLR allele ( 49,56 ), suggesting that nitric oxide also promotes LDLR function. In support of this conclusion, transgenic overexpression of eNOS in apoE-defi cient mice decreases LDL-cholesterol levels ( 57,58 ). Because apoE is not required for LDLR-dependent LDL uptake, the reduction in LDL-cholesterol observed in the transgenic animals suggests that elevated nitric oxide improves LDL uptake by the LDLR. This improvement in LDLR function may involve nitrosylation of ARH.
Under normal conditions, hepatocytes may use nitric oxide to regulate whether the LDLR internalizes LDL or remnant particles. Whole body nitric oxide production | v3-fos-license |
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} | pes2o/s2orc | Absorption and Scattering Properties of Organic Carbon versus Sulfate Dominant Aerosols at Gosan Climate Observatory in Northeast Asia
Carbonaceous and soluble ionic species of PM 1.0 and PM 10 were measured along with the absorption and scattering properties and aerosol number size distributions at Gosan Climate Observatory (GCO) from January to Septem-ber 2008. The daily averaged equivalent black carbon (EBC) measured as aerosol absorption exhibited two types of spectral dependence with a distinct maximum (peak) at either 370 nm or 880 nm, by which two subsets were extracted and classified into the respective groups (370 and 880 nm). The 370 nm group was distinguished by high organic carbon (OC) concentrations relative to elemental carbon (EC) and sulfate, but sulfate was predominant for the 880 nm group. The PM 1.0 OC of the 370 nm group was mainly composed of refractory and pyrolized components that correlated well with PM 1.0 EC1, referred to as char EC, which suggests bio-fuel and biomass combustion as the source of these OC fractions , particularly during winter. The scanning electron microscope (SEM) images and the number size distributions implied that aerosols of the 370 nm group were externally mixed upon transport in fast-moving air masses that passed through the Beijing area in about one day. In contrast, the aerosols of the 880 nm group were characterized by high sulfate concentrations, and seemed to be internally mixed during slow transport over the Yellow Sea region over approximately 2 to 4 days. The absorption and scattering coefficients of the 880 nm group were noticeably higher compared to those of the 370 nm group. The average absorption ångström exponent (AAE) was estimated to be 1.29 and 1.0 for the 370 and 880 nm groups, respectively, in the range 370–950 nm. These results demonstrated that the optical properties of aerosols were intimately linked to chemical composition and mixing state, characteristics determined both by source and atmospheric aging processes. In OC dominant aerosols, absorption was enhanced in the UV region, which was possibly due to refractory and pyrolized OC compounds. Under sulfate dominant conditions, the sulfate coating on BC particles likely contributed to the absorption of the longer visible light. Consequently, single scattering albedo (SSA) was higher for the 880 nm group than for the 370 nm group, emphasizing that the relative abundances of absorbing and scattering constituents are also important in estimating the climate effect of aerosols.
Introduction
Atmospheric aerosols play an important role in climate change by altering the global radiation balance both directly and indirectly.The relative importance of these processes depends on the chemical composition and size distribution of aerosols (IPCC, 2007).Carbonaceous and sulfate particles are the two most important anthropogenic aerosol constituents influencing climate (Chuang et al., 1997;Haywood and Ramaswamy, 1998;Schult et al., 1997).A positive component of this radiative forcing is largely due to black carbon (BC) produced from pyrolysis during incomplete combustion Published by Copernicus Publications on behalf of the European Geosciences Union.
of biomass and fossil fuels (Ramanathan and Carmichael, 2008;Roessler and Faxvog, 1979).The magnitude of the direct radiative forcing from BC exceeds that due to CH 4 , suggesting that BC may be the second most important component of global warming after CO 2 , in terms of direct forcing (Bond et al., 2013).
However, the definition and measurement techniques for atmospheric BC (commonly referred to as soot) and elemental carbon (EC) have long been subjects of scientific controversy.In general, BC and EC are identified by their different analytical methods: BC is measured using its optical properties, with the main property being strong absorption of visible light with a mass absorption cross section (MAC) at a wavelength of λ = 550 nm above 7.5 ± 1.2 m 2 g −1 for freshly produced particles (Bond et al., 2013).In contrast, EC is usually determined by thermal methods based on its chemical properties, and is defined as the carbonaceous fraction of particulate matter that can only be gasified by oxidation starting at temperatures above 340 • C (Petzold et al., 2013).Among the types of BC, the BC derived from the light-absorption coefficient (σ ap ) using a MAC is called equivalent BC (EBC).Given that this measurement technique detects particles that absorb light, EBC measurements could include other absorbing compounds and possibly lead to slightly different mass concentrations than EC measurements.The light absorption of BC is generally considered to be weakly dependent on wavelength (Bergstrom et al., 2002).Conversely, other lightabsorbing aerosol components such as organics and dust show stronger dependence on the absorption spectrum that contributes to absorption at shorter wavelengths (Bergstrom et al., 2007;Kirchstetter, 2004).Recent studies have reported that light-absorbing organic carbon that is not black ("brown carbon"), tends to absorb light strongly in the blue and ultraviolet (UV) spectral regions (Andreae and Gelencsér, 2006;Jacobson, 1998Jacobson, , 1999)).In the atmosphere, fresh BC ages as it is transported, leading to mixing with other aerosol components such as organics, minerals, and sulfates (Hasegawa and Ohta, 2002).It has been found that organic or sulfate coating around an inner BC core enhances the light absorption of BC by a "lensing" effect (Cross et al., 2010;Lack et al., 2009).Thus, the presence of light-absorbing organics and aerosols, either as intrinsic components of or as coatings on the BC particles makes it imperative to reassess and redefine BC and other light-absorbing carbonaceous matter in the atmosphere (Andreae and Gelencsér, 2006).
In regions influenced by various emissions from deserts, oceans, biomass burning, and human and industrial activities, aerosol chemical composition and optical properties are extremely complicated.Therefore, it is critical to understand the link between chemical composition and optical properties, particularly for policy making related to climate change.East Asia is a typical example of one of the regions described above and is one of the strongest BC emission source regions (Bond et al., 2004(Bond et al., , 2013)).Here, we examined light-absorbing and scattering properties of aerosols in relation to their major components including carbonaceous and soluble ions using data obtained at GCO located in northeast Asia.
Measurements
The chemical compositions of PM 1.0 and PM 10 were measured along with aerosol optical properties and particle number distributions from August 2007 to September 2008 at GCO (33.17 • N, 126.10 • E, 70 m a.s.l.) on Juju island, which is known as an ideal location for monitoring Asian outflows.For this study, a subset of 29 samples was chosen from January to September 2008, for which the measurements of chemical composition, aerosol absorption and scattering, and number size distribution were all available.The sample set included five samples from January, three from February, four from March, eight from April, four from May, one from June, two from August, and two from September 2008.Details of the sampling procedures and methods can be found in Lim et al. (2012).
For PM 1.0 and PM 10 , sampling was conducted once every 6 days, starting at 09:00 LST and lasting for 24 h.Eight inorganic species were determined by using ion chromatography (Dionex 4500, Dionex, USA), and EC and organic carbon (OC) were quantified by the Interagency Monitoring of Protected Visual Environments (IMPROVE) thermal/optical reflectance (TOR) protocol, which produced four OC fractions in a non-oxidizing He atmosphere (OC1, OC2, OC3, and OC4 at temperatures of 120, 250, 450, and 550 • C, respectively) and three EC fractions in an oxidizing atmosphere of 2 % O 2 / He mode (EC1, EC2, and EC3 at 550, 700, and 800 • C, respectively).In the He mode, a fraction of OC was pyrolyzed (OP) and this evolved in the oxidizing atmosphere until the reflected light achieved its initial value.OP is assumed to be originally organic carbon, so OC is considered to be the sum of the four OC fractions and the OP fraction, and OP is subtracted from the sum of EC fractions.
Light-absorption coefficients (σ ap ) were obtained every 10 min from the seven wavelength aethalometer measurements (AE-31, Magee scientific Corp., USA) and EBC mass concentrations were derived with the "Magee BC" calibration factor (Hansen, 2005).The aethalometer collects the aerosol sample on a quartz fiber filter tape and measures the light attenuation of samples at wavelengths covering the UV to the near-IR ranges (i.e., 370, 470, 520, 590, 660, 880, and 950 nm).Similar to other filter-based absorption instruments, the aethalometer needs to be corrected for scattering by the fiber filter substrates, scattering of aerosols embedded in the filter, and filter loading by accumulation of light absorbing particles (Collaud Coen et al., 2010).In this study, σ ap was determined by following the correction process presented by Arnott et al. (2005).The absorption ångström exponent (AAE) was determined by statistical regression to fit the σ ap data with a power law equation.The MAC of EBC along wavelengths was calculated by dividing the light absorption coefficient by EC mass concentration.The scattering coefficient was obtained from a nephelometer (model 3563, TSI Inc., USA) every 10 min at three wavelengths: 450 nm, 550 nm, and 700 nm, which were corrected to STP and for truncation according to Anderson and Ogren (1998).The absorption and scattering data were averaged daily for comparison with chemical compositions that were measured daily.Aerosol number size distributions from 10.4 nm to 469.8 nm in diameter were measured every 10 min using a scanning mobility particle sizer (SMPS, model 3034, TSI Inc., USA).The scanning electron microscope (SEM) image was obtained from quartz filters laden with PM 1.0 after being coated with platinum and palladium.
Spectral dependence of absorption on chemical characteristics
We compared daily averaged EBC concentrations with PM 1.0 EC concentrations and examined the spectral dependence of EBC.The EC concentrations of PM 1.0 agreed well with EBC at 880 nm, which corresponds to 92 % of PM 1.0 EC.At other wavelengths, the EBC concentrations were less than 90 % of PM 1.0 EC.The daily averaged EBC concentrations varied along wavelengths, some of which showed clear maxima either at 370 nm or at 880 nm.The EBC concentrations of the others did not significantly change along wavelengths, but tended to be slightly higher at 660 nm.While the maxima at 370 nm were apparent regardless of EBC concentration, they were less distinguished at 880 nm or 660 nm as EBC concentration decreased.Thus, we only considered EC concentrations higher than mean for the classification of the 880 nm maximum.Based on the wavelength peak of EBC, five samples were selected for both the 370 and 880 nm groups.Then, chemical characteristics of each group were compared for major constituents, including EC, OC, sulfate, and inorganic salts.In particular, their relative abundance was distinguished between the two groups such as OC / EC, OC / sulfate, and EC / sulfate ratios.It is noteworthy that all three ratios were higher for the 370 nm group than for the 880 nm group.While the 370 nm group was characterized by relatively higher OC concentration, sulfate was predominant for the 880 nm group.However, there was one exception in the 880 nm group (9 January 2008), in which the OC / EC ratio was high with relatively low ratios of OC / sulfate and EC / sulfate.Its maximum absorption was also not as distinct as the other four in the group.Thus, it was excluded and the other four samples were finally classified as the 880 nm group.The five samples of the 370 nm and the four samples of the 880 nm group were collected in winter and spring, respectively (Table 1).
Figure 1a shows the EBC concentrations relative to PM 1.0 EC concentrations along wavelengths.Although the concen- trations of EBC and PM 1.0 EC were expected to be equal, they were found to be slightly different.The EBC / PM 1.0 EC ratio varied from 0.73 to 1.04 and from 0.76 to 1.18 for the 370 and 880 nm groups, respectively.For the former, the EBC / PM 1.0 EC ratio was the highest at 370 nm and decreased toward 950 nm by 0.07 ∼ 0.18.For the latter, the EBC / PM 1.0 EC ratio was at its minimum at 370 nm and gradually increased by 0.15 ∼ 0.22 to its maximum at 880 nm.The following discussion regarding absorption enhancement along wavelength is based on this analysis.The chemical characteristics of the two groups are summarized in Table 1.While EBC concentrations were lower for the 370 nm group compared to those of the 880 nm group, OC concentrations were similar in the two groups.The difference in meteorological variables and precursor gases reflect seasonal characteristics of each group.b Of the total mass, eight ionic soluble ions, OC, and EC were measured.Detailed information on chemical composition can be found in Lim et al. (2012).
Organic carbon dominant regime
The OC / EC ratio of PM 1.0 was higher for the 370 nm group (2.68) than for the 880 nm group (1.81) (Fig. 1b).The av-erage OC / EC ratio of the total sample set was 2.26.For the 370 nm group, the contribution of OC3, OC4, and OP to PM 1.0 OC was noticeable.The higher PM 1.0 OC / EC ratio of the 370 nm group was mostly due to the greater contribution of refractory OC, including OC3 and OC4 that are resistant to volatilization and evolve at higher temperatures (450 and 550 • C), and OP.The average ratio of PM 1.0 (OC3 + OC4 + OP) / EC for the 370 nm group (2.00) was higher than that of the 880 nm group (1.20) and the total sample set (1.51).The average PM 1.0 OP / EC was also greater for the 370 nm group (0.87) than the 880 nm group (0.53).
In this study, the larger fraction of PM 1.0 refractory OC and OP against EC (e.g., (OC3 + OC4 + OP) / EC and OP / EC in Fig. 1b) was associated with the enhanced ratio of EBC at 370 nm to EC, which implied the strong light absorption of aerosols in the UV region.It has been reported that brown carbon shows steeper wavelength dependence and has various origins in the atmosphere, e.g., soil humics, humiclike substances (HULIS), tarry materials from combustion, and bioaerosols (Andreae and Gelencsér, 2006 and references therein).These components are considered to be the class of HULIS (Hoffer et al., 2006;Lukács et al., 2007) which is associated with the water soluble organic carbon (WSOC) fraction with high molecular weight (Miyazaki et al., 2007;Yu et al., 2004).In addition, it has been reported that the WSOC fraction of ambient aerosols is prone to charring, and it accounts for a significant fraction of overall OP (Andreae and Gelencsér, 2006;Yu et al., 2002).In the recent study by Kondo et al. (2011) that evaluated the charring of four kinds of laboratory HULIS, more than half evolved as the OC fraction resistant to volatilization in the He mode and released in He / O 2 mixtures.Clarke et al. (2007) also argued that the measured refractory OC, for which they used a density of bulk HULIS, remaining at high temperature (400 • C) in thermal analysis was responsible for enhanced shortwave absorption in biomass burning plumes.These studies support the observation that the refractory OC lead to more charring in the He mode and, subsequently, to enhanced OP fractions.In this study, a direct link between refractory OC and HULIS or other light-absorbing organics was not confirmed.However, the higher contribution of refractory OC and OP to total OC was one of the main attributes of the 370 nm group that showed distinctively enhanced absorption at the UV region.
The ratio of PM 1.0 (OC3 + OC4 + OP) / EC was higher than the median not only for the 370 nm group, but also for those samples including 20 and 26 February, 23 and 26 April, and 30 May.The absorption and chemical characteristics of these five samples (hereafter "660 nm group") were compared with that of the 370 nm group to see if refractory OC and OP were responsible for the light absorption at short wavelengths.For the 660 nm group, there were no trends in light absorption with wavelength but the absorption was slightly enhanced at 660 nm.Although the ratio of PM 1.0 (OC3 + OC4 + OP) / EC was similar in the two groups, this ratio in PM 10 was much lower for the 370 nm group (2.60) compared to that of the 660 nm group (3.64).
In the previous study by Lim et al. (2012), OC3 and OC4 were found predominantly in PM 10 when air masses were most likely impacted by soil minerals from dust source regions.For the 660 nm group, the fraction of PM 10 refractory OC was high and the ratios of Ca 2+ / Mg 2+ (1.37) and Ca 2+ / Na + (0.45) in PM 10 were higher than those of seawa-ter, suggesting substantial influence by soils.The influence of soil was also in accordance with a slight increase in absorption at 660 nm for the 660 nm group (Hansen, 2005).
The inorganic salts concentration (Na + + Ca 2+ + Mg 2+ + Cl − ) in the PM 10 of the 370 nm group was 3.3 times higher than that of the 880 nm group and 1.6 times higher than that of the total sample set.For the 370 nm group, the ratio of Cl − / Na + (1.44) was close to that of seawater (1.80) and the ratio of Mg 2+ / Ca 2+ (1.01) was smaller than that of seawater (3.10) but larger than that of aerosols mainly affected by soil.The ratios of Na + / Mg 2+ (3.79) and Cl − / Mg 2+ (5.54) were also less than those of seawater.These ratios indicate that the inorganic ions of the 370 nm group were likely derived from salts deposited in dry lakes in northeastern China (Park et al., 2011;Zhang et al., 2012).The air masses of the 370 nm group were accompanied by strong northerlies or northwesterlies passing the northeastern part of Inner Mongolia (bottom panels in Fig. 4a and b), where salt pans as well as deserts are developed, releasing alkaline soils into the atmosphere.If particular meteorological conditions are met, the inorganic salts deposited onto clay particles could be readily mobilized and transported long distances through the atmosphere by strong winds.Therefore, the coarse particles of the two groups were possibly derived from different sources such as salt deposits and soil minerals, contributing to scattering for the 370 nm group and to absorption for the 660 nm group.In addition, the local emission of these inorganic salts should not be disregarded for all samples because Gosan is located right by the sea.
For the 370 nm group, the inorganic salts of PM 10 (Na + + Ca 2+ + Mg 2+ + Cl − ) showed a good correlation with the light-scattering coefficient at 550 nm (R 2 = 0.85) (Fig. 2a).In addition, PM 10 OC was well correlated with the scattering coefficient, while PM 1.0 OC showed a weak correlation.The coarse OC possibly originated from salt deposits in dry lakes, and it could be agglomerated with inorganic salts.In this group, OC and inorganic salts were considered as main contributors to light scattering.The chemical effect on light scattering was possibly outweighed by the size effect of the aerosols (Tang, 1996).Mishchenko et al. (2004) also reported that when one of the aerosol components is much larger than the others, it dominates the total optical characteristics of the mixture, especially for semi-externally or externally mixed particles.
In the 370 nm group, three types of particles recognized by their distinct shapes and sizes seemed to be mixed externally (Fig. 3a).One type consisted of agglomerates that were more irregular in size and shape and bigger than soot agglomerates shown in Fig. 3b, and were thought to be organic particles coating soot (Adachi and Buseck, 2008;Adachi et al., 2010).It is likely that soot particles, being relatively less aged, increased in size through organic coatings under high OC / EC and OC / sulfate conditions.Two other distinct particle shapes were a thin cube and a sphere, thought to be a sea-salt particle and a tar ball, respectively.In particular, a tar ball is considered to possibly be brown carbon (Alexander et al., 2008) as well as an indicator of biomass combustion (Pósfai et al., 2004).It is distinguished in shape and structure from soot aggregates, typically made up of spherules 20 to 60 nm in diameter, often with an internal structure of curved graphene-like layers (Fig. 3b) (Faeth and Köylü, 1995;van Poppel et al., 2005;Vander Wal and Tomasek, 2004).The air masses of the 370 nm group underwent fast transport, passing through the Beijing area in about one day, and as a result, inorganic particles being externally mixed with EC and organic aerosol might have been able to play a part in light scattering under low sulfate conditions.The majority of samples classified into the 370 nm, 880 nm, or 660 nm groups were collected under the influence of Chinese outflows in winter and spring and thus carbonaceous compounds were mainly associated with coal and biomass combustion for residential heating (Lu et al., 2011).Their study pointed out biofuel from residential sectors as a predominant source of OC, especially in winter.In our study, for the 370 and 660 nm groups, the concentrations of PM 1.0 EC1, referred to as char EC (Han et al., 2010;Lim et al., 2012) were well correlated with the sum of OC3, OC4, and ) is a potential emission sensitivity distribution of 40 000 particles released in a particular grid cell at the measurement location during the measurement interval and followed backward in time.This is considered proportional to the particle residence time in that cell.
OP in PM 1.0 (R 2 = 0.6), whereas this was not the case for the 880 nm group (R 2 < 0.1).The char EC is known to be produced from incomplete combustion at lower temperatures; e.g., biofuel combustion (Han et al., 2010).This implies that the major source of PM 1.0 OC for the 370 and 660 nm groups were biofuel combustion.However, the enhanced absorption of the short wavelengths was observed only for the 370 nm group.This optical trait of the 370 nm group would be intimately coupled with chemical characteristics such as high ratios of OC / EC, (OC3 + OC4 + OP) / EC, OP / EC, OC / sulfate, EC / sulfate in PM 1.0 , in conjunction with meteorological conditions such as low temperature and high wind speed causing aerosols to be less aged and externally mixed.This also represents favorable conditions for semivolatile organics to be partitioned into the particle phase.In particular, the number distributions of the 370 nm group showed a distinct peak between 100 and 200 nm in SMPS measurements (top panels in Fig. 4a and b), which is bigger than freshly produced urban soot particles (< 100 nm) and a size that has been suggested for absorbing particles from biomass burning (100-200 nm) (Schwarz et al., 2008).The agglomerates shown in Fig. 3a could comprise refractory OC or OP as a main component of OC and account for the peak distribution at 100-200 nm for the 370 nm group.The ratio of OP to OC was high, particularly in PM 1.0 at GCO (Lim et al., 2012).Therefore, the PM 1.0 refractory OC and OP of the 370 nm group are most likely to be responsible for enhanced light absorption in the UV region and to be a constituent part of brown carbon.
Sulfate dominant regime
In contrast to the 370 nm group, the 880 nm group was distinguished by higher sulfate concentrations, which resulted in lower ratios of EC / sulfate and OC / sulfate in PM 1.0 (Fig. 1b).The average concentrations of PM 1.0 sulfate were 3.25 µg m −3 , 9.83 µg m −3 , and 5.16 µg m −3 for the 370 and 880 nm groups and the total sample set, respectively.The average ratios of EC / sulfate and OC / sulfate in PM 1.0 were 0.78 and 2.20 for the 370 nm group; 0.23 and 0.42 for the 880 nm group; and 0.37 and 0.90 for the total sample set.The high sulfate concentrations were coupled with stagnant conditions over the Yellow Sea, which supplied sufficient humidity and time for sulfate conversion in Chinese outflows (Lim et al., 2012) (Table 1).
In general, scattering coefficients at 520 nm were well correlated with sulfate concentrations of PM 1.0 , which was more pronounced in the 880 nm group (R 2 = 0.90; Fig. 2b).Although the scattering coefficients and sulfate concentrations of the 880 nm group were greater than those of the 370 nm group, the scattering coefficient per sulfate mass (slope in Fig. 2b) was less for the 880 nm group.This result is in accordance with in the findings of previous studies in which BC incorporated or randomly positioned within the sulfate www.atmos-chem-phys.net/14/7781/2014/Atmos.Chem.Phys., 14, 7781-7793, 2014 aerosol can lead to absorption enhancement and reduce the expected direct cooling effect due to sulfate (Bond et al., 2013;Chýlek et al., 1995;Fuller et al., 1999;Martins et al., 1998).The mixing of BC with non-absorbing materials such as sulfate has been known to alter the scattering properties of aerosols (Bond et al., 2006).
For the 880 nm group, BC was likely to be internally mixed with sulfate during the aging processes, which modified the absorption properties of the aerosols and resulted in spectral dependence of EBC (Fig. 1a).The cluster of soot aggregates in Fig. 3b was bigger and more compacted, compared to that in Fig. 3a, which suggests the soot aggregates of the 880 nm group were more aged (Fu et al., 2012).In other studies, the more aged Chinese air masses were sampled, the more packed soot aggregates were observed (Kang et al., 2012).Although it was not determined if the BC core was surrounded by a well mixed shell including sulfate or simply incorporated into other components, the daily variation of the particle size distribution (top panels in Fig. 4) gives a hint of the mixing state of aerosols, in conjunction with other measurements (e.g., Fig. 3).For the 880 nm group, the mode and number concentration remained unchanged through the day (top panels in Fig. 4c and d), implying that aerosols were rather internally mixed.In general, the atmospheric BC of remote areas is often internally mixed with other materials such as sulfate (Clarke et al., 1997;Pósfai et al., 1999) through intensive processing due to longer residence times (Hasegawa and Ohta, 2002).From observations near megacities in China including Shanghai, the results of single particle analysis indicate that aerosols were internally mixed during serious pollution events such as haze under prevailing stagnant condition (Fu et al., 2012;Tao et al., 2011;Yang et al., 2012).The air masses classified into the 880 nm group were slowly transported over the Yellow Sea over 2 to 4 days (bottom panels in Fig. 4c and d), during which BC particles must have been mixed with other types of aerosols such as sulfateforming BC coatings or intrinsic components.
AAE in relation to organic carbon and sulfate
The change in EBC concentrations with wavelengths (Fig. 1a) is related to the intrinsic properties of BC types such as composition, size, mixing state, and source.AAE, denoting the spectral dependence of light absorption, was derived for each group by fitting the measured σ ap at seven wavelengths from 370 nm to 950 nm with a power law equation (Fig. 5a).While the AAE of the 370 nm group was 1.29 (1.24-1.4) for the averaged σ ap ranging from 19.97 Mm −1 to 5.72 Mm −1 , the AAE of the 880 nm group was close to 1.0 (0.95-1.05), the theoretical value of BC, with higher σ ap from 22.69 Mm −1 to 8.75 Mm −1 .In several laboratory and field studies (Kirchstetter, 2004;Schnaiter et al., 2003Schnaiter et al., , 2005)), it was observed that BC produced from high-temperature combustion processes (e.g., diesel combustion) has a low spectral dependence with AAE ≈ 1.0, whereas BC from low-temperature combustion (e.g., biomass burning) exhibits a much stronger spectral dependence with AAE > 2.0.Moreover, organic aerosols were reported to contribute to strong light absorption at shorter wavelengths (i.e., UV), leading to higher AAE (Dubovik et al., 1998;Kirchstetter, 2004;Schnaiter et al., 2005).Kirchstetter (2004) demonstrated that in addition to BC, OC contributed considerably to measured light absorption in the UV and visible spectral regions.Theoretical and measurement studies also revealed that coating with scattering aerosol increased the absorption of visible light (Chung et al., 2012;Lack and Cappa, 2010;Schnaiter et al., 2005).Coating BC with scattering materials such as sulfate was reported to increase (Lack and Cappa, 2010) or decrease AAE to less than one (Schnaiter et al., 2005).Lack and Cappa (2010) pointed out that AAE was 1 for sufficiently small BC (∼ 10 nm) and was subject to change by techniques and analysis methods, thereby suggesting that AAE estimated from measurements should be discussed in view of the analysis method.
The average AAE of the 370 nm group was 1.29, which was higher than the diesel BC AAE (∼ 1.0) but lower than biomass burning BC AAE (∼ 2.0).As discussed above, these samples were influenced by the Beijing plumes, and open biomass burning events were not detected.Consequently, we consider this group to be OC-rich aerosols transported from densely populated areas.In fact, our results lie in the upper limit of AAE (1.0-1.5)derived from OC-rich pollution cases at GCO (Lee et al., 2012).
The AAE of the 880 nm group had a narrow range between 0.95 and 1.05 with a mean of 1.0, which was measured in regions close to the source of BC emitted from high temperature combustion and where externally mixed BC dominates absorption (Bond et al., 2013;Kirchstetter, 2004;Schnaiter et al., 2003).AAE from measurements was found to be ∼ 1.0 near an urban roadway (Kirchstetter, 2004) and 0.9-1.3 in samples from urban plumes (Gyawali et al., 2009).In a previous study performed at GCO, AAE of 0.8-1.5 were derived for aerosols during sulfate rich pollution (Lee et al., 2012), which is in accordance with our result for the 880 nm group.
To examine the different absorption tendencies of the two groups, we calculated BC MAC along wavelengths for the two groups assuming the measured absorption was attributed solely to BC particles measured as EC (Fig. 5b).Our MAC values were 7.5 ± 3.0 and 7.7 ± 3.5m 2 g −1 at 520 nm for the 370 and 880 nm group, respectively.For the 880 nm group, the mean EC mass concentration was higher by 35 % and MAC was also higher up to 13 % at near-IR than those of the 370 nm group.Bond et al. (2013) adopted a value of 7.5 m 2 g −1 at 550 nm for freshly emitted BC and Bond et al. (2006) suggested ambient MAC values in polluted regions as around 9-12 m 2 g −1 .In the study of Lee et al. (2012) performed at GCO, the MAC was 4.2 ± 1.1 m 2 g −1 for sulfate-pollution event, which is much smaller than that of ours for the 880 nm group.Liu et al. (2008) claimed that the absorption cross section of BC particles changed by their morphology as they were being packed from chain-like shape, depending on the compactness of a cluster and the size and number of individual particles.This morphological change in BC particles is coupled with aggregating or internal mixing with hydrophilic particles and coating during aging processes.In particular, the coating of scattering material is known to enhance MAC of carbonaceous core.At GCO, sulfate-rich aerosols are mostly found in aged air masses of Chinese outflows and therefore, the degree of aging associated with SO 2 availability was likely to modify optical properties of carbonaceous aerosols.Considering these complex facts, our MAC of 7.7 ± 3.5 m 2 g −1 for 880 nm group likely represents the absorption characteristics of aged and sulfate dominant aerosols in the study region.It is also noteworthy to mention that our AAEs were derived from regression method using seven wavelengths, whereas they were calculated using wavelength pairs in Lee et al. (2012).On the other hand, the MAC of the 370 nm group was similar to those of the polluted OC-rich aerosols in Lee et al. (2012).The OCdominant aerosols of 370 nm group were less aged than those collected in other periods, for which absorption was highly enhanced in UV region.These results suggest that the MAC of aerosol is greatly dependent on chemical compositions and atmospheric process during aging.
Considering the argument of Lack and Cappa et al. (2010), we compared AAEs calculated for four wavelength ranges: 370-520 nm, 470-660 nm, and 660-950 nm (Fig. 5c).For the 370 nm group, the AAE was increased only at UV region (370-520 nm) by 15 % relative to that of the visible range (470-660 nm) with no considerable difference in the other ranges.In contrast, the AAE of the 880 nm group was decreased by 6 % at UV region (370-520 nm) but increased by 20 % at IR region (660-950 nm), compared to that of the visible range (470-660 nm).These results imply that the enhanced absorption of UV and longer visible light were responsible for the spectral dependence observed in the EBC of the 370 and 880 nm groups, respectively.
It should also be mentioned that this analysis only considered the most dominant species.Therefore, dominant species such as OC and sulfate are likely responsible for enhanced absorption of UV and longer visible light for the 370 and 880 nm groups, respectively.The enhanced absorption of OC dominant aerosols in the UV region is consistent with what was observed from other measurements, which highlights the contribution of OC to aerosol spectral dependence.When sulfate was predominant, particularly in aged air masses in northeast Asia, sulfate likely contributed to light absorption at longer visible light probably as a coating on BC particles.
SSA in relation to major aerosol composition
In this study, enhanced light absorption at 370 nm was found in a fast-moving winter air masses transported from the Beijing region within approximately one day, whereas the high mass absorption at 880 nm was observed in air masses that slowly passed through the Yellow Sea region (bottom panels in Fig. 4).In the northeast Pacific rim, outflows transported from areas around Beijing tended to show a higher ratio of black carbon to sulfate than those from other parts of China, exerting a strong positive influence on the net warming (Lim et al., 2012;Ramana et al., 2010).
In order to examine how chemical composition alters the optical properties of aerosols, thereby affecting climate, SSA was calculated and compared for the two groups (Fig. 5d).The SSAs for the two groups decreased with increase in wavelength with a greater spectral dependence of the 880 nm group.Bergstrom et al. (2002) discussed that the SSA of a mixture of BC and non-absorbing material decreased with wavelength in the solar spectrum, in contrast to most mineral dusts, of which SSA increased with wavelength increase.For this reason, SSA can be used to distinguish aerosol types.The SSA was higher for the 880 nm group than for the 370 nm group, despite the higher absorption of the 880 nm group.For the 370 nm group, the steady SSA values around 350-500 nm could be associated with enhanced absorption by light-absorbing OC, in view of MACs in Fig. 5.In the same context, the greater spectral dependence of the 880 nm group was likely due to absorption enhanced at longer wavelength.This result highlights the importance of considering relative abundance as well as absolute concentrations when estimating radiative forcing of aerosols.
Conclusions
PM 1.0 and PM 10 samples were collected daily for the analysis of soluble ions, OC, and EC from January to September 2008, in conjunction with continuous measurements of absorption and scattering properties and number size distributions of aerosols at GCO.In the comparison of daily averaged EBC with PM 1.0 EC concentrations, two types of spectral dependence were identified with clear maxima (peaks) at either 370 nm or 880 nm, by which two subsets of each five samples were extracted and classified into 370 and 880 nm groups.The groups were distinguished by the relative abundance of major constituents such as OC / EC, OC / sulfate, and EC/surface ratios.While the 370 nm group was characterized by high ratios of OC / EC, OC / sulfate, and EC / sulfate, sulfate was predominant for the 880 nm group.
In the PM 1.0 of the 370 nm group, the ratios of OC / EC and (OC3 + OC4 + OP) / OC were higher than those of the 880 nm group.The main trait of the 370 nm group was the enhanced light absorption at 370 nm and greater contribution of refractory OC and OP to total OC, as previous studies reported that increased absorption, particularly in the UV regions, was from light-absorbing organic matter, frequently referred to as brown carbon.In addition, the refractory and pyrolized OC were well correlated with PM 1.0 EC1 (char EC), which indicates biofuel and biomass combustion from residential heating as the source, particularly during winter.Contrastively, the refractory OC in PM 10 of the 370 nm group was likely derived from alkaline soil in dry lakes, contributing to light scattering.The SEM images and number size distributions together with trajectory analysis suggested that these aerosols were externally mixed in fast-moving air masses from the Beijing area passing over Mongolia in approximately one day.In comparison, some samples with a chemical composition similar to the PM 1.0 of the 370 nm group but with different PM 10 due to the influence of soil minerals, showed a slight increase in absorption at 660 nm without a clear tendency in spectral dependence.
In the sulfate dominant regime, the chemical characteristics of the 880 nm group resulted in the lowest ratios of EC / sulfate and OC / sulfate in PM 1.0 among all of the measurements.The OC / EC ratio was lower in the 880 nm group than the 370 nm group; while OC was similar in the two groups, EC was higher in the 880 nm group.The scattering coefficients at 520 nm were higher for the 880 nm group owing to its high sulfate concentrations in both PM 1.0 and PM 10 .However, the scattering coefficient per sulfate mass was less in the 880 nm group than in the 370 nm group.In addition, the aerosols were highly likely to be internally mixed because the mode and number distributions of aerosols remained unchanged through the day and air masses were transported slowly over the Yellow Sea.
The average AAE estimated from σ ap at seven wavelengths from 370 nm to 950 nm was 1.29 (1.24-1.4) for the 370 nm and 1.0 (0.95-1.05) for the 880 nm groups, respectively.AAEs calculated for several different wavelengths confirmed that the absorption was enhanced in the UV or longer visible light regions, leading to the maxima at 370 nm or 880 nm in EBC concentrations, respectively.In turn, the dominant species such as OC and sulfate were likely responsible for enhanced absorption of UV and longer visible light for the 370 and 880 nm groups, respectively.In the OC dominant regime, the enhanced absorption of UV highlights the contribution of OC to aerosol spectral dependence.When sulfate was predominant, particularly in aged air masses in northeast Asia, sulfate likely contributed to light absorption at longer visible light probably as a coating on BC particles.Finally, SSA was higher for the 880 nm group compared to that of the 370 nm group, despite the higher absorption of the 880 nm group.The results of this study demonstrate that the optical properties of aerosols are intimately linked with their composition and mixing state and revealed the importance of the relative abundance as well as absolute concentrations of absorbing and scattering constituents in determining the climate effect of aerosols.
Figure 1 .
Figure 1.Comparison of the 370 nm group with the 880 nm group.(a) EBC / PM 1.0 EC ratios as a function of wavelength.(b) Ratios of OC / EC, OC3 + OC4 + OP / EC, OP / EC, EC / sulfate, and OC / sulfate in PM 1.0 .Ratios are shown as mean values and error bars indicate standard deviation (1σ ).
Figure 2. (a) Correlation of PM 10 (Na + + Ca 2+ + Mg 2+ + Cl -) with 550 nm scattering coefficient.(b) Correlation of PM 1.0 sulfate with 550 nm scattering coefficient.Scattering coefficient data obtained by nephelometer were collected at 10-minute intervals, and were averaged for each day to be compared with our observed PM data.Lines show linear regressions, for which slopes and R 2 values are given.
Figure 3 .
Figure 3. SEM images of ambient particles.(a) Particles in Regions and/or particles marked by ⅰ, ⅱ, and ball, respectively.(b) Aged soot aggregates with group, collected on 14 April 2008.
Figure 3 .
Figure 3. SEM images of ambient particles.(a) Particles in the 370 nm group, collected on 3 January 2008.Regions and/or particles marked by i, ii, and iii appear to be organic matter, sea salt, and a tar ball, respectively.(b) Aged soot aggregates with larger size and higher compactness in the 880 nm group, collected on 14 April 2008.
Figure 4 .
Figure 4. Comparison of particle number size distribution (top panels) and air mass trajectories (bottom panels) for the 370 and 880 nm groups.(a) 3 January and (b) 14 February are included in the 370 nm group, and (c) 17 April and (d) 21 May are included in the 880 nm group.Particle number concentrations were measured by SMPS.Air trajectories were obtained by Flexpart model footprint images in (Stohl et al., 2005; http://zardoz.nilu.no/~andreas/STATIONS/GOSAN/index.html).The model output (s kg −1) is a potential emission sensitivity distribution of 40 000 particles released in a particular grid cell at the measurement location during the measurement interval and followed backward in time.This is considered proportional to the particle residence time in that cell.
Figure 5 .
Figure 5. (a) Absorption coefficients (σ ap ) of the 370 and 880 nm groups and fitted absorption ångström exponent (AAE).(b) Mass absorption cross section (MAC) of the two groups (Please see text for details of the calculation).(c) AAE for the two groups in different wavelength ranges.(d) Single scattering albedo (SSA) along the wavelengths for the two groups.
Table 1 .
Comparison of the 370 nm group and the 880 nm group.
a The numbers in parentheses are averaged values. | v3-fos-license |
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} | pes2o/s2orc | Reduced-representation Phosphosignatures Measured by Quantitative Targeted MS Capture Cellular States and Enable Large-scale Comparison of Drug-induced Phenotypes*
Profiling post-translational modifications represents an alternative dimension to gene expression data in characterizing cellular processes. Many cellular responses to drugs are mediated by changes in cellular phosphosignaling. We sought to develop a common platform on which phosphosignaling responses could be profiled across thousands of samples, and created a targeted MS assay that profiles a reduced-representation set of phosphopeptides that we show to be strong indicators of responses to chemical perturbagens. To develop the assay, we investigated the coordinate regulation of phosphosites in samples derived from three cell lines treated with 26 different bioactive small molecules. Phosphopeptide analytes were selected from these discovery studies by clustering and picking 1 to 2 proxy members from each cluster. A quantitative, targeted parallel reaction monitoring assay was developed to directly measure 96 reduced-representation probes. Sample processing for proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment have been fully automated, making possible the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12%. This highly reproducible process allowed ∼95% of the reduced-representation phosphopeptide probes to be detected in ∼200 samples. The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures.
duced-representation phosphopeptide probes to be detected in ϳ200 samples.
The performance of the assay was evaluated by measuring the probes in new samples generated under treatment conditions from discovery experiments, recapitulating the observations of deeper experiments using a fraction of the analytical effort. We measured these probes in new experiments varying the treatments, cell types, and timepoints to demonstrate generalizability. We demonstrated that the assay is sensitive to disruptions in common signaling pathways (e.g. MAPK, PI3K/mTOR, and CDK). The high-throughput, reduced-representation phosphoproteomics assay provides a platform for the comparison of perturbations across a range of biological conditions, suitable for profiling thousands of samples. We believe the assay will prove highly useful for classification of known and novel drug and genetic mechanisms through comparison of phosphoproteomic signatures. Molecular & Cellular Proteomics 15: 10 Our understanding of disease mechanisms and therapeutic opportunities is rapidly expanding because of incredible advances in molecular profiling technologies. Within the last decade, the broad application of high-throughput transcriptional profiling has resulted in rich sets of gene expression data collected from biological samples subjected to drug and genetic perturbations (1,2). As an example, the ambitious Connectivity Map (CMap) 1 project (http://www.lincscloud. org/) collects transcriptional profiles from cells perturbed with biologically active compounds or genetic manipulations and enables cross-comparisons of these profiles to help develop insight into the biological mechanisms at play (3,4).
High-throughput transcriptional profiling represents a novel approach to derive functional associations among drugs, genes, and diseases but only reflects one axis of cellular information (gene expression). The proteomic axis, and particularly the post-translational modifications to the proteome, may provide alternate and complementary information for discovering these connections. Initial and sustained signals to environmental changes (such as drug treatment and neomorphic disease states) are frequently mediated by changes of post-translational modifications on proteins. Protein phosphorylation in particular is known to be a strong mediator of cellular signaling (5,6). Changes in the phosphoproteome can result in subsequent disruptions in protein-protein interactions (7,8), alterations in protein stability, changes in cellular localization of proteins (9,10), and potentiation of novel transcriptional programs. Importantly, dysregulation of phosphosignaling is also known to be involved in multiple diseases, including cancer (11)(12)(13)(14)(15)(16)(17). We propose that profiling phosphosignaling responses to drug treatments and other perturbations can generate cellular signatures that will expose novel functional connections complementary to gene expression profiles. Quantitative, mass spectrometry-based proteomics is one tool of choice for generating these profiles because it can provide direct observation of these post-translational events whereas nucleic acid sequence-based techniques cannot.
The majority of protein kinases are S/T-directed and the levels of phosphoserine (pS) and phosphothreonine (pT) are generally higher in abundance than phosphotyrosine (pY) sites. Although there are Ͼ70,000 known pS/pT sites in the human proteome (8,18,19), protein phosphorylation is typically present at sub-stoichiometric levels. Because of the level of phosphorylation and its role in many cell signaling processes, analytical techniques to enrich for protein phosphorylation have been developed. For example, antibody-based assays have been developed to study tyrosine phosphorylation (14,20,21), and metal affinity-based methods have been used to enrich pS, pT, and pY-containing peptides from proteolytic digests of cells and tissues (22,23). In combination with highly sensitive mass spectrometry workflows, these enrichment techniques have facilitated global phosphoproteomic studies in many biological systems (24 -27).
However, to facilitate modern "omics" analyses and leverage techniques pioneered in gene expression studies, it would be highly desirable to have reproducible observations of phosphopeptide analytes across large numbers of samples generated under different conditions. Yet, even with the fastest and most sensitive MS instruments currently available, it is not possible to reproducibly measure all of the same peptides or modified peptides across multiple experiments using datadependent analysis methods. Comparisons across even small numbers of proteomic experiments are difficult because dif-ferences in sample processing protocols and mass spectrometry data acquisition methods can cause sampling variation (28 -31). Variations in data production may result in the lack of phosphosite detection even when the modification is present. As a result, it is currently not possible to reproducibly and quantitatively monitor all known phosphosites in a large number of human phosphoproteome samples.
To overcome these challenges, we considered that phosphorylation is mediated by just a few hundred protein kinases and phosphatases that can modify hundreds of thousands of amino acid sites on various protein targets. We hypothesized that the implicit one-to-many relationship of kinases to substrates suggests that there is some redundancy in the cellular information conveyed by phosphorylation and that collapsing the number of monitored sites based on their coordinate activity could provide a core set of highly informative phosphopeptide probes. This idea is consistent with work published by Alcolea et al., where phosphosignaling events within acute myeloid leukemia cell lines with different sensitivities to kinase inhibitors were profiled to reveal several hundred correlated phosphorylation sites that were involved in parallel kinase pathways (32). In addition, a similar strategy was used to develop the "L1000" reduced-representation transcriptional profiling assay that is the basis for the transcriptional Connectivity Map project. The L1000 assay retains 80% of information content at Ͻ1% of the cost of microarray or RNA-Seq-based expression profiling (33).
Monitoring a reduced-representation set of phosphopeptide probes using a targeted MS approach could be a time-and cost-effective approach to monitor changes in phosphosignaling in response to multiple drug and genetic perturbations. Such an assay could identify connections between molecular perturbations and elucidate cell type-specific cell signal transduction using analysis methods similar to those utilized in the transcriptional profiling field. A similar approach was recently reported by Picotti and colleagues, who developed a targeted proteomic assay to probe biological processes in Saccharomyces cerevisiae in response to environmental perturbations by selecting sentinel proteins from existing data (34). Such targeted approaches could eliminate stochastic sampling effects and allow for accurate quantification across large sample sets without significant loss in information content relative to a full phosphoproteome.
The work described below explores this possibility and consists of three main sections: (1) a discovery arm where we identify a high-value set of phosphopeptide probes from traditional, data-dependent large scale SILAC-based phosphoproteomic data, (2) a configuration arm where we develop a targeted, internally standardized phosphopeptide assay that generates almost complete data (data that contains observations for all phosphopeptides), and (3) a proof-of-principle arm where we explore the sensitivity of the assay to diverse perturbations and biological systems and demonstrate its general utility. In our discovery arm we produced global phos-phorylation data representing 156 samples that included 26 chemical perturbations in three different cell lines using conventional phosphopeptide enrichment and MS-based techniques. From these data we selected representative phosphosites (and their associated observable peptides) from clusters of sites that exhibited coordinated regulation across discovery experiments. We then configured a targeted parallel reaction monitoring (PRM) assay against these phosphopeptide probes using isotopically-labeled synthetic analogs, and in parallel, developed automated workflows that enabled proteolytic digestion, protein quantification, peptide desalting, and phosphopeptide enrichment from 96 samples simultaneously in anticipation of scaling the assay to generate a large corpus of data. Our initial evaluation of the configured assay was to regenerate samples under similar conditions to those used in our discovery experiments to see if we could recapitulate the previously observed relationships using only the reduced-representation phosphoproteome at a fraction of the effort required for deeper phosphoproteomic profiling. Finally, in our proof-of-principle arm we extended our experimental conditions to vary the treatments, cell types, and time points measured with the explicit goal of demonstrating that the assay could generate phosphoproteomic profiles outside of the parameters under which it was developed and to demonstrate that the assay is responsive to disruptions of signaling pathways of known biological importance.
We call this assay "P100" because it measures ϳ100 phosphopeptide probes in a single 60 min assay. This assay is a phosphosignaling analog of our Global Chromatin Profiling assay (35,36) that has already proven to be of great utility. We believe that the P100 assay will prove highly useful for classification and stratification of drug and genetic mechanisms as facilitated through comparison of phosphoproteomic signatures of known chemical and genetic perturbations to those of novel perturbations or those where mechanistic insight is currently lacking. Together, these proteomic signature generation assays can form the basis for proteomic Connectivity Maps to complement their transcriptional analogs.
Drug Treatment and Harvest-The following applies to discovery, configuration, and confirmation experiments. Compounds were obtained from Sigma (St. Louis, MO) or EMD Millipore (Darmstadt, Germany), with the exception of JQ-1 which was the generous gift of Dr. James Bradner (Dana-Farber Cancer Institute, Boston, MA). Once cells reached ϳ95% confluence, they were treated with the compounds listed in Table I and supplemental Table S1 for 6 h at 37°C. After 6 h, the cells were washed twice with cold PBS (Gibco, 10010 -023) and harvested by scraping. Cells were pelleted at 1000 ϫ g for 2 min. The supernatant was then removed, and the cell pellet was frozen in liquid nitrogen until cell lysis and phosphopeptide enrichment.
For discovery experiments, we used three-state SILAC labeling to determine quantification of phosphoproteomic changes. In these experiments, we held the "light" channel constant as DMSO and varied the drugs in the medium and heavy channels (as schematically depicted in Fig. 1C and listed in Table I). For each drug/cell type combination, two complete biological repeats (grown several weeks apart) were performed. Ratios were determined using MaxQuant (see below) of treatment versus DMSO for each drug. The entirety of the discovery data set, including a table that specifies SILAC labeling states for each of the 26 drug combinations, can be found in ftp:// [email protected]. This data set has been deposited in MassIVE with accession ftp://MSV000079524@massive. ucsd.edu.
For all other experiments, cells were grown in typical growth medium without metabolic labeling of proteins. Instead, the synthetic versions of the P100 assay peptides (probes) are used to derive quantitative information.
Embryonic Stem Cell and Neural Progenitor Cell Culture and Treatment-Individual colonies of H9 human embryonic stem cells (ESCs) were cultured with mouse embryonic fibroblast-conditioned media (MEF-CM; provided by Sanford-Burhnam Research Institute, La Jolla, CA) in matrigel (BD, 356231)-coated plates. For neural progenitor cell (NPC) induction, ESC cell colonies of 60 -80% confluence were incubated in MEF-CM containing each 5 M of dorsomorphin (Sigma, P5499), A83-01 (Sigma, SML0788) and PNU 74654 (Sigma, P0052). Once NPC cells and reached ϳ95% confluence, they were treated with the compounds listed in Supplemental Table S2 for 24 h at 37°C. H9 cells or NPCs were harvested by washing twice with cold PBS (Gibco, 10010 -023) and centrifugation at 1000 ϫ g for 2 min. The supernatant was removed, and the cell pellets were frozen in liquid nitrogen until cell lysis and phosphopeptide enrichment.
Discovery Phosphopeptide Enrichment-Phosphopeptide enrichment was completed using Phos-Select Iron Affinity gel (Sigma, P9740) that were prepared by washing four times with 40% acetonitrile/0.1% formic acid. Prior to enrichment, peptides were reconstituted in 40% acetonitrile/0.1% formic acid. Phosphorylated peptides were enriched for with 15 l IMAC beads for each sample for 30 min. Phosphopeptide enrichment was completed as previously described (23). After enrichment, Phos-Select gel was loaded on Empore C18 silica-packed stage tips (3M, 2315, St. Paul, MN) and desalted (38). Briefly, StageTips were equilibrated with 2 ϫ 100 l washes of methanol, 2 ϫ 50 l washes of 50% acetonitrile/0.1% formic acid, and 2 ϫ 100 l washes of 1% formic acid. Samples were then loaded onto stage tips and washed twice with 50 l of 80% acetonitrile/0.1% trifluoroacetic acid and 100 l of 1% formic acid. Phosphorylated peptides were eluted from IMAC beads with 3 ϫ 70 l washes of 500 mM dibasic sodium phosphate, pH 7.0, and washed twice with 100 l of 1% formic acid before being eluted from stage tips with 100 l 50% acetonitrile/0.1% formic acid. All washes were performed on a tabletop centrifuge at a maximum speed of 3,500 ϫ g.
Phosphopeptide Enrichment-Desalted samples were reconstituted in 80% ACN/0.1% TFA that contained isotopically labeled standards for enrichment quality control and moved to an AssayMAP Bravo robotic system (Agilent, Santa Clara, CA). AssayMAP cartridges containing Ni-NTA-agarose packing material (Qiagen, 1018611) were washed with water, stripped with 100 mM EDTA, and loaded with 100 mM FeCl 3 . Fe-NTA cartridges were primed with 1:1:1 ACN/methanol/0.01% acetic acid, and samples were loaded at 5 l/min. Flow-throughs were re-loaded onto cartridges at a flowrate of 2 l/min. Cartridges were washed with 80% ACN/0.1% TFA, and peptides were eluted with 500 mM K 2 HPO 4 (pH7) at 5 l/min. Eluates were vacuum concentrated to dryness, and subsequently desalted using AssayMAP RP-S cartridges according to the manufacturer's instructions. A detailed protocol is shown as Supplemental Protocol 2.
Phosphopeptide Enrichment-Desalted samples were reconstituted in 80% ACN/0.1% TFA that contained isotopically labeled standards for enrichment quality control and moved to an AssayMAP Bravo robotic system (Agilent). Agilent AssayMAP Fe-(III)-NTA cartridges (note that we changed the enrichment media from the previous experiment) were washed with water, stripped with 100 mM EDTA, and loaded with 100 mM FeCl 3 . Fe-(III)-NTA cartridges were primed with 1:1:1 ACN/methanol/0.01% acetic acid, samples were loaded at 20 l/min and flow-throughs were re-loaded onto cartridges eight additional times. Cartridges were washed with 80% ACN/0.1% TFA, and peptides were eluted with 500 mM K 2 HPO 4 (pH7) at 5 l/min. Eluates were vacuum concentrated to dryness, and subsequently desalted using AssayMAP RP-S cartridges according to the manufacturer's instructions. A detailed protocol is shown in supplemental Protocol S3: Optimized Automated Phosphopeptide Enrichment SOP using Agilent NTA-polymeric resin.
Discovery Mass Spectrometry-Each sample was subjected to 3 consecutive LCMS analyses with the following rationale. First, data were acquired using a typical data-dependent top 12 method (parameters below). These data were searched using MaxQuant version 1.2.2.5 (see below). A second round of acquisition was performed using tailored exclusion lists (AMEx (30)) for each sample to minimize re-acquisition of the same precursors in consecutive analyses. Again, these data were searched in MaxQuant. When the first two rounds of acquisition and search were complete, we determined peptides (and their corresponding precursor m/z and retention times) that had been identified in a majority of the samples but lacked quantitative information in some samples. We rationalized that targeted MS/MS acquisition for these peptides would lead to a more complete data matrix for analysis. Therefore, we created individual precursors lists for each sample to "fill in the blanks" consistent with the principles of AIMS (39) data acquisition.
All three acquisition strategies employed the same LC separation conditions described below. Samples were chromatographically separated using a Proxeon Easy NanoLC 1000 (Thermo Scientific) fitted with a PicoFrit (New Objective, Woburn, MA) 75-m inner diameter capillary with a 10 m emitter tip was packed under pressure to ϳ20 cm with C 18 Reprosil beads (1.9 m particle size, 200 Å pore size, Dr. Maisch GmBH) and heated at 50°C during separation. Samples were loaded in 4 l 3% ACN/1% formic acid and peptides were eluted with a linear gradient from 7-30% of Buffer B (0.1% FA and 90% ACN) over 82 min, 30 -90% Buffer B over 6 min and then held at 90% Buffer B for 15 min at 200 nL/min (Buffer A, 0.1% FA and 3% ACN).
During data-dependent acquisition, eluted peptides were introduced into a Q-Exactive mass spectrometer (Thermo Scientific) via nanoelectrospray (2.15 kV). A full-scan MS was acquired at a resolution of 70,000 from 300 to 1800 m/z (AGC target 1e6, 5ms Max IT). Each full scan was followed by top 12 MS2 scans at a resolution 17,500 (Isolation width 2.5 m/z, ACG Target 5e4, 120 ms Max IT).
For AMEx (30) runs, individual exclusion lists were added and the match tolerance was set as the software default. Dynamic exclusion was set at 20 s.
For AIMS (37) runs, individual inclusion lists were added and the match tolerance was set as the software default. If fewer than 12 inclusion list targets were active, then the balance of MS/MS scans were selected from next most intense precursors subject to dynamic exclusion (20 s).
Targeted Mass Spectrometry-Targeted LCMS Data Acquisition-Samples were chromatographically separated using the same conditions as the Discovery Mass Spectrometry with the following changes. Samples were reconstituted in 10 l 3% ACN/5% formic acid containing isotopically labeled versions of all phosphopeptide probes. Peptides were eluted using a shorter linear gradient from 3-40% of Buffer B over 45 min, 40 -90% Buffer B over 5 min and then held at 90% Buffer B for 10 min at 200 nL/min. Eluted peptides were introduced into a Q-Exactive mass spectrometer (Thermo Scientific) via nanoelectrospray (2.15 kV). A full-scan MS was acquired at a resolution of 35,000 from 300 to 1800 m/z (AGC target 3e6, 50 ms Max IT). Each full scan was followed by fully scheduled, targeted HCD MS/MS scans at resolution 17,500 (Isolation width 2 m/z, ACG Target 2e5, 50 ms Max IT). Each peptide species was subjected to targeting MS/MS for 3-5 min depending on the empirical chromatographic properties, centered on the average observed retention time of two scheduling runs containing synthetic versions of a subset of isotopically labeled phosphopeptide probes.
For the time-course experiments, eluted peptides were introduced into a Q-Exactive Plus mass spectrometer (Thermo Scientific) with the same parameters described above with the following exceptions: full-scan MS was acquired at 35,000 from 300 to 1200 m/z (AGC target 3e6, 20 ms Max IT) and HCD MS/MS scans at 17,500 (Isolation width 1.7 m/z with 0.3 m/z offset, AGC target 2e5, 50 ms Max IT).
The raw mass spectrometry data have been deposited in the public proteomics repository MassIVE and are accessible at ftp:// [email protected]. Skyline files corresponding to the targeted analyses can be accessed at: https://panoramaweb. org/labkey/project/LINCS/AbelinSupplemental/begin.view?.
MaxQuant Data Analysis-Raw MS Data were searched with Max-Quant version 1.2.2.5 against the Uniprot Human Protein Database (Complete Isoforms, download date 20-MAR-2012) containing 81489 entries. Three-state SILAC was specified: Arg0:Lys0, Arg6:Lys4, and Arg10:Lys8, and the following modifications were allowed: Carbamidomethylation of C (fixed); Oxidation of M, Phosphorylation of S, T, and Y, Acetylation of N termini (variable). The mass tolerances allowed were 7 ppm for MS precursors and 20 ppm for MS/MS fragments. Peptide, protein, and site FDRs were 0.01 as calculated using by MaxQuant. H/L and M/L ratios were extracted for phosphosites, and log2-transformed. All MaxQuant tables, including parameters, can be found in ftp://[email protected]. Phosphosites that were observed in at least 75% of all experiments were retained. If a data point was missing for a particular phosphosite in a given condition, we imputed its value by random sampling of a normal distribution based on the mean and standard deviation of all other measurements of that phosphosite in other conditions (6.9% of values were imputed). Subsequently, variance was measured for each phosphosite, and sites with the lowest 15% of variance across all experiments were discarded as uninformative. The final yield of sites used for subsequent calculations and selections was ϳ1000.
We performed hierarchical clustering of phosphosite ratios using GENE-E (http://www.broadinstitute.org/cancer/software/GENE-E/ index.html), clustering both samples and sites using a distance metric of 1-Pearson's correlation. Principal Component Analysis of these data was performed using R. After reducing the data to 55 distinct clusters, representative phosphopeptides were selected from each cluster using an in-house Perl-script that combed through MaxQuant data tables, ranking each by heuristic rules designed to maximize success in automated proteomic workflows and data analysis (i.e. easy site localization, high re-observability, absence of missed tryptic cleavages, etc.). The peptides that were ultimately selected can be found in supplemental Table S3.
Skyline Data Analysis-Data Analysis-Files were imported into a Skyline-daily (40) with pre-selected charge states for all reduced-representation phosphopeptide probes (Supplemental Skyline Document 1) and for the quality control internal standard phosphopeptide probes (Supplemental Skyline Document 2). Transitions were chosen on the basis of selectivity for phosphosite localization and detectability. Each sample and phosphopeptide probe was manually validated using the criteria of retention-time agreement with other samples and the co-eluting presence of all transitions with corresponding isotopically labeled standards. Heavy/light ratios were extracted on the basis of transition area integration using Skyline defaults. Data for each modification were normalized by the median of all samples before clustering. Clustering was performed in GENE-E (http://www.broadinstitute.org/cancer/ software/GENE-E/index.html) using unsupervised hierarchical methods with the following methods: Pearson correlation, row and column clustering.
Computation and Visualization of Connectivity Maps-All possible intersample Pearson correlation coefficients were calculated using P100 profiles. A "sample group" was defined for each set of replicates for a given compound in a given cell type. An intergroup connectivity was assigned as the average correlation between all replicates in one group to another group. As an example, let us assign Group A as the three replicates of digoxin in MCF7 cells and Group B as the 3 replicates of lanatoside C in PC3 cells. It would follow that the intergroup connectivity between Group A and Group B is the average of the nine correlation coefficients possible between replicates of these groups.
Cytoscape (41) was used to visualize sample groups and the computed intergroup connectivity. Node arrangements were computed using preset spring-embedded and/or force-directed layouts options, using the intergroup correlations as the weights. Edge thickness is directly proportional to the intergroup connectivity. RESULTS We set out to develop a rapid and robust targeted proteomic assay that could generate molecular signatures based on the phosphorylation state of cellular proteins. An overview of this process is presented in Fig 1 A. We first identified small molecules that were predicted to modulate phosphorylation in pleiotropic (but non-uniform) ways. We then treated multiple cell types with these compounds and collected large-scale global phosphorylation data. We rationalized that, because a small number of kinases and phosphatases (Ͻ1000) seemingly modulate a large number of phosphosites (Ͼ100,000),
A.
Select Effective Drugs many sites were likely to be coordinately regulated. Thus, traditional "deep" phosphoprofiling may convey redundant information when the levels of two or more sites are modulated similarly across a wide number of perturbations. We hypothesized that a smaller set of sites could serve as surrogate markers for a larger set. From our global phosphorylation data, we were able to identify groups of phosphosites that behaved similarly upon drug treatment, from which we selected highly-observable representative phosphopeptides. We then configured a reduced-representation targeted MS assay for these phosphopeptide probes ("P100"). We executed the P100 assay in new samples under similar treatment conditions used in our discovery experiments to confirm that the assay was functional. We also performed the P100 assay on samples from two new cell types and several additional compounds that were not used for assay configuration to further demonstrate the functionality of the assay under diverse conditions. Finally, we compared phosphosignaling profiles collected from cells treated with compounds of known mechanisms in a time-course experiment to demonstrate that the assay is sensitive to disruptions in multiple common signaling pathways, including MAPK, PI3K/mTOR, and CDK cell cycle. These experiments also allowed us to analyze the time-dependence of emergence of phosphosignatures. Overall, we established a data generation platform that enables the production of large-scale, phosphosignaling signatures that can be used to stratify drugs and other perturbations into classes and to compare known reference drugs to novel compounds, helping to further elucidate their mechanisms. Discovery Experiments-To find small molecules with pleiotropic effects on phosphosignaling, we analyzed gene expression data in the Connectivity Map (CMap, https://www.broad institute.org/cmap/) for compound treatments that positively and/or negatively regulated the expression of groups of kinase and phosphatase genes. The hypothesis was that, if a set of kinases' expression is modulated, then the phosphosites that they regulate should be coordinately modulated as well. We avoided highly specific, single kinase inhibitors, as such compounds would not be likely to generate the desired pleiotropic effects on the phosphoproteome.
At the time of our analyses, the CMap data set consisted of Affymetrix array data for Ͼ700 samples across cell lines representing 4 lineages: breast cancer (with both regular and serum stripped growth conditions), skin cancer, prostate cancer, and leukemia, where each sample is the collapsed gene expression profile (derived from Ͼ6000 individual profiles, or 8 -9 profiles/sample) of a compound treatment in a particular cell type. We began by extracting all Affymetrix probes corresponding to genes annotated as kinases or phosphatases (a total of 317 probes). We then clustered these data in the sample dimension, such that cell/compound treatment combinations with similar kinase/phosphatase gene expression profiles segregated together (Fig. 1B). We scored each cluster for the number of genes positively or negatively regulated. Examples of high scoring clusters are indicated by arrows along the top in Fig. 1B. Representative compounds were selected from high-scoring clusters, with an emphasis on diversity of the compounds (indicated by tick marks in the track below the running average in Fig 1B). In some cases we selected several structural analogs of a compound family to compare their effects on cells. Ultimately, we selected 26 compounds (including 1 broad spectrum kinase inhibitor and 1 broad spectrum phosphatase inhibitor; Table I) that we FIG. 1. Large-scale transcriptional and phosphoproteomic profiling data for identification reduced-representation phosphoproteome probes. A, A schematic depicting the development of the P100 assay. Drug treatments that modulated phosphorylation in pleiotropic ways were selected, and large-scale global phosphorylation data were collected from multiple cell types treated with these drugs. Representative phosphopeptide probes were identified from these data and used to configure the P100 assay. Confirmation and proof-of-principle of the P100 assay's functionality were demonstrated via classification and stratification of samples from multiple biological contexts. Associations among samples were then identified using P100 data. B, Affymetrix gene expression levels corresponding to genes annotated as kinases or phosphatases were extracted from the CMap database and clustered (dendrograms omitted for clarity). Combinations of cell/compound treatments with similar kinase/phosphatase gene expression profiles are illustrated and groups with similar profiles are summarized by the "Running Average" graphic above. Arrows indicate strongly coordinated groups of samples. C, A workflow for the large-scale discovery experiments is depicted. Cells were grown in SILAC medium, lysed, digested, fractionated, and analyzed using high resolution UPLC-MS. D, A clustered heatmap representing the 1,200 commonly observed phosphosites that were present in Ͼ75% of all MS experiments is displayed. Groups of phosphosites with coordinate activity are clustered together along the vertical axis, whereas samples are clustered along the horizontal axis (dendrograms omitted for clarity). Data underlying the figure are available in Supplemental Data Set 1. deemed to be strong candidates for general modulation of kinase and phosphatase genes across a range of cell types. Next, we measured the cellular phosphoproteome after perturbation by these 26 different compound treatments (compared with DMSO control) across three cell lines (MCF7, PC3, and HL60) in biological duplicate, for a total of 156 experiments (scheme in Fig. 1C). The amalgamated data set quantified more than 10,000 unique phosphosites. Importantly, we found over 1200 sites that were quantified in Ͼ75% of all experiments. Using only these 1200 sites, we imputed missing values as described above, and low variance phosphosites were discarded as uninformative (bottom 15%). To better visualize the data set, we performed hierarchical clustering of both the conditions (x axis) and the detected phosphosites (y axis) (Fig. 1D).
We analyzed the discovery data set with an eye toward finding reproducible responses (signatures) of drug treatments coordinately exhibited by multiple phosphosites for single or related conditions (ftp://MSV000079524@ massive.ucsd.edu). If such signatures and coordinated groups could be found, we could then select representative or "proxy" phosphopeptide marker(s) and thereby formulate a condensed phosphoproteomic assay. Selected regions of the heatmap from Fig. 1D are shown in Fig. 2 to illustrate these concepts. We show a unified response to paclitaxel treatment across all three cell lines studied in Fig. 2A, with up-regulation of several phosphosites across a diverse set of proteins. Note that all 6 biological replicates of paclitaxel treatment cluster together in the heatmap regardless of the cell line being studied (MCF7, PC3, or HL60). We call this a lineage-independent signature, and it is notable as these cell lines are of diverse origin (including a hematological line that grows in suspension, HL60). Although many other phosphosites also contribute to these treatments clustering together, these particular sites (shown in Fig. 2A) We demonstrate both lineage-independent and lineagespecific signatures across a set of structural analogs in Fig. 2B and 2C. Digoxin, commonly known as Digitalis, is a cardiac glycoside widely used in the treatment of various cardiovascular conditions (42). Its structural analogs digoxigenin, digitoxigenin, and lanatoside C are reported to have similar bioactivity. Nearly all biological replicates of these compounds cluster together across the MCF7 and PC3 cell lines and elicit a common up-regulation of a series of phosphosites (Fig. 2B). Interestingly, the antibiotic protein synthesis inhibitor anisomycin also clusters together with the cardiac glycosides, although this observation is of unknown significance. In parallel, we can also detect a lineage-specific down-regulation of a series of sites that only seems to occur in MCF7 cells (Fig. 2C). These examples show that phosphoproteomic signatures are potentially useful in recognizing common mechanisms of actions of related compounds yet retain enough information to differentiate subtleties in cellular response.
Although we believe that these phosphoproteomic signatures are generalizable and are useful on their own, the derivation of specific biological information from these data is also desirable. We noticed an interesting cluster of phosphosites in the data set that seemed to be coordinately regulated across a diverse number of conditions (Fig. 2D). By "coordinately regulated," we mean that the group of sites went up or down as a group in response to different treatment contexts and across multiple cell types. We immediately recognized that the parent genes of the sites shown in Fig. 2D seemed to have a strong bias toward chromatin function, and this was borne out by performing a functional enrichment test against the Gene Ontology categories which showed that the parent genes were enriched for Chromatin Binding (GO:0003682) with an FDR-adjusted p value of 7 ϫ 10 Ϫ3 when compared with all parent genes in the data set. We further learned that seven of the eight parent genes in the cluster are known to participate in protein-protein interactions in a tight network (Fig. 2E) in the STRING database (43). This observation raises the intriguing possibility of direct regulation of the chromatin machinery by phosphosignaling in a coordinated manner. Although there are likely numerous other biological insights to be gained from deeper analysis of the discovery data set, this was beyond the scope the present study, the aim of which was to develop an efficient phosphosignaling panel for wider deployment.
Reducing Representation and Configuring the P100 Assay-We employed a reductionist approach to collapse the large universe of phosphosites that could be monitored into a relatively small number of detectable surrogate targets, referred to as the reduced representation set. This innovation will allow retention of important signaling information at a fraction of the effort normally associated with deep phosphoproteomic profiling. This idea draws inspiration from the L1000 assay developed for the Library of Integrated Network-based Cellular Signatures (LINCS) at Broad Institute that reduces the number of transcripts needed for monitoring global expression profiling. LINCS efforts have resulted in a 1000plex Luminex-based gene expression assay that retains 80% of information content at Ͻ1% of the cost of microarray or RNA-Seq-based expression profiling. A similar proteomic approach has recently been illustrated by Soste et al., in the context of signaling in yeast (34).
We began by performing hierarchical clustering on our high quality phosphoproteomic data as depicted in Fig. 1D and Fig. 2. Using principal component analysis, we estimated that it would take at least ϳ40 -50 components to explain 80% of the total variance in the data set. We therefore made simple linear models and measured the sum-squared-error of these models versus a partial hold-out of the data (cross-validation) for increasing numbers of components above 40. We found a local minimum in the error at 55 components and therefore went on to set thresholds on our hierarchical clustering data that divided the phosphosites into 55 different components. We note that this achieves approximately the same degree of compression (1200 sites to 55 sites, or ϳ20 fold) of the L1000 gene expression assay (20,000 genes to 1000 genes). Lastly, we selected 2 phosphopeptide probes from each of the 55 clusters for a total of 110 phosphopeptides (the "P100 probes"). These probes were chosen using heuristic rules designed to maximize mass spectrometric ease of observation (i.e. minimize missed tryptic cleavages, bias toward minimal phosphosite localization possibilities, etc.).
Building on the work above, we developed a high resolution, accurate mass targeted assay to identify and quantify ϳ100 highly informative and representative phosphopeptides across multiple cell types treated with various drugs. Several of our initial 110 probes peptide probes could not be successfully configured into the targeted assay for various reasons (i.e. failed peptide synthesis, poor peptide stability, unacceptable MS performance, etc.), resulting in a final panel of 96 phosphopeptide probes in supplemental Table S3. An analysis of the peptides that we selected (using PhosphoMotif Finder) suggests quite a diversity of possible kinase substrates, with 27 unique known motifs spanning a range of kinases (including CDKs, GSK3, PKA, and PKC) among our 96 probes (44) (supplemental Table S4).
Anticipating deploying this assay at a large scale, we automated the sample processing protocol as depicted in Fig. 3A. After a manual cell lysis step, the automated sample processing protocol encompassed protein quantification (using a colorimetric assay), protein digestion, IMAC phosphopeptide enrichment, and sample desalting using Agilent Bravo liquid handling and AssayMAP platforms. Automation facilitated the simultaneous processing of 96 samples in only 3 days, with a plate phosphopeptide enrichment variance of 12% as determined using stable-isotope-labeled internal standards (supplemental Fig. S1). This highly reproducible process allowed ϳ95% detection of the reduced-representation phosphopep-tides in close to 200 samples. Detailed protocols for the automated portions of the method are provided in supplemental Protocols S1, S2, and S3. We were able to generate phosphosignaling profiles in under 2 weeks from the time of cell harvest to processed results using this assay by directly measuring the reduced-representation set of phosphopeptides across multiple cell lineages and perturbations.
The top signaling pathways from the KEGG database (45) represented by the 1200 phosphopeptides that were used to generate the reduced-representation phosphopeptide probe
C.
FIG. 3. P100 automated sample processing, pathway coverage, and data analysis pipeline. A, A schematic of automated P100 sample processing. On the first day, cells are lysed, subjected to protein quantification and diluted to a uniform concentration. Proteins are then reduced, alkylated, and digested. On the second day, samples are desalted using a 96-well device and dried overnight. On the third day, phosphopeptide enrichment by IMAC and desalting occurs. Finally, samples are analyzed using high resolution UPLC-HCD MS/MS. B, The top signaling pathways represented by the larger set of phosphopeptides used to identify the reduced-representation phosphopeptide probe set are shown. Each signaling pathway is depicted as a circle that is sized to indicate the number of source proteins involved in a specific pathway. A larger size indicates that a signaling pathway is represented by a larger number of phosphopeptides. The color of each pathway is only meant to show the diversity of the signaling pathways represented. C, The data analysis pipeline for the P100 assay is shown. Data are collected in a 96-well plate format, analyzed within Skyline, and exported for summarization. Phosphopeptide probes and sample outliers are removed and the light/heavy peptide ratios are normalized. Quality controlled data are hierarchically clustered and molecular signatures for different perturbations are revealed. set are shown in (Fig. 3B). The abbreviations for each signaling pathway are shown on a circle that is sized to indicate the number of source proteins involved in a specific pathway that are present in the set of 1200 phosphopeptides. A larger size indicates that a signaling pathway is represented by a larger number of phosphopeptides. The edges of the signaling network in Fig. 3B represent source proteins that are shared among different signaling pathways, and thicker lines indicate a higher number of source proteins are shared between two pathways. We believe that these pathways should be covered in the targeted assay by proxy through the reduced-representation set, and indeed demonstrate this for several of them (see results and discussion of Fig. 7 below). All targeted P100 phosphoproteomics data are analyzed using Skyline (40), and subsequent data reduction, QC, and visualizations are achieved through a series of R-scripts to facilitate reproducible research. These R-scripts are automatically executed upon uploading of P100 Skyline data documents into our Panorama server (https://panoramaweb.org/labkey/LINCS. url). A schematic illustrating the final P100 data analysis pipeline is presented in Fig. 3C.
P100 Assay Confirmation and Proof-of-Principle-To confirm that the P100 assay could produce molecular signatures of comparable utility to those from larger scale phosphoproteomic data, we replicated a subset of the perturbational conditions from our discovery experiments (supplemental Table S1 and supplemental Data Set S2). We illustrate the results of these experiments in Fig. 4. Fig. 4A presents an overview of the entire data set, where all of the samples are clustered by their P100 signatures (columns) and all of the P100 probes are also clustered (rows). The value in each cell is the ratio of the endogenous (light) version of the peptide to the internal standard (heavy) version, after row-based normalization and Z-score transformation. Therefore, the value is equivalent to the number of standard deviations away from the mean a given phosphopeptide is in a sample relative to all other samples. Importantly, very few missing values are present in this data set (they appear as gray cells when present). After data QC and filtering, we were able to derive acceptable measurements from 141 of 144 possible samples while retaining sufficient data quality from 92 of the 96 P100 probes. Samples were rejected if less than 80% of the probes were observed, and probes were rejected if they were present in less than 90% of samples. It should be noted that a missing value does not indicate that we failed to detect the peptide via its internal standard, but rather that the endogenous level was too low to detect. For the future, we could either assign these values as zeros or use another technique to substitute a suitable value.
We isolated regions of this heatmap from Fig. 4A, shown as cyan and green boxes, to draw attention to how samples treated with similar compounds clustered together. In general, we observed that biological replicates and structurally related compounds clustered closely together in the assay. For ex-ample, phosphosignaling profiles that arose from digoxigenin, digitoxigenin, digoxin, and lanatoside C treatments all clustered together in the assay (cyan box). We also observed that phosphoprofiles of the same treatments clustered across cell lineages, while retaining some lineage-specific determinants, as shown in the green box for GW8510 treatments. We present an alternative visualization of these results in https:// panoramaweb.org/labkey/project/LINCS/AbelinSupplemental/ begin.view?, with accompanying text in supplemental Methods and Results 1. We think that this alternative "connectivity map" view allows intuitive insight into biological results.
A summary of the pairwise correlation comparisons among non-self (a sample of one cell type treated with one compound compared with other cell line and treatment combinations), the same drug treatments across cell lines, and the same drug treatment within each cell line are presented in Fig. 4B. In the left panel, the distribution of non-self pairwise Pearson correlations among phosphoprofiles is plotted. If a sufficient number of samples are profiled, and the phosphosignaling signatures obtained are not systematically biased in some way, one would expect that comparison of any two random samples (through their signatures) would not yield a strong correlation. Thus, the distribution of pairwise correlations of all samples should be approximately normal and centered on zero. Indeed, we observe such a distribution of non-self pairwise correlations as expected, which demonstrates that the assay is not systematically biased. Intriguingly, the long tail at the right of the distribution shows some samples do demonstrate strong correlations despite being treated with different compounds. Based on the observations from Fig. 4A, these strong correlations likely occur among class members or structural analogs. This suggests that the P100 profiles may be useful in classifying and stratifying novel compounds and perturbations.
The middle panel of Fig. 4B shows the correlations among the phosphoprofiles produced from the same drug treatment across differing cell types, characterizing the lineage-independent response in the assay. If a drug treatment impacts cell signaling pathways similarly across cell types, the distribution of pairwise correlations should shift toward a positive correlation. The distribution of pairwise correlations between the same drug treatments across different cell lines does shift toward 1 indicating that the same drug has similar effects on phosphosignaling across cell types. However, the majority of the pairwise correlations reside between 0 and 0.5, demonstrating that, although detectable, lineage-independent responses to the same drug probably only represent a portion of the signatures obtained through P100.
In the right panel of Fig. 4B, the pairwise correlations among the phosphoprofiles of the same drug treatment within each cell line (biological replicates) are displayed. The distribution of pairwise correlations now shifts even more toward a positive correlation demonstrating the reproducibility of the entire P100 assay process across different drug treatments.
The strong rightward shift toward 1 demonstrates excellent "replicate recall," meaning that biological replicates are likely to be strongly correlated. Taken together, the middle and bottom panels suggest that there is a strong lineage-specific component to signaling responses in cells although some commonality persists at a lineage-independent level.
To confirm that the P100 assay could retain utility in biological systems beyond those used for development, we conducted a study using cell lines and perturbations that were not used during assay configuration. We selected human embry-onic stem cells (ESC) and derived neuronal precursor cells (NPC) from them as these represent extremely active areas of research in developmental biology. We chose to treat the ESCs and NPCs with compounds known to affect levels of chromatin modifications because epigenetic regulation has been shown to be important for both maintenance of and exit from pluripotency (46). Moreover, mutations in some epigenetic modifier genes have been identified as genetic risk factors associated with autism spectrum disorders and other neurodevelopmental etiologies (47). The drug classes represented in this study include a BRD4 inhibitor, an EZH2 inhibitor, and HDAC inhibitors. The P100 phosphosignaling profiles generated from epigenetically active drug-treated ESCs and NPCs are shown in Fig. 6 (see also https://panoramaweb.org/ labkey/project/LINCS/AbelinSupplemental/begin.view?). As we
A. A log2 fold-change vs. median
seem to indicate that the signaling consequences of administering these compounds may vary widely in differing cell types. Notably, the structurally similar SAHA and MS-275 HDAC inhibitors developed visibly distinct signatures in the space of phosphosignaling despite having ostensibly the same target (in contrast, we have shown that their chromatin modification signatures are largely the same using our chromatin profiling assay (35); data not shown). Remarkably, we show that these epigenetically active compounds develop distinct lineage-specific signatures in the space of phosphosignaling, lending further evidence to the notion that there may be direct linkages between the chromatin and signaling machinery in cells, as earlier suggested by the observations in Fig. 2D-2E. Moreover, these observations were garnered using the reduced-representation assay in a biological context that was not considered during its design. Even so, we were able stratify samples and demonstrate good replicate recall, indicating that the P100 assay can be applied to diverse perturbations in multiple cell types, further validating its general utility.
As a final proof-of-principle, we designed an experiment to demonstrate that the P100 assay is responsive to perturbations of known biological pathways. We selected a panel of small molecules that were known to inhibit members of MAPK, PI3K/mTOR, and Cell Cycle signaling pathways. The mapping of the compounds onto the enzymes that they inhibit, along with a partial pathway reconstruction (as assembled from KEGG (45,48)), is shown in Fig. 7A. Additionally, this experiment was designed to measure the time-dependence of P100 signature evolution, and we collected samples after 3, 6, and 24 h of compound treatment. We carried out these experiments in the breast cancer line MCF7, which has the notable characteristics being Her2 Ϫ , ER ϩ , and PR ϩ/Ϫ ; and also bears helical region PIK3CA mutations, increased phosphorylation of AKT, and overall hyperploidy up to 4n (49,50).
The P100 molecular profiles derived from these experiments are shown in Fig. 7B (data are available in https:// panoramaweb.org/labkey/project/LINCS/AbelinSupplemental/ begin.view?). The profiles are ordered according to the linear pathway reconstruction, and within each major column, all of the replicates at each time point are shown (increasing in time from left to right). It is visually apparent from these profiles that the different compound treatments generated a diverse set of molecular responses in the P100 assay. We also saw that most of the drug perturbations have only modest effects with increasing time of treatment, with the greatest effects generally observed at the 24 h time point but clearly observable at the 3 h time point. This is reminiscent of the effect observed with increasing doses of staurosporine (Fig. 5), and thus P100 signatures are both dose-and time-responsive. There a few exceptions, however. The upstream inhibitors Pazopanib (PDGFR) and TG101348 (JAK2) seem to have dramatic alterations to their profiles at 24 h. As well, the CDK4/6 inhibitor PD-0332991 only manifests a strong signature after 24 h of administration, at which time its signature appears largely like the other CDK inhibitors tested. Interestingly, PD-0332991 (now called Palbociclib) was developed as a specific treatment for Her2 Ϫ , ER ϩ breast cancer, matching the genetic status of MCF7 (50). We also note that, despite the cytotoxicity of many of these compounds after 24 h, unique signatures persist for the different drugs. In other words, the profiles do not converge to a universal "signature of death" at the 24 h time point and diverse signaling mechanisms still seem to be at play.
We noticed that, serendipitously, signatures derived from compound treatments that were immediately proximal with respect to their projections onto the reconstructed pathway seemed to be visually similar at most time points. To further investigate this observation, we calculated all of the pairwise correlations of samples at each time point (i.e. all 3 h samples compared with other 3 h samples, 6 h with 6 h, etc.), and then calculated the mean correlation for all compound pairs. The resulting correlation matrix is shown in Fig. 7C. Several interesting features emerged from this analysis. First, the strongest average correlations are on the diagonal, which again demonstrates the good replicate recall of the assay. Second, however, we noticed that there were blocks of off-diagonal correlation that appeared to be immediately adjacent to the diagonal. In fact, when we attempted to draw boundaries around these blocks of off-diagonal correlation (lavender, green, and yellow polygons), we noticed that they almost perfectly corresponded to the pathway modularity that we had assembled from the KEGG database. This is not to say that all of the perturbations within the same pathway module generate the same signature in the P100 assay. However, the correlation matrix suggests that near-neighbors in the pathways share components of the same phosphosignaling response, even with our reduced-representation of the phosphoproteome, thus allowing us to reassemble some notion of pathway modularity from the P100 data alone. DISCUSSION Our goal was to develop a framework for a high-throughput, standardized proteomic assay that could be used to interrogate the effects of molecular perturbations on cell signaling across large sample sets generated under disparate conditions. In general, most proteomic investigations of cellular responses to drugs involve shotgun data acquisition (26, 28 -30, 39). Shotgun proteomics enables the identification of thousands of proteins in complex samples, but large-scale comparisons across disparate conditions are difficult and yield incomplete data sets. In addition, discovery experiments that implement shotgun phosphoproteomic strategies are typically slow and labor intensive.
In order to generate almost "complete" data with few missing values, we configured a targeted proteomic assay for a set phosphopeptide probes that provides a reduced but highly informative representation of the phosphoproteome. We iden-tified these probes from large-scale, discovery cell perturbation studies and optimized the assay for robustness under several biologically relevant paradigms (i.e. multiple cell types, dose response, time-series). We hope that targeted methods for phosphoproteomic investigations, such as our P100 assay, will enable more rapid data generation and will increase the throughput of MS-based phosphosignaling studies at a fraction of the effort of traditional discovery experiments.
The P100 assay enables investigations into how perturbations affect cellular phosphosignaling phenotypes through generation of standardized profiles that can be compared across many conditions. Perturbations may take several forms, such as small molecule treatment, genetic manipulation, and induction of disease or differentiation models. In the most basic sense, the assay can help to stratify compounds into classes based on mechanism of action while denoting cell-type specific responses. For example, we identified strong connectivity among cardiac glycosides across multiple cell types using P100 data but are able to recognize cell-type of origin of each of the samples. Importantly, we can compare
hours
A. C.
B.
FIG. 7. Time-resolved signatures from P100 reveal modularity of biologically important signaling pathways. A, Pathway reconstruction showing the targets of a set of drugs known to inhibit important nodes in key signaling pathways. These drugs were administered for 3, 6, and 24 h to MCF7 cells. B, P100 molecular signatures from samples treated with the drugs. No clustering of samples has been performed, but P100 probes (rows) have been clustered (dendrogram omitted). In each major column corresponding to one drug, individual profiles from each sample are ordered according to time point from left to right. C, Correlation matrix of profiles from comparing profiles of samples at the same time point. The boundaries shown are meant to draw attention to the off-diagonal adjacent enrichment of correlation. These boundaries happen to correspond to the modules in the reconstructed pathway, and are colored by pathway module as in Fig. 7A. profiles obtained from any mode of perturbation to one another, and perhaps generate hypotheses about specific genetic mechanisms through which small molecules are acting (by similarity of profiles), or potential therapeutic options could counter a disease state (by anti-correlation of profiles). Thus, the assay espouses the "Connectivity Map" principle of using molecular profiles as an abstract means of associating cellular phenotypes (3,4).
In alignment with our view of the P100 assay as a secondary screening tool, the robustness and throughput of the assay were enhanced by automating sample processing steps (Fig. 3A). We developed automated protocols for protein reduction, alkylation, digestion, phosphopeptide enrichment, and sample desalting using Agilent Bravo liquid handling and AssayMAP platforms. We also introduced important process monitoring controls, such as enrichment standards, to monitor for peptide losses and systematic errors that are crucial to a production effort. We even demonstrated that we could simultaneous process 96 samples in only 3 days with a plate enrichment CV of 12% and detect ϳ95% of the reduced-representation phosphopeptides in close to 200 samples using our isotopically labeled quality control standards (supplemental Fig. S1). The assay itself is a single injection LCMS experiment with a 60 min nominal run time, enabling significant annual throughput to generate large data sets.
We qualified the targeted P100 assay and the use of a reduced-representation phosphoproteome by comparing measurements of our limited probe set to deeper data acquired under the conditions used for our large-scale discovery experiments (Fig. 4A). Using these data, we were able to replicate sample correlations observed in our discovery experiments demonstrating that a smaller set of phosphopeptide probes can be used as surrogates for a larger phosphopeptide set. These connections are intuitively visualized using a network graph architecture (supplemental Fig. S2) that efficiently summarizes sample correlations and helps to evaluate the biological relevance of results. Above and beyond these re-observations, we systematically evaluated the performance of the assay statistically. We calculated compared pairwise correlations among treatments and cell lines (Fig. 4B) and demonstrated that the null background is in line with expectations while replicate treatment can be detected both within a single cell type and even across cell types.
We went on to show that the strengths of the profiles in the assay have dose-and time-dependent correlations (Figs. 5, 7). Demonstration of dose-responsiveness is important for judging relative efficacies of similar compounds and downstream efforts in lead optimization for compounds in development. Time-responsiveness also demonstrates that there is sufficient dynamic range in the assay to study processes as they evolve, and that cellular responses to drug treatments are sustained over time. Although we could distinguish treatment time (via profile strength) via the P100 profiles, we saw very few instances of radical dynamic shifts in the profiles among the 3, 6, and 24 h time points that we measured. These observations argue against wholesale changes in protein levels over the assay time course. We acknowledge the shortcoming of not measuring the related "nonphospho" form of the P100 probes, but this turned out to be extremely difficult because of the complexity of the "nonphospho" proteome given that we wanted to keep the assay to a single LCMS experiment. In any case, the overall amount of phosphorylation of a given site is still fit-for-purpose as a component of the signature as we define it, despite it reflecting both the sitespecific changes and the overall protein abundance. We do not claim that the assay represents site stoichiometry.
Further analysis of the time course experiments shows that signatures are largely established by 3 h, and that the strongest correlations occur between the 3 and 6 h time points. We therefore believe that this "assay window" is the ideal time to measure phosphosignaling responses, before processes like global changes in gene-or protein-expression have a chance to exert strong effects on the signatures in the assay. We extended the assay window to 24 h to see if secondary effects would dominate the signatures. Surprisingly, most signatures simply increased in strength rather than changed in major ways (with exceptions, discussed in results), despite cytotoxicity of some compounds. When we measured signatures of epigenetically active compounds in ESCs and NPCs (further discussed below), we chose to do so at 24 h with the idea of generating the strongest possible signatures in cell types that we had not yet tested. At the same time, we acknowledge that some of these data may not be ideal because some molecular perturbations, such as treatment with the kinase inhibitor staurosporine, can result in the death of some cell types after 24 h. Therefore, we only presented those data as an illustration of our ability to collect P100 phosphosignaling profiles in diverse cell types that include NPC and ESC.
We believe that the P100 assay will be widely applicable in many areas of biology. We demonstrated that the assay was responsive in diverse biological backgrounds by collecting data from new cell types treated with compounds not used during the assay configuration phase. We were able to measure P100 phosphosignaling profiles in both embryonic stem cells (ESC) and neuronal precursor cells (NPC) in response to different classes of epigenetically active drugs, including a BRD4 inhibitor, an EZH2 inhibitor, and HDAC inhibitors (Fig. 6). As with our cancer cell line validation experiments, biological replicates of drug treatments clustered together by their P100 molecular signatures across NPCs and ESCs. Our ability to generate molecular signatures using P100 data for drug treated ESCs and NPCs illustrates the potential for the P100 assay to be applied in a broad range of cells and perturbation conditions. We also demonstrated that compounds with epigenetic mechanisms of action can elicit cell signaling phenotypes, suggesting that monitoring phosphosites is important for many biological contexts. Furthermore, the phosphosignaling profiles we gen-erated from ESCs and NPCs may provide insight into neuronal lineage development and neuropsychiatric disorders.
Our use of reduced-representation phosphopeptide probes is similar to the use of "sentinel proteins" reported by Soste et al. of the Picotti laboratory. Picotti and colleagues developed a targeted proteomic assay to probe biological responses to environmental perturbations in a less complex biological system, Saccharomyces cerevisiae, by selecting sentinel proteins from existing data (34). Like Soste et al., we selected probes that were highly detectable in our discovery data set. However, we selected probes to specifically monitor signaling through the lens of protein phosphorylation, whereas Soste et al. selected a mix of probes for general protein abundance and phosphoproteins, thus requiring two separate mass spectrometry experiments and biochemical workflows to execute the assay.
Although we selected a specific set of phosphopeptide probes to monitor with the P100 assay, the concept of reduced-representation phosphoprofiling can be extended to different sets of probes as long as they can be reproducibly detected and have varying responses to molecular perturbations. Extending reduced-representation profiling to alternate probes or adding more peptides enables researchers to tailor their assays to investigate specific biological questions, while retaining the ability to reproducibly measure targets across large sample sets. However, tailoring the panel may reduce the general utility of the assay. It is important to note that the selection of the P100 reduced-representation set was completely data driven, without preconceived notions of cellular pathways or biological function. This may seem counterintuitive; as high value is typically placed on readouts of "known pathways." Here, we challenge that notion by demonstrating that many phosphosites with poorly understood or unknown "functions" provide a great deal of information, in the entropic sense, about the state of the cellular response to perturbations that helps to classify responses to drug perturbations. In that way, the P100 is complementary to readouts of "known pathways" and may help to define novel networks of cellular signaling.
Nevertheless, the discovery data used to configure the P100 assay contained a wide selection of phosphoproteins that belong to known pathways. Thus, it can be argued that the P100 assay "projects" into these pathways by the proxy, because the selected probes have correlations to the underlying discovery data. The top 20 pathways described by our coordinately regulated P100 groups are shown in Fig. 3B. Many of these cell signaling pathways have been implicated in disease etiology and are of interest to both biologist and clinicians. For example, dysregulation within the MAPK signaling pathway has been implicated in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple types of cancer (51,52). The AMPK pathway controls metabolic functions within cells, and has been linked to obesity, cardiovascular disease, neurogenesis and stroke (53)(54)(55). In addition, inefficient ErbB signaling is associated with neu-rodegenerative diseases including multiple sclerosis and Alzheimer's disease (56,57).
Perhaps most dramatically, we have demonstrated that the P100 assay is sensitive to disruptions of (at least) the MAPK, PI3K/mTOR, and cell cycle cascades despite not being explicitly designed to monitor these pathways (Fig. 7). In these experiments, we intentionally disrupted important kinases in these signaling pathways and observed unique P100 signatures that simultaneously allowed us to distinguish the treatments while reconstructing the modularity of the linear pathways. For example, phosphoprofiles produced from JAK and PI3K inhibition positively correlated with one another because inhibition of JAK will prevent phosphorylation of insulin receptor substrate and p85, which leads to inhibition of the PI3K pathway (58). These data illustrate how the P100 assay can stratify samples and reinforce our understanding of network architecture according to direct and indirect interactions among the pathways perturbed by drug treatments.
These results validate the idea that the assay is a good proxy for monitoring diverse cellular processes. Furthermore, our ability to monitor alterations in these pathways is in good agreement with the pathways onto which we believe the P100 assay projects (Fig. 3B) based on the underlying configuration data. Of course, there is certainly room for more focused, "known" target-driven phosphosignaling assay panels in the future for specific applications. Another possible improvement might be a hybrid P100-DIA assay that allows focused detection and quantification of our P100 analytes while retaining the ability to mine primary data for novel phosphopeptide analytes of interest in defined pathways.
The P100 assay is a robust screening tool on its own, but is also intended to be used in conjunction with other molecular profiling techniques (such as gene expression profiling) to develop comprehensive pictures of cellular responses to perturbation. For example, it would be interesting to generate chromatin signature data on epigenetically active compounds in our global chromatin profiling assay (35,59) along with P100, thus providing measures of processes that are directly targeted by the molecules together with measures of secondary process that might also be of interest. Our demonstration of signaling responses for these compound classes (Fig. 6) hints at this possibility. We plan to test this hypothesis by collaborating with others under the auspices of the Library of Integrated Network-based Cellular Signatures (LINCS) program to systematically collect molecular profiling data from a diverse set of chemical, genetic, and environmental manipulations across a range of cell types representing important disease models (www.lincsproject.org). A preview of these data has been made available even in advance of publication (https://panoramaweb.org/labkey/LINCS.url). We believe that these types of large-scale data integration efforts will further our understanding of how genomics, epigenetics, and phosphosignaling contribute to disease progression.
In conclusion, we developed and qualified a high-throughput assay that can be used as a tool to interrogate the effects of molecular perturbations on cell signaling in various cell types. Using P100 assay data, we demonstrated that reduced-representation phosphoprofiles are informative by reproducing sample classifications observed in our highthroughput discovery data. We recapitulated prior expected cell signaling correlations in a reduced assay space and demonstrated that signature strength is time-and dose-responsive. We were also able to identify correlations among perturbations that are known to target the same pathways, as well as correlations among perturbations that have overlapping downstream signaling events. Therefore, we believe the P100 assay can produce a large number of foundational data sets that can be queried to identify correlations among phosphosignaling profiles produced by disparate drug treatments even though only ϳ100 phosphopeptide probes are monitored. Overall, we demonstrated the tractability of large-scale phosphoproteomic studies by presenting a generalizable, high-throughput MS assay that can be used as a robust tool that offers the ability to make longitudinal comparisons among thousands of samples. These comparisons, especially when married with other molecular profiling data, should be an important contribution from the phosphoproteomic sphere to our understanding of the molecular underpinning of cellular machinery and its response to biologically-relevant perturbations. | v3-fos-license |
2019-05-24T13:06:21.722Z | 2019-05-24T00:00:00.000 | 162183307 | {
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} | pes2o/s2orc | Evaluation of Molecularly Imprinted Polymers as Synthetic Virus Neutralizing Antibody Mimics
Rapid development of antibody-based therapeutics are crucial to the agenda of innovative manufacturing of macromolecular therapies to combat emergent diseases. Although highly specific, antibody therapies are costly to produce. Molecularly imprinted polymers (MIPs) constitute a rapidly-evolving class of antigen-recognition materials that act as synthetic antibodies. We report here on the virus neutralizing capacity of hydrogel-based MIPs. We produced MIPs using porcine reproductive and respiratory syndrome virus (PRRSV-1), as a model mammalian virus. Assays were performed to evaluate the specificity of virus neutralization, the effect of incubation time and MIP concentration. Polyacrylamide and N-hydroxymethylacrylamide based MIPs produced a highly significant reduction in infectious viral titer recovered after treatment, reducing it to the limit of detection of the assay. MIP specificity was tested by comparing their neutralizing effects on PRRSV-1 to the effects on the unrelated bovine viral diarrhea virus-1; no significant cross-reactivity was observed. The MIPs demonstrated effective virus neutralization in just 2.5 min and their effect was concentration dependent. These data support the further evaluation of MIPs as synthetic antibodies as a novel approach to the treatment of viral infection.
INTRODUCTION
The complex interplay between the environment, the expanding human population and intensified livestock production systems has led to an apparent increase in the frequency of emergence and re-emergence of viral zoonoses. As demonstrated by the recent Ebola outbreak in West Africa, there is potential for new and emerging viral infections to cause large epidemics with significant mortality and morbidity. Antibodies which neutralize viral infectivity are critical for immunological protection and this may be exploited in the context of both passive and active immunization. However, there is currently an unmet need to be able to rapidly and inexpensively produce therapeutic antibodies for new/emergent viral diseases.
Molecularly imprinted polymers (MIPs), often referred to as synthetic antibodies, offer an alternative approach to biomolecular and viral recognition. Molecular imprinting involves the formation of a polymer comprising selective cavities based on a molecular or biomolecular template (Mayes and Mosbach, 1997;Hawkins et al., 2005;Li et al., 2014). It has been demonstrated that polymerization of monomers in the presence of a molecular target causes the formation of corresponding binding sites in the resulting polymer. MIPs for binding small molecules have been extensively researched and successfully commercialized for the solid phase extraction of drugs and pesticides (Sánchez-González et al., 2019) and sample clean-up and improvement of chromatographic analysis (Regal et al., 2012). In recent years there has been an increase in research activity to imprint larger templates using water-based polymers realizing their application for imprinting of more complex biologicals such as proteins and viruses (Stevenson et al., 2016). To date, their potential use has been almost entirely focused on bio-extraction/analysis (Stevenson et al., 2016) and sensor applications (Saylan et al., 2017). More recently, the MIP technology has been applied as a novel nucleant for protein crystallization (Saridakis et al., 2011;Reddy et al., 2012). The strategy relies on the MIP cavities possessing complimentary chemical architecture allowing the template biological to lock into the cavity.
The majority of papers published to date on virus imprinting have been in the development of MIP-based virus diagnostics and sensors (Hayden et al., 2003;Perozo et al., 2006;Malitesta et al., 2012;Altintas et al., 2015;Malik et al., 2017;Ahmad et al., 2019;Tancharoen et al., 2019). Typically, a surface stamping method has been used to imprint for example tobacco mosaic virus, parapoxvirus ovis, and human rhinovirus. The stamping approach produces a thin film directly integrated with a sensor such as electrochemical (Malitesta et al., 2012;Canfarotta et al., 2018), optical (Ahmad et al., 2019), or acoustic (Hayden et al., 2003;Uludag et al., 2007) devices resulting in a concentration dependent signal upon selective binding of virus. Sankarakumar and Tong (2013) performed the first study on the potential antiviral use of MIPs. MIPs imprinted with bacteriophage fr were able to reduce phage titers by approximately 1 log which was significantly greater than the neutralization by non-imprinted polymers (NIPs).
The aim of this study was to evaluate in vitro the antiviral activity of MIPs imprinted with the porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) as a model mammalian virus. PRRSV-1 is an enveloped RNA virus of the Arteriviridae family (Kappes and Faaberg, 2015), which causes the most economically important infectious disease affecting the global pig industry (Holtkamp et al., 2013;Paz, 2015). PRRSV-1 was also selected since it would allow direct follow-on experimental studies to evaluate the safety and efficacy of MIPs in pigs, which serve as an excellent large animal model.
The rationale behind selection of monomers for this study was based on our previous experiences using acrylamide and functionalized acrylamide monomers for protein imprinting (Reddy et al., 2012;El-Sharif et al., 2014a,b, 2015. In our studies to date, we have demonstrated that MIP binding affinity and selectivity for target protein increases in the order N-isopropylacrylamide < acrylamide < N-hydroxymethylacrylamide. This order also represents increasing hydrophilicity and hydrogen bonding capability of the monomer, which we understand to be the predominant non-covalent interaction between template and monomer (El-Sharif et al., 2014a,b). In extending the use of monomers to virus imprinting, and based on hydrogen bonding interactions with the hydrophilic envelope glycoproteins of the virus, we therefore anticipated a similar order of imprinting capability. It should be noted that cavity selectivity would be based on a combination of virion shape but also intermolecular interactions between surface proteins and hydrogen bonding functional groups (associated with monomer) within the cavity.
To produce purified PRRSV-1 virions for molecular imprinting, virus propagation was performed in 850 cm 2 roller bottles (Thermo Fisher Scientific, Loughborough). Roller bottles were seeded with 2.2 × 10 7 MARC-145 cells and incubated in a roller incubator at 37 • C. As monolayers approached confluence, the growth medium was replaced with PRRSV-1 Olot/91 virus suspended in 50 ml DMEM medium supplemented with 1% FBS at a multiplicity of infection of 0.1. Four days post-infection the culture supernatant was collected, pooled with a freeze-thawed cell lysate, clarified by centrifugation at 524 × g for 15 min and stored at −80 • C. PRRSV-1 virions were purified by continuous density ultracentrifugation. Virus-containing supernatants were further clarified by centrifugation at 4,700 rpm (TX-750 rotor, Beckman Coulter, High Wycombe, UK) at 4 • C for 1 h. Polyethyleneglycol (PEG)-6000 (Sigma, Poole, UK) was slowly added (7% w/v) under stirring, the mixture incubated at 4 • C under slow continuous stirring overnight and precipitated virus collected by centrifugation at 4,700 rpm (TX-750 rotor), 4 • C for 30 min. The precipitated virus pellet was resuspended in 7 ml TNE buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 2 mM EDTA) and incubated overnight at 4 • C. Insoluble material was removed by centrifugation (4,700 rpm, 4 • C for 10 min in a TX-750 rotor). The resulting clarified viral supernatants were pelleted through a sterile 30% sucrose in TNE buffer cushion at 28,000 rpm for 4 h at 4 • C using a SW50.1 rotor and XPN-100 Ultracentrifuge (Beckman Coulter). Virus pellets were gently reconstituted in low volumes of TNE buffer before being layered over tubes containing a continuous 15-45% sucrose gradient prepared using a Gradient Master 108 (BioComp, New Brunswick, Canada). Tubes were centrifuged at 28,000 rpm, 4 • C for 4 h using a SW50.1 rotor and XPN-100 Ultracentrifuge. 1.5 ml fractions were collected and PRRSV-1 infectious titers determined. Those containing the major part of infectious virus were dialyzed into PBS using Slide-A-Lyzer TM Dialysis Cassettes (Thermo Fisher). Infectious titers were confirmed post-dialysis and virus was inactivated by incubation with 0.1% β-propiolactone (Sigma). Infectious titers were determined by log-fold serial dilution of viruses, which were added to 96 well flat bottom tissue culture plate containing MARC-145 or FBT cells (5 × 10 3 cells/well).
The plates were incubated at 37 • C with 5% CO 2 for 4 days. Immunoperoxidase staining was performed to determine the number of infected wells and allow calculation of the median 50% tissue culture infective does (TCID 50 ;Finney, 1978). PRRSV-1 was detected using N-protein specific mAb 1AC7 (Ingenasa, Madrid, Spain) and BVDV-1 with the E2-specific mAb WB214 antibody (APHA Scientific, Addlestone, UK).
All work with infectious viruses was risk assessed and conducted under biocontainment level 2 conditions. Polyacrylamide (pAA) MIP was prepared by mixing 13.5 µL of AA (40% (w/v) solution) with 30 µL of MBAm crosslinker. The solution was then added to 50 µL of purified inactivated PRRSV-1 Olot/91 in PBS (10 7.4 TCID 50 /ml) and 2.5 µL of MilliQ water to give 10 7.1 TCID 50 /ml of virus template in monomer solution. The solution was vortexed for 30 s, before the catalyst, TEMED (2 µL) and initiator, APS (2 µL) were added to give a final volume of 100 µL. The solution was vortexed for a further 30 s and then purged with nitrogen for 5 min. Polymerization was allowed to occur overnight at room temperature (∼22 • C).
Production and Evaluation of
Poly-N-hydroxymethylacrylamide (pNHMA) was prepared in a similar fashion using 16 µL of NHMA [48% (w/v) solution] as functional monomer, and 30 µL of MBAm crosslinker, which was then added to 50 µL 10 7.4 TCID 50 /ml of purified inactivated PRRSV-1 Olot/91 in PBS. The solution was vortexed for 30 seconds. TEMED (2 µL) and initiator, APS (2 µL) were then added to give a final volume of 100 µL. The solution was vortexed for a further 30 s and then purged with nitrogen for 5 min and polymerization allowed to occur and left overnight at room temperature (∼22 • C).
Poly-N-isopropylacrylamide (pNIPAM) was prepared using 14 µL of NIPAM [60% (w/v) solution] as functional monomer, and 30 µL of MBAm as crosslinker. The resulting solution was then added to 50 µL of 10 7.4 TCID50/ml of purified inactivated PRRSV-1 Olot/91 in PBS and 2 µL of MilliQ H2O. The solution was vortexed for 30 s. TEMED (2 µL) and initiator, APS (2 µL) were then added to give a final volume of 100 µL. The solution was vortexed for a further 30 s and then purged with nitrogen for 5 min and occurred overnight at room temperature (∼22 • C).
For all three MIP preparations, the molar ratio of monomer to crosslinker was kept constant at 20:1. For every MIP hydrogel created, a non-imprinted control polymer (NIP) was prepared and conditioned in an identical manner, but in the absence of template virus.
Following polymerization, hydrogels were then individually granulated using a 35 µm sieve and conditioned by washing 100 mg of granulated gel with five 0.4 mL volumes of MilliQ H 2 O followed by elution of the template, using five 0.2 mL volumes of 10% (w/v):10% (v/v) SDS:acetic acid (pH 2.8) and another five 0.4 mL volume washes of MilliQ H 2 O to remove any residual eluent, and finally with a further two washes with PBS to condition the gels. Each wash step was achieved by vortexing followed by a centrifugation for 3 min at 2419 × g. The PBS conditioned hydrogels were then diluted 1:1 (w/v) in PBS (150 mM, pH 7.2 ± 0.2) and stored at room temperature for virus neutralization study purposes.
Non-purified PRRSV-1 was diluted to 2 × 10 5 TCID 50 /50µl and mixed with an equal volume of MIP or NIP suspension and incubated at room temperature for 1 h with mixing every 5 min. After incubation, the mixture was centrifuged at 1,500 × g to pellet the MIP/NIP. Twenty five microliter of the resulting supernatant was removed and the infectious PRRSV titer determined as described above. The neutralizing capacity of PRRSV-1 imprinted MIPs was also similarly tested on BVDV-1. An experiment was additionally performed by varying the incubation time of the virus with the MIP, from 1 h to 30, 15, 7.5, 5, 2.5, and 1.5 min. The effect of varying the concentration of the MIPs was tested by performing a serial 1:3 dilution of the MIPs in PBS prior to incubation with virus.
Electron Microscopy (EM)
Virus suspensions post-dialysis and post-inactivation were prepared for negative stain electron microscopy as follows. Seven microliters of each sample were incubated at room temperature on glow discharged, Formvar coated EM grids (Agar Scientific, Stansted, UK) for 2 min. Excess sample was gently removed and following a brief water wash, grids were placed onto 3% aqueous uranyl acetate (Agar Scientific, Stansted, UK) droplets for 1 min. Excess uranyl acetate was removed and grids were allowed to dry before being imaged in a FEI T12 transmission EM at 100 kV with a Tietz F214 camera.
Statistical Methods
Data were graphically plotted and statistically analyzed using GraphPad Prism v7.03 (GraphPad Software, La Jolla, United States). A one-way ANOVA with Tukey's multiple comparisons tests was performed on log transformed infectious viral titers. For assessment of the neutralizing capacity of MIPs, mean data from 3 independent batches of MIPs were compared. Datasets for other assays were technical replicates within single experiments.
RESULTS
To produce a high quality template for this exploratory study, PRRSV-1 virions were purified by density gradient ultracentrifugation (Figures 1A-C). Whilst infectious virus was detected in all sucrose gradient fractions, the highest titers were detected, as predicted, in the central fractions of the gradient, i.e., pools E and F ( Figure 1A). Testing of the individual sucrose gradient fractions confirmed the highest titers in fractions 16-18 ( Figure 1B) and these fractions were dialyzed into PBS and inactivated prior to imprinting. Infectious virus titers postdialysis and post-inactivation were determined (data not shown) and electron microscopy confirmed the structural integrity of the template virus and the absence of significant cellular debris ( Figure 1C). Three separate assays were performed (each in triplicate) to assess the PRRSV-1 neutralizing properties of independently produced batches of MIPs, all imprinted with purified PRRSV-1 virions ( Figure 1D). Each of the MIPs (pAA, pNHMA, and pNIPAM) were tested alongside their corresponding NIP. A PBS control treatment was used as a further negative control in each assay. Both pAA MIP and pNHMA MIP produced a highly significant reduction in infectious titer of PRRSV-1 (p < 0.0001) when compared with their respective NIPs and the PBS control, both reducing the infectious titer to the point of or below the limit-of-detection (LoD) of the assay. The titers of virus recovered from pAA NIP and pNHMA NIP treatments were statistically comparable to the PBS control treatment. There was no inter-batch variation in this effect suggesting a reproducible imprinting-related neutralization of virus. However, pNIPAM produced a very similar reduction in infectious viral titer in both its MIP and NIP forms, suggesting that molecular imprinting was not entirely responsible for the neutralization event in the case of this polymer.
The specific binding capacity of the pAA and pNHMA MIPs (both imprinted with PRRSV-1) was tested by comparing their neutralizing effects on PRRSV-1 to the effects on BVDV-1 (Figure 2A). Neither pAA MIP nor pNHMA MIP showed significant reduction in BVDV-1 infectious viral titer. pNHMA imprinted with PRRSV-1 was selected for an assay evaluating the effect of decreasing incubation time on the neutralizing capacity of the MIP on PRRSV-1 virus ( Figure 2B). Significant reduction in infectious viral titer to the assay LoD (p < 0.001) was demonstrated at 60, 30, 15, 7.5, 5, and 2.5 min incubation time, with no significant differences in effect between the different incubation times. A 1.5 min incubation time caused a significant (p < 0.05), but far less pronounced reduction in infectious titer. With a view to assessing the binding capacity of virusimprinted MIPs, pAA and pNHMA were selected for a trial of the effect of decreasing MIP concentration on neutralizing capacity ( Figure 2C). Both MIPs showed a broadly similar effect with decreasing concentration, providing complete neutralization of infectious virus to the LoD of the assay at both neat and 1:5 dilutions. Lower concentrations of MIP showed a dose dependent reduction in neutralizing effect, which was more pronounced with the pAA MIP.
DISCUSSION
This is the first study to demonstrate that MIPs imprinted with a clinically relevant virus can exert potent antiviral effects, reducing the infectious viral titer recovered to below the LoD of the assay used. Both pAA and pNHMA MIP imprinted with PRRSV-1 were consistently able to neutralize a high quantity of infectious PRRSV-1 (> 4 log reduction). A lack of neutralizing effect on BVDV-1 suggests that a specific binding interaction is taking place between the target molecule (virus, in this case PRRSV-1) and the MIP. The concentration and time dependent effect of neutralization further supports a rapid and specific binding. That there was no discernible difference in the neutralization capacity between pNIPAM MIP and NIP is interesting and suggests an alternative non-specific mechanism for virus neutralization. We have shown previously with protein imprinting that pNIPAM is unable to demonstrate selective protein rebinding in either the MIP or NIP form (Reddy et al., 2012). That there is a virus neutralization event in this study, regardless of imprinting taking place, points to the potential toxic nature of pNIPAM. Alternatively, pNIPAM MIPs and NIPs may behave the same because they both hydrophobically bind the viruses. It has been established in this study with an animal virus and by others with bacteriophages (Sankarakumar and Tong, 2013;Li et al., 2017) that MIPs can successfully bind and neutralize viruses under in vitro conditions. Whereas we have demonstrated complete neutralization of PRRSV-1 (> 4 log reduction in titer) with a single MIP dose, in contrast, in the bacteriophage studies, it was observed, that a single dose of MIP was not sufficient to completely neutralize viral infection (∼1 log reduction in titer at best).
The results of our incubation time trial showing that complete neutralization could be achieved in as little as 2.5 min, is very promising from the perspective of clinical application. However, this would need to be re-assessed in the context of plasma proteins and other potentially interfering molecules that would be present in vivo. In terms of suitability for in vivo testing, further work is needed to ensure suitable biocompatibility of our MIPs. Li et al. (2017) produced dopamine-based MIPs that showed no significant cytotoxicity on human hepatoma cells (Li et al., 2017) but of particular relevance, was the successful systemic application of acrylamide-based MIPs, to mice without notable adverse effects (Hoshino et al., 2010). The latter demonstrated the ability of MIPs to bind the cytotoxic peptide melittin, the principle component of bee venom, in the bloodstream of mice, which significantly reduced the mortality and morbidity associated with melittin envenomation.
The data from this study has demonstrated a highly effective and specific neutralization of virus infectivity with certain hydrogel-based MIPs. Whilst promising, it is possible that the destructive method used to produce these cavity-containing hydrogel-MIPs leads to the majority of the material comprising redundant unselective particles, devoid of template-specific cavities. Further studies will evaluate virus imprinting of nanoparticle-based MIPs (nanoMIPs) for the efficient production of high bioaffinity materials (Canfarotta et al., 2016;Xu et al., 2017). MIPs imprinted with virions may be produced according to a variety of methods, giving nanoscale shells with cavity populated surfaces.
In conclusion, hydrogel-based MIPs are capable of specifically neutralizing virus infectivity in vitro within a short enough incubation time to be clinically relevant. The findings of this study support the further evaluation of MIPs as alternative, stable, and economical "plastic" antibodies that could be used to treat and prevent viral infections.
DATA AVAILABILITY
All datasets generated for this study are included in the manuscript and/or the supplementary files.
AUTHOR CONTRIBUTIONS
SG, HE-S, and SR contributed conception and design of the study. SG, HE-S, SH, RF, RM, PH, and MS performed the study and analysis. SG, RF, and SR wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.
FUNDING
This study was supported by a Research Pump Priming Fund award from the University of Surrey, a Seed Award from the Wellcome Trust (108003/A/15/Z) and UK Biotechnology and Biological Sciences Research Council (BBSRC) awards BBS/E/I/00007031 and BBS/E/I/00007039. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. | v3-fos-license |
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} | pes2o/s2orc | Temporal Changes of Human Breast Milk Lipids of Chinese Mothers
Fatty acids (FA), phospholipids (PL), and gangliosides (GD) play a central role in infant growth, immune and inflammatory responses. The aim of this study was to determine FA, PL, and GD compositional changes in human milk (HM) during lactation in a large group of Chinese lactating mothers (540 volunteers) residing in Beijing, Guangzhou, and Suzhou. HM samples were collected after full expression from one breast and while the baby was fed on the other breast. FA were assessed by direct methylation followed by gas chromatography (GC) analysis. PL and GD were extracted using chloroform and methanol. A methodology employing liquid chromatography coupled with an evaporative light scattering detector (ELSD) and with time of flight (TOF) mass spectrometry was used to quantify PL and GD classes in HM, respectively. Saturated FA (SFA), mono-unsaturated FA (MUFA), and PL content decreased during lactation, while polyunsaturated FA (PUFA) and GD content increased. Among different cities, over the lactation time, HM from Beijing showed the highest SFA content, HM from Guangzhou the highest MUFA content and HM from Suzhou the highest n-3PUFA content. The highest total PL and GD contents were observed in HM from Suzhou. In order to investigate the influence of the diet on maternal milk composition, a careful analyses of dietary habits of these population needs to be performed in the future.
Introduction
Human milk (HM) is considered the optimal form of nourishment for infants during the first six months of life [1] and among its macronutrients, the lipid fraction is crucial, representing approximately 50% of the energy supplied to the newborn infant [2]. Lipids (2%-5%) occur in milk in the form of fat globules mainly composed of triacylglycerols (TAG) (~98% of total lipids) surrounded by a structural membrane composed of phospholipids (PL) (0.8%), cholesterol (0.5%), enzymes, proteins, glycosphingolipids (e.g., gangliosides (GD)), and glycoproteins [3,4].
Subjects
This study was part of MING, a cross-sectional study designed to investigate the dietary and nutritional status of pregnant women, lactating mothers, and young children aged from birth up to three years living in urban areas of China [26]. In addition, the HM composition of Chinese lactating mothers was characterized. The study was conducted between October 2011 and February 2012. A multi-stage milk sampling from lactating mothers in three cities (Beijing, Suzhou, and Guangzhou) was performed for breast milk characterization. In each city, two hospitals with maternal and child care units were selected and, at each site, mothers at lactation period 0-240 days were randomly selected based on eligibility criteria. Subjects included in the period 0-5 days were recruited at the hospital, whereas the other subjects were requested by phone to join the study; if participation was dismissed a replacement was made. The response rate was 52%. Recruitment and milk, as well as baseline data collection, were done in separate days. A stratified milk sampling of 540 lactating mothers in six lactation periods of 0-4, 5-11, and 12-30 days, and 1-2, 2-4, and 4-8 months was obtained in the MING study.
Inclusion and Exclusion Criteria
Eligibility criteria included women between 18-45 years of age giving birth to a single, healthy, full-term infant and exclusive breastfeeding at least until four months of age. Exclusion criteria included gestational diabetes, hypertension, cardiac diseases, acute communicable diseases, and postpartum depression. Lactating women who had nipple or lacteal gland diseases, who had been receiving hormonal therapy during the three months preceding recruitment, or who had insufficient skills to understand study questionnaires were also excluded.
Ethical and Legal Considerations
The study was conducted according to the guidelines in the Declaration of Helsinki. All of the procedures involving human subjects were approved by the Medical Ethics Research Board of Peking University (No. IRB00001052-11042). Written informed consent was obtained from all subjects participating in the study. The study was also registered in ClinicalTrials.gov with the number identifier NCT01971671.
Data Collection
All subjects responded to a general questionnaire including socio-economic and lifestyle aspects of the mother. Self-reported weight at delivery, number of gestational weeks at delivery, and delivery method were also recorded. Additionally, a physical examination evaluated basic anthropometric parameters (height, weight, mid-arm circumference) blood pressure, and hemoglobin. Data collection was done through face-to-face interviews the day of HM sample collection. In addition, the date of birth and gender information of the baby was collected after the data collection, since the data was not included in the initial questionnaires. Subjects were contacted by phone and were asked to clarify these two aspects retrospectively.
HM Sampling
Breast milk sampling was standardized for all subjects and an electric pump (Horigen HNR/X-2108ZB, Xinhe Electrical Apparatuses Co., Ltd., Beijing, China) was used to sample the milk. Samples were collected at the second feeding in the morning (9:00-11:00 a.m.) to avoid circadian influence on the outcomes. A single full breast was emptied and aliquots of 10 mL for colostrum and 40 mL for the remaining time points was secured for characterization purposes. The rest of the milk was returned to the mother for feeding to the infant. Each sample was distributed in freezing tubes, labelled with subject number, and stored at −80 • C until analysis. Figure 1 shows the study flowchart for the subjects' recruitment.
FA Quantification
FA profile was determined by preparing the methyl esters of FA (FAMEs). A direct transesterification of HM was performed with methanolic chloridric acid solution, as described by Cruz-Hernandez et al. [27]. Briefly, into a 10 mL screw cap glass test tube, milk (250 µL) was added and mixed with 300 µL of internal standard FAME 11:0 solution (3 mg/mL) and 300 µL of internal standard TAG 13:0 solution (3 mg/mL). After addition of 2 mL of methanol, 2 mL of methanolic chloridric acid (3 N), and 1 mL of hexane, the tubes were heated at 100 • C for 90 min. To stop the reaction 2 mL of water were added and after centrifugation (1200× g for 5 min) the upper phase (hexane) was transferred into gas chromatography vials. The analysis of FAMEs was performed by GC using a CP-Sil 88 capillary column (100 m, 0. 25
Gangliosides Quantification
GD were quantified as previously described by Giuffrida et al. [2]. Briefly, HM (0.2 mL) was dissolved in water (1 mL) and mixed with 4 mL methanol/chloroform (2/1). After centrifugation (3000× g, for 10 min), the upper liquid phase was quantitatively transferred into a 15 mL centrifuge tube. The residue was mixed with water (1 mL), 2 mL of methanol/chloroform (2/1), shaken, put into an ultrasonic bath at 25 • C for 10 min, centrifuged (3000× g, for 10 min), and upper liquid phases polled together; the volume was adjusted to 12 mL with methanol 60% and pH to 9.2 by adding Na2HPO4 30 mmol/L (0.2 mL). The extract solution was loaded on an Oasis HLB VAC RC SPE cartridges (30 mg, 15 mL, Waters) previously conditioned with methanol (2 mL) and methanol 60% (2 mL). The sample was passed through the cartridge at maximum flow rate 2-3 mL /min. The sorbent was washed with 2 mL of methanol 60% and dried by vacuum suction for a few seconds; the analyte was eluted with methanol (2 mL). Solvent was evaporated to dryness under a nitrogen flow at 30 • C and the residual lipids were re-dissolved in 0.2 mL of methanol 70% and analysed by liquid chromatography (LC) coupled with quadrupole time of flight (QTOF), using an Aquity BEH C18 column (1.7 µm; 150 × 2.1 mm i.d.; Waters). All chromatography was performed at 50 • C. Solvent A was composed of water/methanol/ammonium acetate (1 mmol/L) (90/10/0.1 v/v/v) and solvent B of methanol/ammonium acetate (1 mmol/L) (100/0.1 v/v). Gradient conditions were as follows: time = 0 min 10% solvent A; time = 0.2 min 10% solvent A; time = 8.2 min 5% solvent A; time = 12.2 min 5% solvent A; time = 12.4 min 0% solvent A; time = 18.4 min 0% solvent A; time = 18.6 min 10% solvent A; time = 21 min 10% solvent A. Flow rate was 0.2 mL/min. Injection volume was 0.01 mL for GD3 and 0.005 mL for GM3. The mass spectrometer was equipped with an electrospray ionization (ESI) ion source. The ESI mass spectra were recorded in the negative ion mode under the following conditions: ion spray voltage (IS) −4000 V, temperature of the source 400 • C, declustering potential (DP) −40 V, ion source gases one and two at 40 and 35 psi, respectively, curtain gas at 15 psi, collision energy −40 V. GD3 and GM3, were monitored by transitions of the precursor ions to the m/z 290. Quantification was performed by the standard addition method.
Demographics and Anthropometrics of Study Subjects
In the current study we analyzed HM from 539 mothers (Figure 1), collected in a cross-sectional design over eight months postpartum. Milk obtained for analyses was a single, whole breast milk sample to have a comprehensive view on nutrient content. The details of the demographics and anthropometrics of the study subjects are outlined in Table 1. Groups of mothers, which delivered either a male or a female infant, were comparable for their age and anthropometric and demographic characteristics. Gestational age at birth (average 39 weeks) were also comparable between groups. The details of demographics and anthropometrics of the study subjects for the time period 0-4 days are not available.
FA
FA were determined by gas chromatography coupled with flame ionization detector (GC-FID), as previously described by Cruz-Hernandez et al. [27] and the results are listed in Table 2.
In our study total SFA content increased significantly from colostrum (35.7% of total FA) to transitional milk (38.9% of total FA) and decreased in mature milk (36.2% of total FA), with palmitic acid (16:0) being the most abundant FA and decreasing significantly (p < 0.05) from 23.2% in colostrum to 19.8% of total FA in mature milk ( Table 2). Stearic acid (18:0) content was constant along the lactation period, i.e., colostrum, transitional, and mature milk, at about 5% of total FA, and medium-chain (MC) FA (10:0-14:0) content was low in colostrum (6.8% of total FA) compared to transitional (13.1% of total FA), and mature milk (11.0% of total FA) ( Table 2). Arachidic (20:0) and lignoceric acids (24:0) were constant along the lactation time at about 0.2 and 0.1% of total FA, respectively. No significant differences (p > 0.05) on total SFA content were observed among cities in colostrum, and transitional milk ( Table 2). SFA content was significant lower (p < 0.05) in mature milk from Suzhou (34.5% of total FA). Palmitic (22.5%, 19.4%, and 18.5% of total FA in colostrum, transitional, and mature milk, respectively) and stearic (4.9%, 4.5%, and 4.8% of total FA in colostrum, transitional and mature milk, respectively) FA also showed the lowest content in mature milk from Suzhou.
In the total population the MUFA content of HM decreased from 40.7% in colostrum to 36.9% of total FA in mature milk, with oleic acid (18:1n-9) being the most abundant FA and decreasing along the lactation time from 34.2% in colostrum to 31.9% of total FA in transitional and mature milk. Other MUFA (i.e., 17:1n-7, 20:1n-9, 22:1n-9. and 24:1n-9) also decreased over the lactation period ( Table 2). The highest level of total MUFA content was found in colostrum (43.1% of total FA), transitional (39.3% of total FA), and mature milk (38.3% of total FA) from Guangzhou ( Table 2). The lowest level of total MUFA content was found in colostrum (38.4% of total FA), transitional (34.7% of total FA), and mature milk (34.3% of total FA) from Beijing ( Table 2). HM samples obtained from mothers in Guangzhou contained the highest level of Oleic acid whereas milk obtained from mothers in Beijing contained the lowest level, respectively: colostrum (37.1% vs. 32.6% of total FA), transitional (34.0% vs. 30.3% of total FA), and mature milk (33.4% vs. 30.1% of total FA).
In the total population, total PUFA n-6 increased from 21.7% in colostrum to 24.1% of total FA in mature milk with linoleic acid (18:2n-6) being the most abundant FA and increasing along the lactation time from 18.9% in colostrum to 22.8% of total FA in mature milk. ARA (20:4n-6) content decreased from 0.9% to 0.5% of total FA from colostrum to mature milk. Beijing and Suzhou showed higher total PUFAn-6 content in colostrum (23.3% and 22.8% of total FA, respectively), transitional (22.5% and 22.9% of total FA, respectively), and mature milk (26.6% and 25.3% of total FA, respectively) than Guangzhou (Table 2).
Total PUFA n-3 in HM from total population slightly increased from 1.4% in colostrum to 1.9% of total FA in mature milk with linolenic acid (18:3n-3) being the most abundant and increasing along the lactation time from 0.9% in colostrum to 1.5% of total FA in mature milk. DHA (22:6n-3) slightly decreased over lactation period from 0.5% in colostrum to 0.3% of total FA in mature milk, and EPA (20:5n-3) was present in a small amount (<0.1% of total FA in colostrum, transitional, and mature milk). The highest level of total PUFA n-3 content was found in colostrum (1.8% of total FA), transitional (2.1% of total FA), and mature milk (2.4% of total FA) from Suzhou (Table 2), which, as a consequence, showed the lowest n-6 to n-3 ratio (12.7% in colostrum, 10.9%in transitional milk, and 10.5% of total FA in mature milk). Table 2. Median fatty acid composition of HM expressed as g/100 g of total FA.
Phospholipids
PL classes were determined by LC-ELSD, as previously described by Giuffrida et al. [28] and the results are listed in Table 3.
We did not measure minor constituents, such as lysophosphatidylcholine, which may contribute only to small amounts of the infant's diet.
From the total population, total PL content in HM decreased along the lactation period from 33.0 in colostrum to 24.2 mg/100 mL in mature milk, being significant lower (p < 0.05) in mature milk (Table 3). PtdCho was the most abundant PL in HM (from 12.0 mg/100 mL in colostrum to 8.2 mg/100 mL in mature milk) followed by CerPCho (from 9.1 mg/100 mL in colostrum to 7.2 mg/100 mL in mature milk), PtdEtn (from 8.5 mg/100 mL in colostrum to 6.4 mg/100 mL in mature milk), PtdIns (from 1.8 mg/100 mL in colostrum to 1.5 mg/100 mL in mature milk), and PtdSer (from 1.5 mg/100 mL to 1.0 mg/100 mL in mature milk). The PL class distribution was similar in colostrum, transitional, and mature milk (Figure 2).
Gangliosides
Gangliosides were determined by LC-MS/MS as described by Giuffrida et al. [29] and the results are listed in Table 4.
From the total population, the amount of GD changed during the lactation period (Table 4), with GM3 significantly increasing (p < 0.05) from 3.8 mg/mL in colostrum to 10.1 mg/L in mature milk and GD3 significantly decreasing (p < 0.05) from 4.1 mg/mL in colostrum to 1.0 mg/mL in mature milk. Total gangliosides increased significantly (p < 0.05) from 8.0 mg/L in colostrum to 11.0 mg/L in mature milk (Table 4). However, variability was high and total ganglioside content ranged from 1. Among the different cities, GM3 content was comparable (p > 0.05) in colostrum; GM3 highest content (p < 0.05) in transitional milk (7.7 mg/L) was observed in HM of lactating mothers from Guangzhou and in mature milk in lactating mothers from Guangzhou and Suzhou, at 10.5 and 10.8 mg/L, respectively (Table 4). Within mature milk (Figure 4) Beijing, Guangzhou, and Suzhou showed the highest GM3 content at 4-8 months (11.0 ± 3.9, 12.3 ± 5.5, and 15.6 ± 6.1 mg/L, respectively). The highest content (p < 0.05) of GD3 was observed in colostrum of lactating mothers from Suzhou (8.6 mg/L); GD3 content was comparable (p > 0.05) in transitional milk among the different cities and between Guangzhou and Suzhou in mature HM (Table 4). However, when considering mature milk at different lactation stages (Figure 4), Beijing, Guangzhou, and Suzhou showed the highest GD3 content at 12-30 days (0.9 ± 1.3, 1.1 ± 1.1, and 1.5 ± 2.2 mg/L, respectively). Suzhou showed the highest content (p < 0.05) of total GD in colostrum and mature milk (12.6 and 11.9 mg/L, respectively), the Among the different cities, GM3 content was comparable (p > 0.05) in colostrum; GM3 highest content (p < 0.05) in transitional milk (7.7 mg/L) was observed in HM of lactating mothers from Guangzhou and in mature milk in lactating mothers from Guangzhou and Suzhou, at 10.5 and 10.8 mg/L, respectively (Table 4). Within mature milk (Figure 4) Beijing, Guangzhou, and Suzhou showed the highest GM3 content at 4-8 months (11.0 ± 3.9, 12.3 ± 5.5, and 15.6 ± 6.1 mg/L, respectively). The highest content (p < 0.05) of GD3 was observed in colostrum of lactating mothers from Suzhou (8.6 mg/L); GD3 content was comparable (p > 0.05) in transitional milk among the different cities and between Guangzhou and Suzhou in mature HM (Table 4). However, when considering mature milk at different lactation stages (Figure 4), Beijing, Guangzhou, and Suzhou showed the highest GD3 content at 12-30 days (0.9 ± 1.3, 1.1 ± 1.1, and 1.5 ± 2.2 mg/L, respectively). Suzhou showed the highest content (p < 0.05) of total GD in colostrum and mature milk (12.6 and 11.9 mg/L, respectively), the Among the different cities, GM3 content was comparable (p > 0.05) in colostrum; GM3 highest content (p < 0.05) in transitional milk (7.7 mg/L) was observed in HM of lactating mothers from Guangzhou and in mature milk in lactating mothers from Guangzhou and Suzhou, at 10.5 and 10.8 mg/L, respectively (Table 4). Within mature milk (Figure 4) Beijing, Guangzhou, and Suzhou showed the highest GM3 content at 4-8 months (11.0 ± 3.9, 12.3 ± 5.5, and 15.6 ± 6.1 mg/L, respectively). The highest content (p < 0.05) of GD3 was observed in colostrum of lactating mothers from Suzhou (8.6 mg/L); GD3 content was comparable (p > 0.05) in transitional milk among the different cities and between Guangzhou and Suzhou in mature HM (Table 4). However, when considering mature milk at different lactation stages (Figure 4), Beijing, Guangzhou, and Suzhou showed the highest GD3 content at 12-30 days (0.9 ± 1.3, 1.1 ± 1.1, and 1.5 ± 2.2 mg/L, respectively). Suzhou showed the highest content (p < 0.05) of total GD in colostrum and mature milk (12.6 and 11.9 mg/L, respectively), the highest content (p < 0.05) of total GD in transitional milk was observed in Guangzhou (10.7 mg/L) ( Table 4).
highest content (p < 0.05) of total GD in transitional milk was observed in Guangzhou (10.7 mg/L) ( Table 4).
Discussion
This study measured the FA, PL, and GD content and the profile of 539 HM samples from Beijing, Guangzhou, and Suzhou.
Discussion
This study measured the FA, PL, and GD content and the profile of 539 HM samples from Beijing, Guangzhou, and Suzhou.
Among different cities, over lactation time, HM from Beijing showed slightly higher SFA content ( Table 2), Guangzhou the highest MUFA content (Table 2), and Suzhou the highest n-3PUFA content ( Table 2).
It is known that the type of fat/oil in the maternal diet influences the FA composition of breast milk. Francois et al. [22] showed that the consumption of six different dietary fats, each providing a specific FA, caused an acute response in HM FA composition, especially within 24 h, and that the response remained significantly elevated for 1-3 days after consumption of dietary fat. Therefore, difference observed in HM FA composition may reflect variation in maternal diet [33].
However, a careful analyses of dietary habits of Guangzhou, Beijing, and Suzhou needs to be performed for correlating to HM composition.
Phospholipids
Several studies have recognized the importance of PL for infant growth [39][40][41]. At the same time, PL are involved in immunity and inflammatory responses [42], and in neuronal signaling [43].
PL content in HM significantly (p < 0.005) decreased along the lactation period from 33.0 in colostrum to 24.2 mg/100 mL in mature milk, in agreement with previous studies performed elsewhere [12,44]. The PL class distribution was similar in colostrum, transitional and mature milk ( Figure 2). PL as emulsifiers are essential for the solubilization of dietary fats and as a consequence for their digestion and absorption. In this regard, the higher content of PL in colostrum and transitional HM compared to mature milk might explain the good fat absorption from HM by the newborn, despite poor pancreatic secretion, as suggested by Harzer et al. [11]. A decrease in PL content in HM along the lactation stage might occur because the diameter of the milk fat globule membrane increases [11,45], decreasing the PL/TAG ratio [7,8].
Our study showed that PtdCho was the most abundant PL in HM (Figure 2), followed by CerPCho and PtdEtn, and PtdIns and PtdSer, in agreement with previous studies [3,11,12,44,46]. PtdCho and CerPCho are important sources of choline considered as an essential nutrient for infants. Choline is a precursory amino alcohol of the neurotransmitter acetylcholine, it acts by regulating the transduction signal, and serves as a source of methyl groups in intermediate metabolism, being considered essential for optimum development of the brain [7,8]. In addition, CerPCho can reduce cholesterol absorption between 20.4%-85.5%, depending on the ingested dose (0.1% and 5.0%, respectively) [47], being possibly involved in cholesterol regulation programming.
Among the different cities, Suzhou showed the highest total PL and PtdEtn levels in colostrum, transitional, and mature milk (Table 3). Dietary sources of PtdEtn may be lecithin from rapeseed oil, whose consumption may explain also the higher content of ALA in HM from Suzhou. However, a careful analyses of dietary habits of this region needs to be performed for correlating to HM composition.
It is well known [58] that lipid and liposoluble nutrients content increases towards the latter part of a feeding session, a phenomenon that has been corroborated by biochemical analyses of total milk fat in fore-milk, and hind-milk [59,60]. Therefore, in order to assure sample homogeneity in our study all efforts have been made to collect fully-expressed milk. Among the cited studies, only Bitman et al., Thakkar et al., Holmes et al., and Fischer et al. [44,48,54,56] refer to full breast milk samples, Sala-Vila et al. [12] to fore-milk, and no detailed sampling procedure is described in the other studies. Analysis performed in fore-milk and hind-milk rather than fully-expressed milk could explain the discrepancy among results.
Gangliosides
GD are widely distributed in almost all human tissues, with the highest amount found in neural tissue and extra-neural organs, such the lung, spleen, and gut. It has been reported that during the first stages of life, dietary GD may have an important role in preventing infections [61] and in cognitive development functions [10,62].
Ma et al. [64] suggested that the ganglioside concentrations in HM at any time point may be influenced by the mother's dietary intake of gangliosides or their precursors. It was demonstrated [67] that GD3 and GM3 are transferred across the human placenta using an ex vivo model of dually-perfused isolated human placental lobules, suggesting that they are available to the developing fetus. Therefore, a careful analysis of dietary habits in this region needs to be performed for correlating to HM GD composition.
Conclusions
In this study, FA, PL, and GD contents and compositions of HM from lactating women living in Suzhou, Guangzhou, and Beijing were evaluated.
HM was collected over a period of eight months, allowing the observation of lipid compositional changes during lactation.
SFA, MUFA, and PL content decreased during lactation, PUFA and GD content increased. Among different cities, over lactation time, HM from Beijing showed the highest SFA content, HM from Guangzhou showed the highest MUFA content, and HM from Suzhou showed the highest n-3PUFA content. The highest total PL and GD contents were observed in HM from Suzhou. In order to investigate the influence of the diet on maternal milk composition, a careful analysis of dietary habits of these population needs to be performed in future work. | v3-fos-license |
2017-10-01T15:52:46.912Z | 2014-03-13T00:00:00.000 | 42451360 | {
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} | pes2o/s2orc | The Ionic Composition of Nasal Fluid and Its Function
The aim of the experiments reported here is to increase our understanding of the function of the nasal fluid. It is generally accepted that the nasal fluid assists in the humidification of the inspired air. It also assists in the capture of inspired particles such as pollen, preventing them getting lodged in the lungs. It is also known to contain antibacterial substances which keep the nose, nasopharynx and respiratory passages relatively free of infection. There are other features of the nasal fluid that are not understood. In cold weather, is it the fluid that collects in the nostrils pure water or nasal fluid? Why does nasal fluid have an exceptionally high potassium concentration? Does nasal fluid secreted during the common cold have the same composition as at other times? My objectives are to try to answer these questions. My method is to collect my nasal fluid in several different ways and have the ionic composition of each determined accurately. My findings are that nasal fluid is similar in composition however it is secreted. In cold weather, if expiration is via the nose, the nasal fluid is diluted by condensed water. The high concentration of potassium in the nasal fluid is not a way of controlling the level of potassium in the body but I suggest that it may assist in maintaining the antibacterial property of the nasal fluid.
Introduction
The nose has many functions and for several functions it relies on its nasal fluid.The nose warms the inspired air and also humidifies it by means of the nasal fluid and by transudation from the superficial capillaries in the nostrils [1].Probably, a more important role of the nose is to filter the incoming air and prevent small particles W. Burke getting into the lungs.Among the most important small particles are pollen grains.In the lungs, pollen can cause allergic reactions and difficulty in breathing.Small particles are trapped by nasal fluid, especially mucus secretion, and these particles are transported by the mucosal ciliary flow [2].It's possible that admixture with the serous secretion prevents the nasal fluid becoming too viscous.A very important function of nasal fluid is its antibacterial property [3] [4].
There is a major problem in determining the exact composition of nasal fluid and this accounts for the fact that there is often disagreement between different reports in the literature.Because of the unavoidable evaporation of the nasal fluid, the concentration of its constituents increases over time.There will be more evaporation if breathing is through the nose than through the mouth.There will be more evaporation if the inspired air is dry.There is a continuous secretion of nasal fluid but we don't know precisely how this varies.Even if it compensates for the loss of water, it cannot restore the original concentrations unless it also varies the concentrations in the secretions.A common feature is the creation of solid deposits in the nostrils as a result of evaporation.Such deposits will tend to increase the concentrations in the overlying fluid.Obviously, when collecting a sample of nasal fluid, one should try to avoid fluid that has been in contact with deposits for any appreciable time.However, removing deposits from the nostrils, say with a cotton bud, is liable to stimulate secretion and for some experiments this may be undesirable.Even clipping the nostrils in order to accumulate nasal fluid may also stimulate secretion.Another important feature is that periodically we swallow our nasal fluid and we do this subconsciously.The swallow may contain nasal deposits and so the next secretion may be markedly different.It is often quite difficult to inhibit a swallow.
It is clear from all these considerations that the composition of the nasal fluid is never constant but varies with time and conditions, and, of course, with the method of collection.One of the best ways of collecting a sample of nasal fluid is to place a weighed piece of absorbent paper (e.g.filter paper) in the nostrils and remove after a particular time [5].However, if secretion is slow, evaporation will occur and concentrations will rise.Probably the best estimate of the composition of nasal fluid is from a sample of nasal fluid immediately after a sneeze because this secretion is relatively large and occurs very rapidly, e.g.0.2 ml in a few seconds [6].
The aim of this project is to answer the following questions.In all the functions of the nasal fluid, what is the relevance of the ionic composition?Does this vary under different circumstances?Why is there an exceptionally high concentration of potassium in nasal fluid?What is the composition of nasal fluid secreted at low temperature?
Methods
The results in this paper were obtained solely from the author.Nasal fluid was collected mostly by the use of a small spoon of either plastic or stainless steel, about 10 mm × 5 mm × 2 mm, and placed as soon as possible in a microcentrifuge tube with lid of 1.5 ml volume or less which is kept closed.Occasionally fluid was collected by means of a small device made of stainless steel which clipped onto the septum between the nostrils and allowed the fluid to siphon into a microtube.This was only used to collect fluid from a sneeze, otherwise too much fluid was lost by evaporation.
All samples of nasal fluid were analysed by PaLMS (Pacific Laboratory Medicine Services), Royal North Shore Hospital, Sydney.Sodium, potassium and chloride were analysed by ion-selective electrodes (Roche Modular or Abbott Architect).Biarbonate, urea, calcium, magnesium and phosphate were analysed by the methods designed by ARCHITECT eSystens and AEROSET System JCT.
Nasal fluid secreted at low temperature was collected either in a cold room at ~4˚C in the Department of Physiology, University of Sydney or in the Climate Chamber at ~4˚C in the Faculty of Health Sciences, University of Sydney.
A total of 21 samples were collected, distributed as shown in the various Tables.The only criterion for accepting a sample was that its volume should be at least 0.25 ml.There were no other criteria.
Wilcoxon signed rank tests were used for all statistical tests.The project was approved by the Human Ethics Administration, University of Sydney (Project No: 2012/2479).
A comparison of Nasal Fluids Obtained in Different Ways
Regardless of how nasal fluid is created, does it always have the same composition?I collected nasal fluid in W. Burke four different ways: 1) as a spontaneous secretion; 2) resulting from a sneeze; 3) as secreted during a common cold; 4) during a low environmental temperature.Table 1 shows the results from these four procedures.The differences between these four sets of results were tested by the Wilcoxon signed rank test.Table 2 shows the ratios of the four methods for each of the elements in the composition of the fluid.
There was no significant difference between any pair of the methods except for the difference between the fluid collected spontaneously and the nasal fluid collected at 4˚C.This difference is most probably explained by the fact that 4˚C fluid was collected over a period of about one hour and this would have allowed more time for evaporation, so giving higher values for almost all the elements.However, the absence of a significant difference between the other pairs does not necessarily mean that there is no real difference.That is because the number of samples is low and, as explained previously, collection of nasal fluid is subject to several complications (evaporation, swallowing, etc).Samples relating to the common cold were collected at an early stage of the infection.At a later stage all the values were elevated but further samples could not be obtained, so it is necessary to perform more experiments to check this observation.However, my preliminary observation is consistent with the results of Vanthanouvong and Roomans (2004) reporting elevated ion concentrations in patients with common cold or rhinitis [7].The spontaneous (spont) values are from samples of nasal fluid for which there was no known stimulus.The data in the last column (4˚C) are the values obtained at 4˚C when expiration was via the mouth, not the nose.All values are expressed as mmol/l (means +/− SEM); number of samples in brackets.See Table 2 for statistical analysis of results.It is relevant to note that the papers do not employ the same method for estimating the ionic composition.Indeed, each paper has a different method, except that the data in columns F and G are from the same laboratory using the same method [7] [11].Even in such ideal circumstances there may be a difference of 30% or more (e.g. the values for potassium).
Effect of Low Environmental Temperature on the Secretion of Nasal Fluid
It has frequently been noticed that on a cold day there is often an accumulation of nasal fluid in the nostrils.This is usually very watery and has led to the belief that it is simply a condensation of water from the warm expired air passing through the cold nose [1].However, it is now believed that the cold environment dilates the blood capillaries supplying the nasal glands and this leads to a nasal secretion above normal.I investigated this phenomenon by collecting nasal fluid over a period of about one hour in a cold room or climate chamber at 4˚C.On three occasions I breathed out through the mouth, on three other occasions I breathed out through the nose.The results of the analyses are shown in Table 4.
It is clear that the concentrations of all the substances measured are much lower when the expired air goes through the nose.The difference is significant (Wilcoxon signed ranks test P = 0.008 for experiment A; P = 0.023 for experiment B).Thus, the nasal fluid collected when expiration is via the mouth is closer to normal, whereas nasal fluid when expiration is via the nose is more dilute.I draw two conclusions from this experiment.The first is a confirmation that a cold environment induces a secretion of nasal fluid.The second is that expired air passing through the nostrils loses some of its water content as condensate, as would indeed be required by the laws of physics, and this, added to the nasal secretion, gives a more dilute sample.The results of such an experiment as this will vary according to several factors: the environmental temperature, the anatomy and physiology of the capillaries in the nostrils, the anatomy and physiology of the nose, especially the size of the nose and the sensitivity of the temperature receptors.However, it does not seem to matter whether inspiration is via the mouth or via the nose.
Why is there an increased blood supply to the nose in cold weather?Apart from the increase in nasal fluid, this will also tend to warm the inspired air and maintain a comfortable environment in the chest.The importance of this is not clear.
Relevance of High Potassium Concentrations in the Nasal Fluid
The concentration of potassium n the nasal fluid is 7 -10 times of that in the blood plasma [5] [7] [12] but also varies a lot, both within an individual (this paper) and between individuals [5].Is there any correlation between the concentrations of sodium and potassium in the various samples of nasal fluid?In answering this question it was important to use as broad a selection of data as possible.Figure 1 plots the distribution of sodium and po 1) and in cited papers (filled circles; data in columns C, E, F, G in Table 3).The regression line is based on all the nasal fluid data; note that this is close to the horizontal, indicating an independence of the secretions of sodium and potassium.A single sample from the author depicts the sodium and potassium in his plasma; data similar to textbook values (open rectangle).Note the radical difference between plasma and nasal fluid.tassium in the nasal fluid obtained in the current experiments (open circles) and also taken from other reports (filled circles); complete data in Tables 1 and 3.The regression line is drawn through all the nasal fluid data in the graph.It is clear from this that there is no correlation between the concentrations of sodium and potassium, Thus, the factor determining the secretion of potassium is different to that regulating the secretion of sodium.Also relevant to this conclusion is the difference between the concentrations of these ions in plasma and in nasal fluid.This is illustrated by plotting in the graph the Na/K relation for the plasma of the author on one occasion, a value close to the textbook value (open rectangle).The large difference between this and the nasal fluid is clear and this emphasises the particular importance of the potassium in the nasal fluid.
What is the role of the potassium in the nasal fluid?This question will be taken up in the Discussion.
Discussion
The results reported here are from a single person, the author.Obviously I cannot claim that they are characteristic of a large population.As I explained in the Introduction there are serious difficulties in getting accurate estimates of the volume and composition of nasal fluid.Therefore, this paper is to a large degree an account of all these difficulties and perhaps some advice about how to avoid serious errors.Although I have tried to avoid such errors, I have not paid any attention to variations in environmental temperature or humidity nor to many subconscious aspects of breathing such as swallowing, sniffing, clearing the throat, etc, all of which might affect the composition of nasal fluid.
A Comparison of Nasal Fluids Obtained in Different Ways.
The results show no convincing evidence that the composition of nasal fluid collected in different ways differed significantly.Even though one pair of methods (spont/4˚C) reached a significant difference, this most probably reflects the problems associated with the collection of nasal fluid referred to in the Introduction, in this case the long time required to collect the secretion at 4˚C with the resultant rise in evaporation.However, I think it is unlikely that there is any dramatic difference between any pair of the secretions.
There is probably no reason why nasal fluid should vary greatly in ionic composition.On the other hand, it would not be surprising if the organic constituents of nasal fluid might vary in concentration.I have examined only urea.Urea is a metabolite of nitrogen-containing foodstuffs and has no physiological function; although it is mainly excreted in the urine, its concentration in nasal fluid might depend on the amount of nitrogen in the diet.
Effect of Low Environmental Temperature on the Secretion of Nasal Fluid
The results obtained here show clearly that the fluid collected from the nostrils in cold weather contains nasal fluid similar to that obtained at normal temperatures.If expiration is via the nose there is a dilution of the nasal fluid, as would be expected if warm air from the lungs passing through a cold nose condenses to water.
Is there a greater secretion of nasal fluid at low temperatures than at room temperatures?I am unable to answer this question.I can collect nasal fluid at room temperatures and such samples are shown in Table 1 as 'spontaneous'.However, I do not know what caused these secretions.I also note, as other people do, that quite often one sneezes, and produces a lot of nasal fluid, for no obvious reason.It is generally believed that such sneezes are caused by an irritation of the nasal mucosa but that such stimulation is not consciously detected.If one breathes always through the nose it is probable that one's nasal secretions pass to the nasopharynx and are swallowed.This would tend to reduce the observed amount of nasal secretion.This factor and all the others mentioned in the Introduction mean that there is considerable uncertainty as to whether there is a difference between the amounts or compositions of nasal fluids collected in different ways.If the cold environment causes a dilation of blood capillaries in the nostrils, the effect on the glands may be fortuitous, not the main effect.
Relevance of Ionic Concentrations in the Nasal Fluid
It is possible that the high potassium fills a role within the nose or the upper airway but such a role has not yet been discovered.It has been known for a long time that the concentrations of the major ions in the nasal fluid are not arbitrary.Fleming (1922) [3] noted that the lysozyme, the antibacterial substance that he discovered in nasal fluid (and other secretions and tissues) acted most rapidly if a small amount of salt (less than 0.1% NaCl) was present but not if the concentration exceeded 5.0%.This combination of facilitatory and inhibitory effects of salt concentrations has been confirmed many times [13]- [15].It is clear that nasal fluid has appropriate ionic concentrations to facilitate an antibacterial influence.However, it is possible that some species of ion may play a specific role.For example, cystic fibrosis (CF) is caused by a mutation in the gene regulating chloride conductance in the apical epithelia, leading to elevated chloride concentration in the nasal fluid (182 mmol/l.vs 132 mmol/l in non-CF nasal fluid) [8].This gene also affects the sodium channel, and the increased chloride concentration, or perhaps the increased NaCl concentration act specifically to reduce or abolish the bactericidal activity of the CF nasal fluid [8] Most of the discussion on this matter has concentrated on sodium and chloride.The relative importance of other ions does not appear to have been studied.The concentration of potassium in nasal fluid is unusually high at about 35 mmol/l (Figure 1).This raises the question of the relevance of potassium in nasal fluid.
Relevance of High Potassium Concentrations in the Nasal Fluid
It is possible that the high potassium fills a role within the nose or the upper airway but such a role has not yet been discovered.Is it possible that this is a way of excreting potassium from the body?From Harada et al. (1984) [16] one can calculate that in each day about 36 ml of nasal fluid is secreted.Let's assume that all of this swallowed.This contains about 1.4 g of potassium (about 1.3 mmol).This is below the lowest amount found in the faeces in cases of low potassium intake [17] and is a small amount compared with the normal excretion of potassium in the urine (40 -66 mmol/day) or faeces (4 -16 mmol/day) [17].Thus it is unlikely that the high concentration of potassium in the nasal fluid is there as a method of controlling the level of potassium in the body.The main control obviously remains with the kidney.
Another possibility is that the potassium contributes to an antibacterial role.It is well known that the nasal fluid has such a property [3] [4] and this must be partly due to the presence of lysozyme and the immunoglobulins, but there are many other antibacterial substances in the nasal fluid [14].In a different context, potassium is used to reduce infection in food preparations such as processed poultry [18] [19], where the potassium is present as a fatty acid salt.The applied solutions are strongly alkaline (pH about 12) and it is possible that the antimicrobial effect is partly due to the high pH.In recent years potassium diformate has been used in pigs and other animals as a growth promoter alternative to antibiotics [20] [21].Potassium diformate is also antibacterial but it is not known whether this effect us due to the low pH, resulting from the free formic acid, or to formate or to the potassium.
In recent years, foods have been coated with edible films that act partly to prevent contamination and to varying degrees are antibacterial.One substance used in these films is potassium sorbate.This seems to work best at low pH such as 3.0 -4.5 [22].Again, it is uncertain what the role of the potassium is, and whether this has any relevance to the high potassium in the nasal fluid.
It remains a possibility that potassium has a special influence as an antibacterial supplement, greater than that of the other ions.A recent paper has data that may be relevant [23].These authors were concerned with the occurrence of ventilator-associated pneumonia (VAP) and chronic lung disease in neonates resulting from airway suctioning, assisted by preliminary instillation of normal saline.They found that replacing the normal saline with a low-sodium solution reduced the incidence of VAP and lung disease.However the low-sodium solution contained 24.5 mmol/l potassium, i.e. an amount similar to that found in normal nasal fluid (see Table 3).They do not comment on the role of the potassium but its presence (and the absence of any bactericide) prompts the question: does potassium have any antibacterial function in the low-sodium solution and, correspondingly, in nasal fluid.
Conclusion
However generated, the ionic composition of nasal fluid is similar.The ionic composition is always appropriate for its antibacterial action.Of particular interest is the relatively high concentration of potassium.One possible role for potassium (as a method of controlling the level of potassium in the body) has been rejected.A second role as a facilitator of antibacterial action should be tested.
Figure 1 .
Figure 1.Sodium/potassium relations in nasal fluid and in plasma.The graph plots sodium and potassium concentrations in nasal fluid obtained in the current experiments (open circles; data in Table1) and in cited papers (filled circles; data in columns C, E, F, G in Table3).The regression line is based on all the nasal fluid data; note that this is close to the horizontal, indicating an independence of the secretions of sodium and potassium.A single sample from the author depicts the sodium and potassium in his plasma; data similar to textbook values (open rectangle).Note the radical difference between plasma and nasal fluid.
+/− SEM of nasal fluid components (mmol/l) obtained at ~4˚C when inspiring through the Mouth and expiring through the Nose (2nd column) or inspiring through the Nose and expiring through the Mouth (3rd column).The 4th column shows the ratios of the values in the 2nd and 3rd columns.Application of the Wilcoxon signed rank test gives a significant difference ( ** ) between the two conditions.(P = 0.008), Figures in brackets are the number of samples.B. In the 5th column are shown the Nose/Mouth ratios from another similar experiment in which inspiration was always through the Mouth: expiration through the Mouth 3 samples; expiration through the Nose 3 samples.Applying the Wilcoxon test showed a significant difference ( * ) between the two conditions (P = 0.023).
Table 3
summarizes data published in this and six other papers.Some papers express their data as a range of
Table 1 .
Analyses of nasal fluids collected in different ways.
Table 2 .
Statistical analysis of results in Table1.
* ns ns nsThis Table contains the ratios of all pairs of Means for each component.spont = spontaneous; snz = sneeze; cc = common cold; 4˚C = samples collected at 4˚C.Analysis by Wilcoxon signed rank test, each condition grouped.No test was significant (ns) except for the spont/ 4˚C comparison (P = 0.0234; * ).
Table 1 .
Data in the other columns are the Means +/− SEM.values, most as means +/− SEMs, one as approximate values.Because I have used four ways of obtaining the data, I have used the range of means from the four ways (column H).As explained in the Introduction, it is not surprising that there is a wide range of values.Nevertheless, allowing for this, there is fair agreement between the eight reports.My values for calcium and phosphate are lower than in two other papers but this may be because these two species of ion are present in low amounts and the estimates are therefore less reliable.
Table 4 .
Effect of low environmental temperature on nasal secretion. | v3-fos-license |
2016-05-12T22:15:10.714Z | 2015-12-01T00:00:00.000 | 17400434 | {
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} | pes2o/s2orc | Multiple Temperature-Sensing Behavior of Green and Red Upconversion Emissions from Stark Sublevels of Er3+
Upconversion luminescence properties from the emissions of Stark sublevels of Er3+ were investigated in Er3+-Yb3+-Mo6+-codoped TiO2 phosphors in this study. According to the energy levels split from Er3+, green and red emissions from the transitions of four coupled energy levels, 2H11/2(I)/2H11/2(II), 4S3/2(I)/4S3/2(II), 4F9/2(I)/4F9/2(II), and 2H11/2(I) + 2H11/2(II)/4S3/2(I) + 4S3/2(II), were observed under 976 nm laser diode excitation. By utilizing the fluorescence intensity ratio (FIR) technique, temperature-dependent upconversion emissions from these four coupled energy levels were analyzed at length. The optical temperature-sensing behaviors of sensing sensitivity, measurement error, and operating temperature for the four coupled energy levels are discussed, all of which are closely related to the energy gap of the coupled energy levels, FIR value, and luminescence intensity. Experimental results suggest that Er3+-Yb3+-Mo6+-codoped TiO2 phosphor with four pairs of energy levels coupled by Stark sublevels provides a new and effective route to realize multiple optical temperature-sensing through a wide range of temperatures in an independent system.
Introduction
Optical temperature-sensing devices have been widely researched to promote their application in electrical power stations, oil refineries, coal mines, and fire detection, as they have been shown to overcome the interference of strong electromagnetic noise, hazardous sparks, or corrosive environments inaccessible to traditional temperature-measurement methods such as thermocouple detectors [1][2][3][4][5]. Sensors built based on the fluorescence intensity ratio (FIR) technique have attracted particular attention due to their ability to reduce dependence on measurement conditions and improve accuracy and resolution. FIR functions independent of fluorescence loss or fluctuations in excitation intensity can be applied to fluorescence systems in which two closely spaced energy levels with separations of the order of thermal energy are involved, following a Boltzmann-type population distribution [1,6,7]. Optical temperature sensors using the FIR technique are mainly focused on fluoride and oxides matrixes [8][9][10][11][12][13][14]. The fluoride matrixes possesses higher fluorescence efficiency and lower excitation power; however, the maximum operating temperature is usually low. On the contrary, the oxides matrices can operate at high temperature, although the fluorescence intensity is lower.
Upconversion emissions of rare earth ion-doped materials are typically utilized to realize FIR measurement because of the large amount of coupled energy levels in many rare earth ions and the easily accessible upconversion luminescence with near-infrared radiation from low-cost, commercially available diodes. Xu et al. [8], for example, reported the FIR of Ho 3+ using two blue emissions from coupled energy levels of 5 G 6 / 5 F 1 and 5 F 2,3 / 3 K 8 and found that Ho 3+ -Yb 3+ -codoped CaWO 4 possessed higher absolute sensitivity due to a larger energy gap between the thermally coupled 5 G 6 / 5 F 1 and 5 F 2,3 / 3 K 8 levels of Ho 3+ ions. The paired energy levels of 3 F 2 and 3 F 3 in Tm 3+ ions have also been used to investigate temperature-dependent red upconversion emissions and corresponding FIR properties [9]. The FIR properties of green upconversion emissions ascribed to paired energy levels of 2 H 11/2 and 4 S 3/2 in Er 3+ -doped materials, in particular, have been quite widely studied [10][11][12][13][14].
In addition to the intrinsic thermally coupled energy levels of rare earth ions, the pair energy levels of Stark sublevels can also be thermally coupled and used to investigate FIR versus temperature characteristics [15][16][17][18]. Baxter et al. [17], for example, used the coupled energy levels of 2 F 5/2(a) and 2 F 5/2(b) by Stark split of 2 F 5/2 levels in Yb 3+ ions to study FIR properties of Yb 3+ -doped silica fiber. Feng et al. [18] investigated the FIR properties of Er 3+ -doped fluoride glass using coupled Stark sublevels of 4 S 3/2(1) and 4 S 3/2 (2) in Er 3+ ions.
In this study, four thermally coupled energy levels of Er 3+ ions based on the Stark sublevels were simultaneously observed in Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphors. FIR properties of the four coupled energy levels from green and red emissions in Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphors were studied as a function of temperature in the range of 307-673 K. The effects of the energy gap of thermally coupled energy levels, FIR value, and upconversion emission intensity on the sensitivity and accuracy of the optical temperature sensor are discussed in an effort to explore potential developments in optical temperature-sensor technology based on different FIR routes in an independent system.
Experimental Section
The sol-gel method was used to prepare Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphors. The rare earth nitrates Er(NO 3 ) 3¨5 H 2 O (99.99%) and Yb(NO 3 ) 3¨5 H 2 O (99.99%) were purchased from Aladdin. Other chemicals including Iso-Propanol (i-PrOH), n-butyl titanate (Ti(OBu) 4 ), acetylacetone (AcAc), and concentrated nitric acid (HNO 3 ) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals are of analytical reagent and were used without any further purification. i-PrOH was first added as a solvent to modified titanium(IV) n-butoxide by facilitating a chelating reaction between Ti(OBu) 4 and AcAc under agitation for 1 h at room temperature. Next, a mixture of deionized water, i-PrOH, and HNO 3 was slowly added into the solution. The mixed solution was stirred for 6 h to form a clear and stable sol. The molar ratios of Ti(OBu) 4 The codoped sols were dried at 373 K for 8 h to remove the solvent. The xerogels were then heated at a rate of 4 K/min and maintained at the sintering temperature of 1073 K for 1 h, then cooled to room temperature in the furnace. The sintered 2 mol % Er 3+ -20 mol % Yb 3+ -2 mol % Mo 6+ -codoped TiO 2 phosphors were finally milled into powders for structural analysis and spectral measurement.
The phase structures of Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphor samples were analyzed by SHIMADZU XRD-6000 X-ray diffractormeter (XRD) with Cu-Kα radiation. A homemade temperature control system, which was composed of a small stove and an intelligent digital-display-type temperature control instrument, was used to adjust sample temperature from 307 to 673 K, at measurement and control accuracy of about˘0.5 K. Temperature-dependent upconversion emissions from each sample were focused onto a Jobin Yvon iHr550 monochromator and detected with a CR131 photomultiplier tube by 976 nm laser diode (LD) excitation. The LD pump current varied from 0 to 2 A, and the spectral resolution of the experimental set-up was 0.1 nm. Figure 1 shows XRD patterns of the Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphor samples. The XRD pattern observed was characteristic of the anatase phase of TiO 2 (JCPDS No. 21-1272) and the face-centered cubic phase of Yb 2 Ti 2 O 7 (JCPDS No. 17-0454) referenced below. There was no diffraction peak of Mo compounds, and the main diffraction peak shifted toward small angles, indicating Mo 6+ stochastically located at the interstitial sites of the matrix lattice as a solution element.
Sensors 2015, 15, page-page 3 peak of Mo compounds, and the main diffraction peak shifted toward small angles, indicating Mo 6+ stochastically located at the interstitial sites of the matrix lattice as a solution element. Figure 2 shows the upconversion emission spectra of Er 3+ -Yb 3+ -Mo 6+ -codoped TiO2 under different pump currents. Green and red upconversion emissions were observed in the wavelengths of 500-540 nm, 540-580 nm, and 620-710 nm, corresponding to 2 H11/2 → 4 I15/2, 4 S3/2 → 4 I15/2, and 4 F9/2 → 4 I15/2 transitions of Er 3+ ions, respectively. Each transition ( 2 H11/2 → 4 I15/2, 4 S3/2 → 4 I15/2, and 4 F9/2 → 4 I15/2) was divided into two emission peaks, which indicated 2 H11/2, 4 S3/2, and 4 F9/2 levels of Er 3+ split into three coupled Stark sublevels of 2 H11/2(I)·(HI) and 2 H11/2(II)·(HII), 4 S3/2(I)·(SI) and 4 S3/2(II)·(SII), and 4 F9/2(I)·(FI) and 4 F9/2(II)·(FII), respectively, due to the effect of crystal field environment on Er 3+ ions. As the LD pump current increased from 0.8 to 2.0 A, the position and number of upconversion emission peaks did not change, whereas the intensity of green and red emissions markedly increased due to the increase in excitation power. The inset in Figure 2 shows the upconversion emission intensity ratios of H I /H II , S I /S II , F I /F II , and (H I + H II )/(S I + S II ) versus the pump current. All intensity ratios of H I /H II , S I /S II , F I /F II and (H I + H II )/(S I + S II ) increased alongside the pump current, implying that the nonradiative processes of Er 3+ in Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphor can partially transform pump energy into heat energy, therefore elevating the phosphor temperature. The temperature variation induced by increasing the pump current caused changes in the intensity ratio [19]; this suggests that the temperature-dependent intensity ratio for the four coupled energy levels of H I /H II , S I /S II , F I /F II , and (H I + H II )/(S I + S II ) can be utilized for optical temperature sensing. Figure 3 shows a schematic energy level diagram of the Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphors under 976 nm LD excitation. The upconversion mechanism of Er 3+ after the addition of Mo 6+ was reported in a previous study on the sensitization of the Yb 3+ -MoO 4 2´d imer to Er 3+ [20][21][22]. The inset in Figure 2 shows the upconversion emission intensity ratios of HI/HII, SI/SII, FI/FII, and (HI + HII)/(SI + SII) versus the pump current. All intensity ratios of HI/HII, SI/SII, FI/FII and (HI + HII)/(SI + SII) increased alongside the pump current, implying that the nonradiative processes of Er 3+ in Er 3+ -Yb 3+ -Mo 6+ -codoped TiO2 phosphor can partially transform pump energy into heat energy, therefore elevating the phosphor temperature. The temperature variation induced by increasing the pump current caused changes in the intensity ratio [19]; this suggests that the temperature-dependent intensity ratio for the four coupled energy levels of HI/HII, SI/SII, FI/FII, and (HI + HII)/(SI + SII) can be utilized for optical temperature sensing. Figure 3 shows a schematic energy level diagram of the Er 3+ -Yb 3+ -Mo 6+ -codoped TiO2 phosphors under 976 nm LD excitation. The upconversion mechanism of Er 3+ after the addition of Mo 6+ was reported in a previous study on the sensitization of the Yb 3+ -MoO4 2− dimer to Er 3+ [20][21][22]. Through a cooperative sensitization process in the Yb 3+ -MoO4 2− dimer, two excited Yb 3+ ions nonradiatively transfer their energy to MoO4 2− . This process is followed by a high excited state energy transfer (HESET) to the 4 F7/2 level of Er 3+ ions. After nonradiative relaxations from 4 F7/2 to the Stark sublevels of HI, HII, SI and SII, green upconversion emissions are produced by transitions of HI/HII/SI/SII → 4 I15/2. The nonradiative relaxation from SII to FI and FII levels and subsequent transitions of FI/FII → 4 I15/2 generate red emissions. In order to distinguish the effects of temperature from the pump current on the intensity ratio (Figure 2), the upconversion emission properties of Er 3+ -Yb 3+ -Mo 6+ -codoped TiO2 were measured under different temperatures. Figure 4 shows the upconversion emissions spectra of Er 3+ -Yb 3+ -Mo 6+codoped TiO2 at measured temperatures between 307 and 673 K. Changes in temperature had no influence on the bands of green and red emissions from 2 H11/2/ 4 S3/2 → 4 I15/2 and 4 F9/2 → 4 I15/2 transitions of Er 3+ between 500 to 580 nm and 620 to 700 nm, respectively; the intensity varied with temperature, however. The inset in Figure 4 shows the intensity of green and red emissions and the intensity ratio of green to red emissions as a function of temperature. The intensity of red emissions decreased with increasing temperature, in accordance with the classical theory of thermal quenching. Temperature-dependent intensity of the red emissions can be expressed as follows [23]: In order to distinguish the effects of temperature from the pump current on the intensity ratio (Figure 2), the upconversion emission properties of Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 were measured under different temperatures. Figure 4 shows the upconversion emissions spectra of Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 at measured temperatures between 307 and 673 K. Changes in temperature had no influence on the bands of green and red emissions from 2 H 11/2 / 4 S 3/2 Ñ 4 I 15/2 and 4 F 9/2 Ñ 4 I 15/2 transitions of Er 3+ between 500 to 580 nm and 620 to 700 nm, respectively; the intensity varied with temperature, however. The inset in Figure 4 shows the intensity of green and red emissions and the intensity ratio of green to red emissions as a function of temperature. The intensity of red emissions decreased with increasing temperature, in accordance with the classical theory of thermal quenching. Temperature-dependent intensity of the red emissions can be expressed as follows [23]: where T is the absolute temperature, and I(T) and I(0) are the fluorescence intensities at temperatures of T and 0 K, respectively; ∆E 1 is the activation energy, k is the Boltzmann constant, and A is a constant. The temperature-dependent intensity of red emissions fits well to Equation (1), where ∆E 1 (FI+FII) = 0.074 eV. Conversely, the intensity of green emissions increased with increasing temperature, which does not satisfy the classical theory of thermal quenching, likely due to the increased Yb 3+ absorption cross-section at elevated temperatures [22,24]. A general theoretical description of the green upconversion emission can be given by [22]: where B is a constant, and hν is the phonon energy participating in the multiphonon-assisted excitation. The dependence of green upconversion emissions on temperature fits well to Equation (2). The I green /I red value increased with temperature, causing the color to turn from red to green with elevated temperature. where T is the absolute temperature, and I(T) and I(0) are the fluorescence intensities at temperatures of T and 0 K, respectively; ΔE′ is the activation energy, k is the Boltzmann constant, and A is a constant. The temperature-dependent intensity of red emissions fits well to Equation (1), where ΔE′(FI+FII) = 0.074 eV. Conversely, the intensity of green emissions increased with increasing temperature, which does not satisfy the classical theory of thermal quenching, likely due to the increased Yb 3+ absorption crosssection at elevated temperatures [22,24]. A general theoretical description of the green upconversion emission can be given by [22]: where B is a constant, and hν is the phonon energy participating in the multiphonon-assisted excitation. The dependence of green upconversion emissions on temperature fits well to Equation (2). The Igreen/Ired value increased with temperature, causing the color to turn from red to green with elevated temperature. According to previous research [1], the relative population of two "thermally coupled" energy levels with separation of the order of thermal energy follows a Boltzmann-type population distribution, causing variation in the transitions of two closely spaced levels at elevated temperature if pumped through a continuous light source. After populations are thermalized at two closely spaced levels, the FIR of upconversion emissions (R) related to the transitions of both levels can be written as follows: where Iupper, Ilower, Nupper, and Nlower are the fluorescence intensity and number of ions for the upper and lower thermalizing energy levels, respectively; ΔE is the energy gap between two coupled levels, and C is a constant relative to the degeneracy, emission cross-section, and angular frequency of corresponding transitions. Equation (3) suggests that FIR is related to the energy gap ΔE and temperature T. Figure 5 shows FIR plots of (HI + HII)/(SI + SII), HI/HII, SI/SII, and FI/FII as a function of (1) and (2).
According to previous research [1], the relative population of two "thermally coupled" energy levels with separation of the order of thermal energy follows a Boltzmann-type population distribution, causing variation in the transitions of two closely spaced levels at elevated temperature if pumped through a continuous light source. After populations are thermalized at two closely spaced levels, the FIR of upconversion emissions (R) related to the transitions of both levels can be written as follows: where I upper , I lower , N upper , and N lower are the fluorescence intensity and number of ions for the upper and lower thermalizing energy levels, respectively; ∆E is the energy gap between two coupled levels, and C is a constant relative to the degeneracy, emission cross-section, and angular frequency of corresponding transitions. Equation (3) suggests that FIR is related to the energy gap ∆E and temperature T. Figure 5 shows FIR plots of (H I + H II )/(S I + S II ), H I /H II , S I /S II , and F I /F II as a function of inverse absolute temperature from 307 to 673 K. The inset shows corresponding upconversion emission intensity and the intensity ratio relative to temperature. The experimental data fits well to Equation (3). Energy gaps ∆E of the four coupled energy levels of (H I + H II )/(S I + S II ), H I /H II , S I /S II , and F I /F II are calculated in Table 1. The decreased intensity of two red emissions with elevated temperature, shown in the inset of Figure 5d, can also be fitted to Equation (1). The activation energy of F I and F II levels is calculated as ∆E 1 FI = 0.069 eV and ∆E 1 FII = 0.080 eV, which is consistent with the average activation energy of (F I + F II ) level (∆E 1 (FI+FII) = 0.074 eV) shown in Figure 4.
Sensors 2015, 15, page-page 6 inverse absolute temperature from 307 to 673 K. The inset shows corresponding upconversion emission intensity and the intensity ratio relative to temperature. The experimental data fits well to Equation (3). Energy gaps ΔE of the four coupled energy levels of (HI + HII)/(SI + SII), HI/HII, SI/SII, and FI/FII are calculated in Table 1. The decreased intensity of two red emissions with elevated temperature, shown in the inset of Figure 5d, can also be fitted to Equation (1). The activation energy of FI and FII levels is calculated as ΔE′FI = 0.069 eV and ΔE′FII = 0.080 eV, which is consistent with the average activation energy of (FI + FII) level (ΔE′(FI+FII) = 0.074 eV) shown in Figure 4. Table 1. Energy gap of coupled energy levels ΔE, pre-exponential factor C, maximum sensitivity Smax, temperature of maximum sensitivity Tmax and upconversion emission intensity for the four coupled energy levels of (HI + HII)/(SI + SII), HI/HII, SI/SII and FI/FII. For optical temperature-sensing applications, it is crucial to know the rate at which the FIR changes with temperature, known as the absolute sensitivity Sa, which is expressed as follows [1]:
Coupled Energy Levels (HI + HII)/(SI + SII) HI/HII SI/SII FI/FII
Equation (4) makes clear that the appropriate selection of two thermally coupled energy levels with a suitable energy difference ΔE is very important. Larger ΔE benefits absolute sensitivity and accurate measurement of emission intensity, due to the decrease of fluorescence peak overlap originating from the two individual thermally coupled energy levels. Knowing this, the absolute sensitivity Sa when using coupled energy levels of (HI + HII)/(SI + SII) (with the largest possible ΔE = 0.0558 eV) is higher than those using the other three coupled levels, as shown in Table 1. The energy gap ΔE must be not too large, though, or thermalization no longer occurs.
Considering practical applications, it is extremely useful to be aware of variations in sensitivity with temperature. Relative sensitivity Sr is expressed [25]: dT kT (5) Compared to absolute sensitivity Sa, relative sensitivity Sr is dependent on not only energy gap ΔE, but also the intensity ratio FIR. Equation (3) indicates that larger FIR causes larger C. Thus, larger ΔE and FIR (or C) contribute to higher Sr. Table 1 also shows pre-exponential factor C values for the four pair energy levels (HI + HII)/(SI + SII), HI/HII, SI/SII, and FI/FII. The coupled energy levels of (HI + HII)/(SI + SII) processed larger relative sensitivity Sr than those of HI/HII, FI/FII, or SI/SII. Sr as a function Table 1. Energy gap of coupled energy levels ∆E, pre-exponential factor C, maximum sensitivity S max , temperature of maximum sensitivity T max and upconversion emission intensity for the four coupled energy levels of (H I + H II )/(S I + S II ), H I /H II , S I /S II and F I /F II . For optical temperature-sensing applications, it is crucial to know the rate at which the FIR changes with temperature, known as the absolute sensitivity S a , which is expressed as follows [1]:
Coupled Energy Levels (H I + H II )/(S I + S II ) H I /H II S I /S II F I /F II
Equation (4) makes clear that the appropriate selection of two thermally coupled energy levels with a suitable energy difference ∆E is very important. Larger ∆E benefits absolute sensitivity and accurate measurement of emission intensity, due to the decrease of fluorescence peak overlap originating from the two individual thermally coupled energy levels. Knowing this, the absolute sensitivity S a when using coupled energy levels of (H I + H II )/(S I + S II ) (with the largest possible ∆E = 0.0558 eV) is higher than those using the other three coupled levels, as shown in Table 1. The energy gap ∆E must be not too large, though, or thermalization no longer occurs.
Considering practical applications, it is extremely useful to be aware of variations in sensitivity with temperature. Relative sensitivity S r is expressed [25]: Compared to absolute sensitivity S a , relative sensitivity S r is dependent on not only energy gap ∆E, but also the intensity ratio FIR. Equation (3) indicates that larger FIR causes larger C. Thus, larger ∆E and FIR (or C) contribute to higher S r . Table 1 also shows pre-exponential factor C values for the four pair energy levels (H I + H II )/(S I + S II ), H I /H II , S I /S II , and F I /F II . The coupled energy levels of (H I + H II )/(S I + S II ) processed larger relative sensitivity S r than those of H I /H II , F I /F II , or S I /S II . S r as a function of temperature for the four coupled energy levels calculated by Equation (5) is shown in Figure 6, in accordance with the above results in the measured temperature range 307-673 K.
Sensors 2015, 15, page-page 8 of temperature for the four coupled energy levels calculated by Equation (5) is shown in Figure 6, in accordance with the above results in the measured temperature range 307-673 K. Maximum sensitivity Smax and temperature Tmax, at which the sensor has maximum sensitivity Smax, are of utmost importance because these two parameters indicate the highest sensitivity properties and optimum operating temperature range of optical thermal sensors. According to Equation (5) (7) Equation (6) indicates that a larger pre-exponential factor C and smaller energy difference ΔE of coupled energy levels help to increase Smax. Equation (7) shows that Tmax is relative to the energy difference ΔE, in which the sensor with a larger ΔE has a higher Tmax. Smax and Tmax for the four coupled energy levels are shown in Table 1. The highest Tmax was found for (HI + HII)/(SI + SII) coupled energy levels used for thermal sensing, due to a larger ΔE. The relatively larger C and smallest ΔE in FI/FII coupled energy levels used for thermal sensing resulted in the highest sensitivity Smax.
Temperature measurement error can be calculated using the relation [8,26]: Larger Sr and smaller ΔR imply better accuracy. As shown in Figure 6, larger Sr at a higher temperature for coupled energy levels of (HI + HII)/(SI + SII) led to a better accuracy in the high temperature range. Likewise, better accuracy can be expected in the low temperature range using HI/HII, SI/SII and FI/FII coupled energy levels for thermal sensing.
The separation of two coupled energy levels ΔE should be large enough to avoid overlap of the two fluorescence emissions and to produce efficient luminescence for feasible and accurate intensity measurement. The efficient luminescence of Er 3+ -doped materials also contributes to the ready detection of luminescence and ΔR accuracy, where only low excitation power is needed. Table 1 Maximum sensitivity S max and temperature T max , at which the sensor has maximum sensitivity S max , are of utmost importance because these two parameters indicate the highest sensitivity properties and optimum operating temperature range of optical thermal sensors. According to Equation (5), S max and T max can be calculated by dS r {dT " 0 as follows: Equation (6) indicates that a larger pre-exponential factor C and smaller energy difference ∆E of coupled energy levels help to increase S max . Equation (7) shows that T max is relative to the energy difference ∆E, in which the sensor with a larger ∆E has a higher T max . S max and T max for the four coupled energy levels are shown in Table 1. The highest T max was found for (H I + H II )/ (S I + S II ) coupled energy levels used for thermal sensing, due to a larger ∆E. The relatively larger C and smallest ∆E in F I /F II coupled energy levels used for thermal sensing resulted in the highest sensitivity S max .
Temperature measurement error can be calculated using the relation [8,26]: Larger S r and smaller ∆R imply better accuracy. As shown in Figure 6, larger S r at a higher temperature for coupled energy levels of (H I + H II )/(S I + S II ) led to a better accuracy in the high temperature range. Likewise, better accuracy can be expected in the low temperature range using H I /H II , S I /S II and F I /F II coupled energy levels for thermal sensing.
The separation of two coupled energy levels ∆E should be large enough to avoid overlap of the two fluorescence emissions and to produce efficient luminescence for feasible and accurate intensity measurement. The efficient luminescence of Er 3+ -doped materials also contributes to the ready detection of luminescence and ∆R accuracy, where only low excitation power is needed. Table 1 shows where (H I + H II )/(S I + S II ) coupled energy levels had the highest accuracy of all samples, due to a larger ∆E and the strongest luminescence intensity; conversely, S I /S II coupled energy levels had the lowest accuracy, evidenced by a smaller ∆E and the lowest luminescence intensity, which are altogether consistent with the results shown in Figure 5.
Conclusions
The green and red upconversion emissions by transitions of Er 3+ Stark sublevels were observed in Er 3+ -Yb 3+ -Mo 6+ -codoped TiO 2 phosphors in this study. There are four coupled energy levels of Er 3+ ions due to the effect of the crystal field environment on Er 3+ , each of which was utilized to study temperature-dependent upconversion emission properties. Based on the FIR technique, the optical temperature-sensing behaviors of sensing sensitivity, measurement error, and operating temperature for the four coupled energy levels were discussed in detail, with all closely related to the energy gap of the coupled energy levels, FIR value, and luminescence intensity. High sensitivity and negligible error are obtainable through the use of different coupled energy levels for optical sensing, throughout a wide range of temperature in an independent system. The utilization of coupled energy levels by Stark split is a new and effective method in the realization of multiple optical temperature measurement. | v3-fos-license |
2018-03-28T05:38:08.121Z | 2017-10-25T00:00:00.000 | 4402216 | {
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} | pes2o/s2orc | Liquid Biofuel Production from Palm Oil Using Dual-Function of Zn / HZSM-5 Catalyst
To produce high yield and quality hydrocarbon biofuel, HZSM-5 zeolite loaded with different Zn concentrations (5, 10, 15 and 20wt.%) was used to palm oil cracking in batch reactor. Reaction temperature and reaction time were optimized. The products contained liquid hydrocarbon biofuel, distillation residual and coke. The physical properties of biofuel products were determined. The results presented that 15% Zn/HZSM-5 exhibited higher hydrocarbon biofuel yield (20.27%) and their excellent product properties with higher heating value (HHV), low viscosity and moisture content. A highest hydrocarbon biofuel yield and good alkane range hydrocarbons distribution from palm oil were obtained under reaction temperature of 420°C for 5 hours.
INTRODUCTION
Due to an increase in energy demand and lacking of world's energy resource, the developments of renewable energies have attracted a great interest.The biomass conversion technology is one of the wonderful candidates to resolve the problem of an insufficient energy.In modern science, the production of green fuels has been focused on vegetable oils as raw renewable fats.Generally, trans-esterification method was used for producing of biofuel from the oils 1 .However, the biofuel production from transesterification is lower oxidative and ambient stability.In order to solve this issue, a catalytic hydro-treatment and cracking processes have been developed to produce green biofuel from vegetable oils.The advantage of this biofuel has low greenhouse gas and toxic components such as nitrogen and sulfur compounds.The hydrotreatment products were obtained from hydrogenation by acid function of catalyst, which were greatly assisted to increase the products stability due to an increasing of isomerization reaction 2,3 .
Recently, the variety of Zn loaded on NaZSM-5 catalyst was used for the production of liquid hydrocarbon from camelina oil 4 , but maximum yield of the liquid hydrocarbon was 19.56%.And over 0.5% (w/w) of Zn added into ZSM-5 catalyst provided a high yield aromatic hydrocarbon production from pyrolysis of Douglas fir sawdust pellets 5 .However, few researchers have done some progress on the liquid hydrocarbon biofuel production over Zn/HZSM-5 catalyst.
From this point of view, no study on Zn doped on HZSM-5 catalyst for the cracking of palm oil has been reported.Therefore, the aim of this work is to produce a high hydrocarbon biofuel yields from the cracking of palm oil over Zn doped on HZSM-5 supports catalyst.Several affecting parameters such as concentration of zinc doped zeolite catalyst, reaction temperature and reaction time were investigated.
EXPERIMENTAL
Composition of palm oil was analyzed using Agilent GC-7890A.The HZSM-5 catalysts doped with different concentrations of Zn (5, 10, 15 and 20wt.%) were prepared using a wet impregnation method.The metal loading was carried out by adding a solution of zinc(II) nitrate hexahydrate to the zeolite in a rotary evaporator.After the impregnation, the catalysts were dried at 80°C for 2 h and then all catalysts were calcined at 500 °C under air for 3 h.XRD pattern of catalysts were recorded on Rigaku RAD-C diffractometer with continuous scanned from 5°-50° (2θ).The surface area measurements and porous properties of the catalysts were analyzed using a Quantacrhome-Autsorb Analyzer.
The hydrocarbon biofuel production was carried out in 1000-ml batch reactor Amar Instrument (India) equipped with an automatic stirrer.In the experimental series, 300 ml of palm oil and 10 g catalyst were loaded in the reactor chamber, sealed and filled with hydrogen to pressure of 3 MPa under ambient temperature.The reaction was performed at 400°C for 3 h.with stirring 500 rpm over HZSM-5, 5% Zn/HZSM-5, 10% Zn/ HZSM-5, 15% Zn/HZSM-5 and 20% Zn/HZSM-5 catalysts.The upgraded palm oil was collected at ambient temperature and it was then distillated at 300 o C, which aimed to obtain the liquid biofuel based hydrocarbon.The main components of liquid biofuel via hydrocracking process were characterized by gas chromatography-mass spectrometry (GC-MS) and simulated distillation gas chromatography (Sim-Dis GC).The physicochemical properties of hydrocarbons biofuel such as high heating value (HHV), density, dynamic viscosity and moisture content were characterized.The HHV was evaluated based on ASTM D4809 using C2000 calorimeter system (IKA-Work, Inc.).Density was examined by ratio of mass to volume of samples at ambient temperature.Dynamic viscosity was measured by using Viscosity Measuring Site VM 30150 (PSL systemtechnik).The moisture content was determined by using Karl Fischer instrument V10S Compact Volumetric KF Titrator.
Feedstock and catalyst characterization
From the GC-MS analysis, the compositions of fatty acid from palm oil show palmetic acid (40.65%), oleic acid (37.87%), linoleic acid (8.43%), stearic acid (5.69%) and myristic acid (2.31%).The XRD patterns of various Zn concentration catalyst supported on HZSM-5 are presented in Fig. 1.The XRD patterns of the fresh catalysts were similarity indicating that the framework of HZSM-5 was still kept after loading Zn(II).The ZnO phase was not observed for fresh catalysts suggesting that the metal oxide species were well dispersed on surface of the support HZSM-5 6,13 .The BET surface areas and pore volumes of fresh catalysts are listed in Table 1.The surface area and total pore volume of catalyst decreased when adding Zn to the HZSM-5 supports.This situation was due to pore blocking by metal oxide species dispersed in the channels of supports material 7,14 .The effect of catalyst on product yield Figure 2 shows the yield of palm oil upgrading product that includes hydrocarbon biofuel, distillation residual and coke.The mass of coke was determined by difference weight of reactor before and after the cracking.The hydrocarbon biofuel yield increases with increased concentration of zinc.The increase in ZnO concentration to the HZSM-5 supports could enhance the yield of hydrocarbon biofuel due to the increase of Lewis acid site, which will do transform fatty acid to hydrocarbon based biofuels 6 .The yield of hydrocarbon biofuel over 20% Zn/HZSM-5catalyst decreased, which this might be indicated that the aggregation of ZnO particles.The aggregation of ZnO that results from decreasing micropores and mesopores volume of the catalyst (see in Table 1) leading to pores blocking and reduce the active sites of catalyst.The distillation residual yields of Zn loaded to HZSM-5 supports were higher than that HZSM-5 without Zn.This fact results in acid site on the catalysts promoted the chemical reaction such as cracking and deoxygenation during palm oil cracking led to conversion of triglyceride molecules to hydrocarbon based biofuel 16 .The coke yield produced with zinc loaded HZSM-5 catalysts was higher than that HZSM-5 without zinc load catalyst.This fact might be due to that the metals loaded HSZM-5 with high activity of producing aromatic hydrocarbon and polymerized to form coke 8 .
The effect of catalyst on physical properties of liquid hydrocarbon product
Table 2 shows the physical properties of hydrocarbon biofuel produced without and with Zn loaded catalyst.Hydrocarbon biofuel had much lower viscosity than that of raw palm oil.The reason is that some large and complicated palm oil molecules had been cracked into smaller and simpler molecules.However, there was no significant difference between the viscosities of all hydrocarbon biofuels produced without and with Zn loaded catalyst.After the palm oil upgrading, the density of all hydrocarbon biofuels decreased to 0.76-0.84g/ml, which was lower than the density raw palm oil.During the upgrading process, palm oil triglyceride molecules were decomposed to fatty acids that then underwent chemical reactions such as deoxidation and decarboxylation to form hydrocarbons and/or alcohols 9 .In this study, there was obvious water phase separated from the oil phase while collecting liquid oil, although some chemical reactions such as dehydration could produce water during the cracking of palm oil.The water content of hydrocarbon biofuel produced with Zn loaded catalyst was lower than that of hydrocarbon biofuel produced without Zn loaded catalyst.This indicates that the use of zinc loaded HZSM-5 inhibited the dehydration during the palm oil cracking process, which led to the lower water content of the hydrocarbon biofuel.The HHVs of all hydrocarbon biofuels increased after upgrading, compared to raw palm oil.During the palm oil upgrading, oxygen atoms were removed through the reactions such as deoxidation, decarboxylation, decarbonylation and dehydration, which led to the increase of HHV.One possible reason is that the water content of hydrocarbon biofuel produced without Zn loaded catalyst was higher than that of hydrocarbon biofuel produced with Zn loaded catalyst, as shown in Table 2.The higher water content would result in the lower HHV of the hydrocarbon biofuel 9,15 .As mentioned above, the 15% Zn/HZSM-5 catalyst evidently presented the highest hydrocarbon biofuel yield and excellent properties of biofuel products.Therefore, this catalyst was selected as a catalyst in the following experiments to produce high hydrocarbon biofuel yield.
Effect of reaction temperature to yield and quality hydrocarbon biofuel
Figure 3 presents the resulted of cracking reaction temperature over 15% Zn/HZSM-5 catalyst to produce high yield and quality hydrocarbon biofuel with high composition of alkane.At 360°C, low yield of liquid hydrocarbon biofuel and high distillation residual yield.This result indicates that the transformation of triglyceride to long chains fatty acid with dehydrogenation and dehydration.Yield of hydrocarbon biofuel further increased to 20.78 wt.% when increase the reaction temperature up to 420°C.Increase in the reaction temperature that affected to enhance the activities of catalyst led to low distillation residual yield.At 440°C, hydrocarbon biofuel and distillation residual yield decreased to 19.81 wt.% and 48.89 wt.% which was due to severe cracking reaction.Liquid hydrocarbons and triglycerides were cracked into gaseous products 10 .Another possible reason is the formation of coke from aromatic hydrocarbons through polymerization and aromatization that result in high coke yield 8 .The selectivity to alkane range hydrocarbons with different carbon numbers is shown in Figure 4.At 360°C, tetradecane (C14) and pentadecane (C15) presented higher selectivity than the other hydrocarbons.In contrast, the short chain hydrocarbons (C6-C12) presented higher selectivity than long chain hydrocarbons (C13-C15), when using the reaction temperature of 440°C.This result illustrated that transformation of heavy hydrocarbons into lighter hydrocarbons by severe cracking.At 380, 400 and 420°C, the selectivity of alkane range hydrocarbons with different carbon numbers increased with reaction temperature.The high hydrocarbon biofuel yield and a good alkane distribution were observed at 420°C.
Effect of reaction time to yield and quality hydrocarbon biofuel
Figure 5 shows the products yield during palm oil cracking over 15% Zn/HZSM-5 catalyst at 420°C under various reaction times.At the first hour, low yield of hydrocarbon biofuel due to oligomerization occurred.Increased hydrocarbon biofuel yield from 14.58 wt.% to 20.67 wt.% with increasing reaction time from the first to the fourth hour.This phenomenon indicates that the remaining triglycerides and/or fatty acids further decomposition into hydrocarbons led to decreased distillation residual yield.From 4 h to 5 h, the products trend was slightly changed might due to same cracking reaction pathway.In the 6 h, the yields of hydrocarbon biofuel and distillation residual decreased as well.This result suggests that the formation of gas and aromatics, which then produced coke led to high coke yield.The selectivity to gasoline, kerosene and diesel with different carbon numbers of alkane is shown in Fig. 6.From 1 h to 2 h, high selectivity to diesel due to hydrogenation and decabonylation of fatty acids 11 .In the third h, the fatty acids and long chain hydrocarbons must be cracked into short chain hydrocarbons led to high selectivity to gasoline and kerosene.The kerosene was slightly changed with increased reaction time from 4 h to 6 h.However, higher selectivity to gasoline than kerosene and diesel was observed when increased reaction time.This characteristic was due to severe cracking and influence of Lewis acid sites on catalyst promoted several reaction pathways to convert triglyceride and fatty acids into gasoline fraction 12 .
CONCLUSIONS
The catalytic effects of HZSM-5 and Zn loaded HZSM-5 catalysts on the product yields and distribution were operated in the batch reactor system.The introduction of Zn on HZSM-5 catalyst was significantly increase yield and improve the properties hydrocarbon biofuel.The 15% Zn/ HZSM-5 catalyst exhibited a good ability to produce the highest amount of gasoline and high liquid hydrocarbon biofuel range alkane yield more than 20% from palm oil at 5 h under reaction temperature of 420°C.
Fig. 2 .
Fig. 2. The product yield of palm oil upgrading over different catalysts.
Fig. 3 .Fig. 4 .
Fig. 3.The product yield of palm oil upgrading at different reaction temperatures | v3-fos-license |
2019-12-05T09:09:47.255Z | 2019-10-30T00:00:00.000 | 214463356 | {
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} | pes2o/s2orc | Evaluation of Singlet Oxygen Scavenging Capacity of Peppermint (Mentha Piperita L.), Marjoram (Origanum Majorana L.), Rosemary (Rosmarinus Officinalis L.) And Sage (Salvia Officinalis L.) on Fatty Acid Photooxidation
Lipid photooxidation is the undesirable chemical process in which singlet oxygen result in the peroxidation of fatty acids. In this study leaves methanolic extracts of peppermint (Mentha piperita L.), marjoram (Origanum majorana L.), rosemary (Rosmarinus officinalis L.) and sage (Salvia officinalis L.) were applied as the natural singlet oxygen scavenger. Amount of flavonoid compounds as the singlet oxygen scavenger agent in these plant species were decreased in the order of peppermint > marjoram> sage > rosemary. Also, The rate of quenching of singlet oxygen in the presence of 1,4-Diazabicyclo[2.2.2]octane (DABCO) as a well-known singlet oxygen scavenger and highly effective synthetic antioxidants in food industry such as Butylated hydroxyanisole (BHA), tert-Butylhydroquinone (TBHQ) and peppermint decreased in the order of peppermint > BHA > TBHQ > DABCO >. Furthermore, photooxidation of oleic acid as an unsaturated fatty acid in the presence of DABCO, peppermint, BHA and TBHQ indicated a preservation of 82.77%, 73.39%, 71.57% and 53.10% on peroxidation of oleic acid, respectively which reveals peppermint has an efficient role on protection of fatty acids from photooxidation. Practical application: In this study, it was confirmed that peppermint (Mentha piperita L.) performs an effective role in restricting or limitation of singlet oxygen generation and fatty acid photooxidation. In vitro study of scavenging effect of peppermint can correlate laboratory results to commercial scale up. However, this would also necessitate the progress of improved methods for the measurement of lipid peroxidation in vivo in the presence of peppermint. © 2019 Mahdi Hajimohammadi. Hosting by Science Repository. All rights reserved.
Introduction
Molecular oxygen in its ground state has two unpaired electrons and when oxygen molecules have excess energy, singlet oxygen can be produced as the result of pairing of these unpaired electrons existent in external orbital [1]. Applying photosensitizer is one of the physical methods, which used for producing singlet oxygen. Light illumination can easily produce singlet oxygen in food systems, particularly in the presence of photosensitizers such as riboflavin and chlorophylls [2]. Lipids can be a target of singlet oxygen because of their electrophilic inherent and produce lipid hydroperoxides [3]. DABCO recognized as very efficient quencher of singlet oxygen in the organic media [4] and synthetic antioxidants such as TBHQ, BHA and BHT have been found to have a strong singlet oxygen quenching ability [5]. People receive antioxidant supplements directly from fresh fruits and vegetables and plants. Peppermint is a medicinally important plant belongs to the family Lamiaceae and commonly known as peppermint is a hybrid of spearmint and watermint. The ancient Egyptians cultivated and documented it in the Icelandic pharmacopoeia of the thirteenth century [6]. It is widely grown in temperate areas of the world, particularly in Europe, North America and North Africa but nowadays cultivated throughout all regions of the world. Peppermint is a perennial 50-90 cm high, normally quadrangular and a prototypical member of the mint family [7,8]. Marjoram, of Lamiaceae family was known to the ancient Egyptians, Greeks and Romans [9]. The high antioxidant capacity of marjoram's methanolic extract has been reported by several studies [10,11].
Marjoram is traditionally administered, orally, for symptomatic treatment of gastrointestinal disturbances and cough. Its spasmolytic and antimicrobial effects are used to treat bronchial diseases. Marjoram is also applied topically to relieve symptoms of the common cold, such as nasal congestion and in mouthwashes for oral hygiene [12]. Also, leaves of rosemary and sage are popular herbal teas and essential-oil containing drugs which are rich sources of di-and triterpenoids, phenolic acids, and flavonoids [13]. There are few studies on the efficacy of natural antioxidants as a O2 ( 1 Δg) quenchers and their roles in the prevention of lipid oxidation because scavenging of DPPH free radical is the basis of a common antioxidant assay and most often an overall antioxidant effect was measured [14,15]. However, singlet oxygen has not radical nature [16]. This project was designed to characterize antioxidant potential of peppermint, marjoram, rosemary and sage as the natural antioxidants in compare with well-known singlet oxygen scavenger such as DABCO and highly effective antioxidants such as BHA and TBHQ.
I Materials
Leaves of peppermint, marjoram, rosemary and sage were collected from Zarandyeh Mamuniya in Iran on August 6 th , 2017. Anthracene, Oleic acid, acetonitrile, MB (methylene blue), DABCO, BHA and TBHQ were purchased from Fluka and Merck and used without further purification. Tetrphenyl porphyrin (H2TPP) was synthesized according to the literatures [17].
II Extraction method
The leaves of peppermint, marjoram, rosemary and sage were dried under vacuum completely. 0.5 gr of dried powder of each leaf was added to 50 ml of acidic methanol (contains 1% hydrochloric acid) and the mixture was stirred for 48 hours in non-light condition. Extract of leaves were immediately used for the next steps.
III Determination of total flavonoid content
The total flavonoid content was determined by the aluminum chloride colorimetric method [18]. Briefly, 0.5 ml of methanolic extract was separately mixed with 1.5 ml of 95% ethanol, 0.1 ml of 10% aluminum chloride, 0.1 mL of 1M potassium acetate and 2.8 mL of distilled water. After incubation at room temperature for 30 min using UV-Vis method the absorbance of the reaction mixture was measured at 415 nm. Sample blank for all the dilution of standard quercetin and all the three methanolic extracts were prepared in similar manner by replacing aluminium chloride solution with distilled water. It was used quercetin solutions at concentrations ranging 25, 50 and 100 ppm to build up the calibration curve. The total flavonoid content was calculated from a calibration curve 0.99 (Y=0.004X-0.0505, R 2 =0.99), and the result was expressed by ppm.
IV Determination of optimal antioxidant using oleic acid photooxidatin 1 ml of extracts (peppermint, marjoram, rosemary and sage) separately was added to 7 ml acetonitrile solution of oleic acid (4.6×10 -3 M) and H2TPP (1×10 -3 M). The continuous irradiation of samples was carried out using solar simulator light (288 power LED lamps, 1 W, 2.3 V (59660 LUX)) for 120 min at room temperature under 1 atm of bubbling of air in the solution. The compositions of products were determined by proton nuclear magnetic resonance ( 1 H NMR) spectroscopy and iodometric titration method. 1 H NMR spectroscopy was analyzed on a Bruker AMX 300 MHz spectrometer using TMS as internal standard. Also, with iodometric titration method peroxide value (PV (meq O2/kg) of samples was determined according to the literature [19].
VI Statistical Analysis
In all analyses, three replicates were applied, and analysis of the results was achieved using SAS software, version 3.9 and then average the results were compared using Duncan test. Also, with Excel software diagrams were drawn.
I Evidences for singlet oxygen generation in the photooxidation of oleic acid
In this work the oxidative alterations of oleic acid as a result of oxidation with singlet oxygen were analyzed in the presence and absence of methanolic extracts of peppermint, marjoram, rosemary and sage as the natural antioxidants. Our target was fatty acid oxidation by singlet oxygen as a noble species which has worked few studies on it [14]. Photooxygenation of oleic acid with H2TPP photosensitizer was investigated as a typical standard sample to evaluate singlet oxygen production ( Figure 1) It is important to note that 1 H NMR spectroscopy (see supporting information (SI and SII)) and iodometric method ( Table 1, entry 1) revealed oxidation of oleic acid to peroxide product stopped in the absence of photosensitizer or when the irradiation was interrupted (Table 1 entry 2). Accordingly, the presence of a porphyrin, light and O2 are essential for the conversion oleic acid to corresponding products (Table 1 entry 3). According to the literature, there are two major pathways for photooxygenation reactions in the presence of non-metal photosensitizers, Type and Type [20]. Singlet oxygen generation (Type ) and its reaction with the substrates is the foremost mechanism that occurs in our circumstances, since conversions of oleic acid obey the order of H2TPP > ZnTPPCl > MgTPPCl (Table 1 entry 3, 4 and 5). Paramagnetic metals are claimed to quench singlet oxygen by energy transfer mechanism from oxygen to the low-lying electron levels and have very short triplet lifetimes (Table 1, entry 4) also diamagnetic metals quench singlet oxygen by a charge transfer mechanism (Table 1, entry 5) [21]. In addition, in the presence of DABCO, which is a wellknown singlet oxygen scavenger, photooxidation of oleic acid was inhibited ( [22][23][24]. Table (Table 1 entry 3, 7 and 8) indicates that conversion of oleic acid in acetonitrile as solvent is higher than ethanol and dimethyl sulfoxide (DMSO) that correlated with singlet oxygen lifetimes in these solvents. For investigation of the type mechanism (generation of superoxide anion radical), we performed oleic acid reaction in the presence O2 •-. In the presence of superoxide anion radical, the rates of oxidation reaction significantly decreased (Table 1entry 9).
II Evaluation of singlet oxygen scavenging capacity of peppermint, marjoram, rosemary and sage
In this work the oxidative alterations of oleic acid as a result of oxidation with singlet oxygen were analyzed in the presence and absence of peppermint, marjoram, rosemary and sage. Flavonoid compounds widely present in plants have been reported to act as singlet oxygen scavenger (see supporting information (SIII)) [25]. Interestingly, the rate of oleic acid oxidation by singlet oxygen reduced in the presence of peppermint, marjoram, rosemary and sage in order of peppermint > marjoram> > rosemary that correlated with total flavonid compounds of these type of plants (Table 2).
III Effect of peppermint on Anthracene photooxygenation
Spectrophotometry is a more convenient option for detection of excited oxygen molecules. A chemical probe is usually used to trap the singlet oxygen and then detection and quantification can be based on absorbance. A very characteristic reaction of singlet oxygen is the [4+ 2] cycloaddition to conjugated cyclic dienes and polycyclic aromatic hydrocarbons such as anthracene [26]. Anthracene traps reversibly singlet oxygen. Singlet oxygen generation by methylene blue (MB) is evidenced by chemical trapping of 1 O2 with anthracene. The UV-Vis spectra of anthracene as function of time irradiation by using of MB as photosensitizer are displayed in (Figure 2A). A reduction of the emission intensity absorption band of anthracene (λmax=375 nm) was observed with increase of irradiation time. This response is a consequence of the anthracene-9,10-endoperoxide formation (see Figure 2). During the phtooxygenation of anthracene, the addition of DABCO, BHT, BHA, TBHQ and peppermint inhibited the oxidation of anthracene in the order of peppermint > BHA > TBHQ> DABCO (Figure 2 A , B). Moreover, the oxidation reaction did not occur under dark conditions. These results confirm that the anthracene oxidation occurs by singlet oxygen under visible irradiation and peppermint because of its flavonoid compounds acts as a very efficient singlet oxygen scavenger.
IV Effect of peppermint on fatty acid photooxgenation
The photosensitized production of singlet oxygen has significance in the areas of the photooxidation of organic compounds and food chemistry [27][28][29][30]. Photooxygenation of oleic acid as one of the targets of singlet oxygen was investigated as a typical standard sample to evaluate the antioxidant effect of peppermint. Figure 3 shows the conversion of oleic acid in an oxygenated solution of acetonitrile under visible light in the presence of peppermint, well-known singlet oxygen (DABCO) and highly effective synthetic antioxidants in food industry such as BHA and TBHQ. The rate of oleic acid oxidation by 1 O2 as a very reactive ROS after 120 min irradiation was reduced to 27% in the presence of peppermint (contains 0.4mg flavonoid) that shows peppermint can be used as an effective additive to fatty acid for preservation of it.
Conclusion
Due to the increase of diseases such as cancer, Alzheimer's disease, skin disorders and etc. with ROS especially singlet oxygen and light, finding efficient antioxidant is very important. The overall evaluation of this study concludes that four species of peppermint, marjoram, rosemary and sage have good antioxidant potential, particularly peppermint. Antioxidant capacity of these species and synthetic polyphenolics against singlet oxygen was comprehensively assessed by anthracene oxidation assay and evaluation of fatty acid oxidation. It was showed peppermint has an efficient role on restricting or limitation of singlet oxygen generation and photooxidation of fatty acid by singlet oxygen. | v3-fos-license |
2020-09-19T17:22:27.610Z | 2020-09-18T00:00:00.000 | 221797361 | {
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} | pes2o/s2orc | Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells
Background Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). Methods Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. Results Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). Conclusions Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract
In mature red blood cells (RBCs), CD47 is associated with different membrane proteins forming linkages with both cytoskeletal and non-cytoskeletal cellular components [11]. CD47 is pivotal in inhibiting RBC phagocytosis via binding to signal regulatory protein α (SIRPα) on macrophages, which counteracts phagocytosis of non-opsonized as well as IgG or complement-opsonized RBCs [13,14]. A decline of cell surface CD47 expression during RBC aging in vivo is believed to promote the clearance of senescent RBCs [15]. Furthermore, microparticle release during RBC storage has been reported to favor CD47 loss during storage of RBCs for transfusion [16]. Beyond its significance in RBC aging, CD47 mediates the interaction of fibrinogen with the RBC membrane [17], and may, therefore, contribute to RBC hyperaggregation and altered hemorheology in inflammatory conditions [18,19].
Similar to apoptosis of nucleated cells [10], ligation of CD47 with monoclonal antibodies, TSP-1, or its derivative peptides has been shown to trigger phosphatidylserine (PS) exposure on RBC cell surface with a concomitant loss of their viability [20]. The mechanisms underlying this phenomenon in RBCs, however, remain elusive. PS externalization, a cardinal morphologic sign of cell death (sometimes also referred to as eryptosis), is stimulated by activation of Ca 2+ -sensitive scramblases [21][22][23]. Influx of extracellular Ca 2+ into the RBC cytoplasm is mediated by voltage-gated and voltageindependent non-selective cation channels (NSCC [24,25]), which are activated by pathophysiologic cell stressors such as hyperthermia, oxidative stress, extracellular hyperosmolality, and starvation [21,26]. Supraphysiologic Ca 2+ overload in RBCs induces metabolic reprogramming [27], and activation of multiple enzymes [21], thereby eliciting cellular dysfunction and death. PSexposing RBCs are rapidly cleared from the circulation and catabolized by macrophages of the reticuloendothelial system in the spleen and liver [21,28].
In the present study, using cytofluorometric and electrophysiological approaches we examined the influence of CD47-dependent signaling, evoked by exogenous TSP-1 or antibody-mediated CD47 ligation, on Ca 2+ dynamics in human RBCs. We further studied the effect of TSP-1 exposure on multiple parameters of RBC deformability and cell death.
RBCs and reagents
Leuko-depleted RBC concentrates were provided by Canadian Blood Services (CBS) Network Centre for Applied Development (netCAD, Vancouver, BC, Canada) after prior approval from the CBS Research Ethics Board (#2015.022). For some experiments, these concentrates were provided by the blood bank of the University of Tübingen (#184/2003 V), Germany, or by the blood bank of Norrlands University Hospital, Umeå, Sweden. Donor RBCs, drawn from refrigerated blood bags containing SAG-M additive solution, were washed twice in PBS (1000×g for 10 min) and subsequently incubated in vitro (1% hematocrit unless indicated otherwise) at 37°C in Ringer's solution (pH 7.4) containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 32 mM HEPES, 5 mM glucose, and 1 mM CaCl 2 . Sample sizes (number of RBC units; n) for control and treatment groups used in individual experiments are indicated in the figure legends. Where indicated, RBCs were incubated with recombinant human thrombospondin-1 (1-50 μg/mL; R&D Systems, Minneapolis, MN, USA) or with anti-human CD47 mAb 1F7 (mouse IgG1), which was purified from hybridoma supernatants [29][30][31]. In some experiments, RBCs were treated with sodium nitroprusside (Sigma Aldrich, Taufkirchen, Germany) or amiloride (Sigma Aldrich), as described in the figure legends.
Electrophysiology
Patch-clamp measurements were performed with a NPC-16 Patchliner (Nanion Technologies, Munich, Germany). The internal and external solutions were as follows: KCl 70 mM, KF 70 mM, NaCl 10 mM, HEPES 10 mM, MgATP 2 mM, EGTA 3 mM, and CaCl 2 1.2 mM to give 120 nM free [Ca 2+ ] i , pH = 7.2 adjusted with KOH (internal) and NaCl 140 mM, KCl 4 mM, MgCl 2 5 mM, D-glucose 5 mM, HEPES 10 mM, CaCl 2 2 mM, pH = 7.3 adjusted with NaOH (external). In these solutions, the resistance of the chips was between 5 and 8 MΩ. Gigaseal formation was facilitated using a seal enhancing solution as recommended by the Patchliner manufacturer and containing: NaCl 80 mM, KCl 3 mM, MgCl 2 10 mM, CaCl 2 35 mM, HEPES 10 mM, pH = 7.3 adjusted with NaOH. Whole-cell configuration was achieved by negative pressure suction pulses between − 45 mbar and − 150 mbar and its formation judged by the appearance of sharp capacitive transients. Whole-cell patch-clamp recordings were conducted at room temperature using voltage steps from − 100 mV to 80 mV for 500 ms in 20 mV increments at 5 s intervals, the holding potential being set at − 30 mV. Whole-cell currents were assessed before (control) and after adding 50 μg/mL TSP-1. To reduce inter-cell variability, data are expressed as normalized current, which is the ratio of the current under specified experimental conditions, i.e. before and in the presence of 50 μg/mL at the membrane potentials used in the protocol, to the current at + 80 mV determined 30-60 s before starting the control measurement.
RBC deformability measurement
RBCs were incubated (40% hematocrit) for 48 h at 37°C. After incubation time, 250 μL of control or TSP-1treated RBCs were washed once with Ringer's solution. Ten μL of the aliquot was transferred into 1 mL viscous PVP (polyvinylpyrrolidone; RR Mechatronics, The Netherlands) for RBC deformability measurements. RBC deformability was measured using the laser assisted optical rotational red cell analyzer (LORRCA; RR Mechatronics, The Netherlands). The two parameters used to describe RBC deformability are EI max and K EI . The EI max is defined as the maximum elongation index predicted at an infinite shear stress. The K EI is the shear stress required to elongate to half the EI max . These parameters are obtained using an Eadie-Hofstee linearization, which plots the measured EI values versus the EI/respective shear stress (EI/SS) [36]. The slope of the best fit line provides the K EI and the y-intercept corresponds to the EI max .
Statistical analysis
Data are expressed as arithmetic means ± SEM. n denotes the number of different donor RBCs studied. Statistical analysis was performed using ANOVA with Tukey's test as a post-test, t test or non-parametric Wilcoxon signed rank test by GraphPad Prism Version 8.4.3 (GraphPad Software, La Jolla, CA). A P-value less than 0.05 was considered statistically significant.
Effect of thrombospondin-1 on Ca 2+ homeostasis in human red blood cells
The impact of TSP-1 treatment on RBC intracellular Ca 2+ levels was examined using Fluo3 fluorescence in flow cytometry analysis. As shown in Fig. 1a and b, exposure of RBCs to TSP-1 (50 μg/mL) for 48 h significantly enhanced the percentage of RBCs with increased Fluo3 fluorescence indicating increased cytoplasmic Ca 2+ concentration. Whole-cell patch-clamp experiments were performed to elucidate whether TSP-1 influences cation channel activity. As illustrated in Fig. 1c and d, exposure of RBCs to 50 μg/mL TSP-1 using physiological internal and external solutions induced an increase in an inward conductance, indicating a possible cation flux into the cells, that may be related to the increase in the intracellular Ca 2+ concentration. Furthermore, treatment with 1 mM amiloride, a cation channel inhibitor [28], abrogated the TSP-1-induced increase in the inward conductance (Fig. 1e). In addition, amiloride also blocked an outward current that was not induced by TSP-1.
Effect of thrombospondin-1 on phosphatidylserine externalization, sphingomyelinase activation, and the generation of reactive oxygen species in human red blood cells Enhanced cytosolic Ca 2+ content is expected to activate scramblases which, in turn, elicit cell membrane PS externalization. We observed that a 48-h incubation of RBCs in the presence of TSP-1 (50 μg/mL) significantly increased the percentage of annexin V-positive RBCs, reflecting cell membrane PS exposure ( Fig. 2a and b). We then interrogated the extent to which Ca 2+ influx contributes to PS externalization triggered by TSP-1. Blocking Ca 2+ entry via NSCC using amiloride (1 mM [26]; Fig. 2c) or removal of extracellular Ca 2+ (Fig. 2d) significantly blunted, but did not abolish, TSP-1-induced PS exposure suggesting that increased Ca 2+ entry participates in, but does not completely account for, TSP-1induced cell death. As TSP-1-induced PS exposure was not abolished by extracellular Ca 2+ removal, we hypothesized that non-Ca 2+ -dependent mechanisms may contribute to the breakdown of phospholipid asymmetry. Multiple recent studies have shown the role of TSP-1 in modulating NO signaling in various cell types (reviewed in [3]). We, thus, explored whether NO-mediated signaling similarly modulates TSP-1-induced alterations in RBCs. As shown in Fig. 2e, treatment of RBCs with the NO donor sodium nitroprusside (1 μM) significantly reduced TSP-1-induced PS externalization, suggesting the involvement of this mechanism in concert with Ca 2+dependent signaling leading to RBC cell death. We then examined whether TSP-1 elicits oxidative stress and sphingomyelinase activation, putative RBC cell death effectors [21]. As shown in Fig. 2f and g, a 48-h exposure of RBCs to 50 μg/mL TSP-1 significantly enhanced DCFDA fluorescence, reflecting ROS production, but did not significantly enhance ceramide abundance suggesting that TSP-1 affects RBC redox balance favoring their suicidal death.
Since CD47 is a receptor for TSP-1 and CD47signaling can regulate cytoplasmic Ca 2+ dynamics [12], an anti-CD47 mAb could induce an increased RBC intracellular Ca 2+ level. For this, anti-CD47 mAb 1F7 was used as it has been shown to induce apoptosis in other cell types [29,31]. As illustrated in Fig. 3a and b, exposure of RBCs to mAb 1F7 (10 μg/mL) for 1-4 h significantly increased Fluo3 fluorescence. However, such an effect of mAb 1F7 was absent after a 24-h incubation (Fig. 3b). We observed that mAb 1F7 (0.1-10 μg/mL) dose-dependently increased the percentage of annexin Vpositive RBCs after a 24-h incubation (Fig. 3c). In addition, there was a time-dependent increase in the percentage of annexin V-positive RBCs in response to mAb 1F7 (Fig. 3d). Interestingly, similar levels of RBC PS exposure in response to mAb 1F7 were also seen in the absence of extracellular Ca 2+ during incubation (Fig. 3e).
Effect of thrombospondin-1 on morphology and deformability of human red blood cells
The effect of TSP-1 on RBC morphology and deformability was determined. In imaging flow cytometry analyses, it was observed that the proportion of RBCs with Fig. 2 Effect of thrombospondin-1 on phosphatidylserine externalization, sphingomyelinase activation, and the generation of reactive oxygen species in human red blood cells. Representative histogram (Black line: 0 μg/mL TSP-1, orange line: 50 μg/mL TSP-1; a and means ± SEM. b of annexin V positive RBCs (n = 7) following 48-h incubation at 37°C in Ringer's solution containing 0-50 μg/mL TSP-1. *** indicate significant difference (P < 0.001) from the absence of TSP-1. Means ± SEM of annexin V positive RBCs following 48-h incubation in 50 μg/mL TSP-1 in the absence or presence of 1 mM amiloride (n = 27; c), 1 mM CaCl 2 (n = 8; d) or 1 μM sodium nitroprusside (n = 12; e). *** indicates significant difference (P < 0.001) from the absence of amiloride, CaCl 2 or sodium nitroprusside. Means ± SEM of the geometric means of DCFDA (n = 9; f) or ceramide-dependent (n = 14; g) fluorescence of RBCs following 48-h incubation without or with 50 μg/mL TSP-1. * indicates significant difference (P < 0.05) from the absence of TSP-1 smooth disc shape was significantly reduced and RBCs with crenated sphere shape were significantly increased after a 48-h incubation in the presence of TSP-1 (Fig. 4a). Accordingly, as shown in Fig. 4b, TSP-1 treatment significantly reduced the morphology index of RBCs. Ektacytometry analyses revealed that TSP-1 treatment affected RBC deformability (Fig. 4c). As depicted in Fig. 4d, in comparison to untreated RBCs, TSP-1 (50 μg/mL) exposure for 48 h significantly reduced maximum elongation index (EI max ) suggesting that TSP-1 induces increased RBC stiffness. Furthermore, TSP-1 (50 μg/mL) treatment tended to increase K EI reflecting RBC rigidity (Fig. 4e). Thus, the ability of RBCs to adopt a new shape in response to deforming forces, which dictate their rheological properties, is affected by TSP-1.
Effect of thrombospondin-1 on microvesiculation of human red blood cells
The impact of TSP-1-induced RBC dysfunction on microvesiculation was assessed. As shown in Fig. 5b and c, incubation of RBCs with TSP-1 for 48 h significantly increased CD47 + /CD235a + MPs relative to RBC count, as compared to untreated RBCs indicating that TSP-1 promotes MP shedding.
Discussion
Compelling molecular evidence points to an essential role for CD47-dependent TSP-1 signaling in the pathophysiology of a wide range of systemic diseases [3][4][5][6]. However, little is known about this signaling mechanism in influencing anucleate RBC functions. Increase of cytoplasmic Ca 2+ levels is a vital element in potentiating premature cell death and clearance of circulating RBCs [21,37]. In the current study, we demonstrate, for the first time, that exogenous TSP-1 causes RBC dysfunction evoking an increase in intracellular Ca 2+ levels, triggering cell death, and altering cell morphology and rheological properties.
Increased intracellular Ca 2+ concentration in RBCs, triggered by the opening of NSCC, stimulates phospholipid scrambling, bleb formation, and vesiculation of the cell membrane [21,28]. Enhanced cytosolic Ca 2+ is further involved in the activation of multiple Ca 2+ -sensitive enzymes such as transglutaminases, phospholipases, calpains, protein kinases and phosphatases [21]. While the molecular identity of the cation channels remains incompletely characterized, it is believed to involve the TRPC6 channel [21,28]. According to our data, the TSP-1-elicited increase in the cytosolic Ca 2+ concentration could be corroborated using whole-cell patch-clamp recordings which showed the presence of a TSP-1induced inward current, alluding to a possible Ca 2+ Fig. 3 Effects of the anti-CD47 mAb 1F7 on cytosolic Ca 2+ levels and phosphatidylserine exposure in human red blood cells. Representative histogram (Black line: 0 μg/mL mAb 1F7, orange line: 10 μg/mL mAb 1F7, following a 4-h incubation; a) and means ± SEM. b of Fluo3-positive RBCs (%) (n = 6; b) following incubation for 0-24 h at 37°C in the presence of 10 μg/mL mAb 1F7. ** indicate significant difference (P < 0.01) from the zero time-point. c Means ± SEM of annexin V positive RBCs (n = 3) following 24-h incubation with 0-10 μg/mL mAb 1F7. * indicates significant difference (P < 0.05) from the absence of mAb 1F7. d Means ± SEM of annexin V positive RBCs (n = 5) following incubation with 10 μg/ mL mAb 1F7 for 0-24 h. * and ** indicate significant difference (P < 0.05 and P < 0.01, respectively) from the zero time-point. e Means ± SEM of annexin V positive RBCs (n = 7) following 24-h incubation with 10 μg/mL mAb 1F7 in the absence or presence of 1 mM CaCl 2 influx. In addition, we also observed that ligation of the anti-CD47 mAb 1F7 induced an increase in RBC cytosolic Ca 2+ levels. These findings are consistent with previous studies in nucleated cells, which suggest that CD47/TSP-1 signal transduction impacts cellular Ca 2+ homeostasis [38][39][40]. Notably, intact TSP-1 was previously demonstrated to upregulate intracellular Ca 2+ levels in fibroblasts; this effect was recapitulated by the TSP-1derived peptide RFYVVMWK underlining the primordial role of TSP-1/CD47 signaling in regulating cytoplasmic Ca 2+ levels [38]. Furthermore, cardiac myocytes treated with 7 N3, a peptide derived from the C-terminal of TSP-1, displayed acutely elevated intracellular Ca 2+ levels through the release of Ca 2+ from the sarcoplasmic reticulum [39].
Ample evidence underscores the role of oxidative stress in modulating RBC Ca 2+ homeostasis and survival by stimulating NSCC conductance [41]. In accordance, our data reveal that TSP-1 treatment stimulated a subtle increase in RBC ROS production, which, in turn, may favor Ca 2+ entry and promote cell death. TSP-1 has previously been shown to potentiate ROS generation in vascular smooth muscle cells via CD47-dependent activation of NADPH oxidase 1 [42]. TSP-1 has further been implicated in oxidative stress-mediated renal ischemia-reperfusion injury by stimulating ROS production in renal tubular endothelial cells [43]. In addition, the present study also revealed that pharmacological NO supplementation significantly blunted TSP-1-induced PS externalization in RBCs. NO was previously shown to influence RBC survival by modulating cell death pathways downstream of intracellular Ca 2+ increase, but not by directly influencing Ca 2+ entry per se [44]. In purview of these findings, NO has previously been documented to be an essential effector of TSP-1 signaling in a wide range of cell types, and is associated with various clinical conditions [3,45].
RBC CD47 serves as a putative molecular switch in erythrophagocytosis [46]. Through activation of signaling mediated by tyrosine phosphatases, downstream of its interaction with SIRPα, CD47 inhibits phagocytosis, and thereby functions as a "do not eat me" signal [11]. Paradoxically, however, CD47 in experimentally aged RBCs was shown to undergo a conformational change and increased binding to TSP-1, which, in turn, promoted phagocytosis [46]. It is, therefore, possible that PS externalization during RBC cell death induced by TSP-1/CD47 signaling contributes, at least in part, to this "eat me" response.
RBCs exhibit an extraordinary ability to deform which facilitates their smooth passage in the microcirculation and, thus, aids in maintaining optimal rheology [47]. Increased RBC stiffness facilitates the elimination of senescent and Fig. 4 Effect of thrombospondin-1 on morphology and deformability of human red blood cells. Means ± SEM showing distribution of RBC morphology (n = 6; a) following a 48-h incubation at 37°C in Ringer's solution in the absence (Control) or presence of TSP-1 (50 μg/mL). RBC morphology was assessed using Bright Field images from ImageStream X MkII (60x magnification). Morphology index (n = 6; b) following a 48-h incubation of RBCs at 37°C in the absence (Control) or presence of TSP-1 (50 μg/mL). c Representative deformability curve (for RBCs from a single donor) of untreated (Control; black line) and TSP-1-treated (red line) RBCs obtained from LORRCA prior to Eadie-Hofstee linearization. Maximum elongation index (EI max ; n = 6; d) and rigidity (K EI ; n = 6; e) following a 48-h incubation of RBCs in the absence (Control) or presence of TSP-1 (50 μg/mL). * and ** indicate significant difference (P < 0.05 and P < 0.01, respectively) from the absence of TSP-1. Gray lines indicate means injured RBCs from the circulation in the spleen [48]. Previous studies have elucidated the pivotal role of RBC NO synthase-derived NO in the regulation of RBC deformability [49,50]. On the other hand, elevated cytoplasmic Ca 2+ levels in RBCs are associated with reduced deformability [51]. Along these lines, we observed that TSP-1 treatment altered the indices of RBC deformability at exposure durations, which also elicited both enhanced cellular Ca 2+ concentration and cell death. As RBC rigidity is an important hemorheological parameter leading to reduced blood viscosity, our findings may explain the occurrence of vaso-occlusive events associated with enhanced TSP-1 plasma levels [52].
TSP-1 has previously been implicated in the pathophysiology of vascular occlusion and pulmonary hypertension associated with sickle cell disease (SCD) [53,54]. Increased prothrombotic risk in SCD is linked to elevated TSP-1 levels, which not only inhibit ADAMTS13 proteolysis of von Willebrand Factor [55], but also provoke RBC MP shedding; this process, in turn, favors RBC adhesion to endothelial cells as well as stimulation of endothelial cell apoptosis [56]. Our data confirm MP shedding from RBCs following exposure to TSP-1 in vitro. Mechanistically, MP shedding by RBCs, as elucidated during their storage under blood banking conditions, may be a consequence of ATP depletion, K + leakage, and elevation of intracellular Ca 2+ [16] It may, therefore, be inferred that TSP-1-induced increase of intracellular Ca 2+ concentration leads to activation of Ca 2+ -dependent proteases leading to cytoskeletal damage and MP shedding [16]. Intriguingly, both TSP-1 and the CD47 agonist 4 N1-1 have been shown to potentiate the transformation of cell shape in SCD from discocytes to echinocytes [56]. It is well established that an increased proportion of RBCs in SCD patients expose procoagulant PS on their surface, which may lead to thrombosis [57]. It is, therefore, reasonable to conjecture that hyperactive cation currents in RBCs are an important underlying mechanism of RBC dysfunction and thrombosis in SCD patients. TSP-1 may contribute to this channel activation.
Accelerated cell death of RBCs has been shown to occur in a variety of systemic conditions and may contribute to anemia, thrombosis, and impaired microcirculation in these disorders [21]. At least in theory, increased TSP-1 levels, encountered in these conditions, may aggravate RBC Ca 2+ entry leading to RBC cell death [21]. Remarkably, TSP-1 serum concentrations were documented to be 100-fold higher than plasma concentrations indicating TSP-1 release by platelets [58]. TSP-1 concentrations used in this study (1-50 μg/mL) are well in the range of plasma and serum levels achieved in conditions such as SCD [53], and interstitial pneumonia [59], respectively. Furthermore, 100 μg/mL of TSP-1 was previously used to demonstrate the impact of CD47 ligation on RBC viability in vitro [20].
Conclusions
Taken together, our data unravel that TSP-1/CD47 signaling mediates enhanced RBC Ca 2+ concentration contributing to cell death. Targeting this signaling pathway may represent a possible therapeutic option in mitigating RBC-related pathophysiology in different clinical conditions associated with elevated TSP-1 levels. | v3-fos-license |
2019-04-09T13:09:04.129Z | 2018-01-01T00:00:00.000 | 104210487 | {
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} | pes2o/s2orc | Antibacterial and free radical scavenging activity of a medicinal plant Solanum xanthocarpum
ABSTRACT The present study describes the phytochemical profiles, antibacterial, and antioxidant properties of different solvent extracts of Solanum xanthocarpum leaves. Phytochemical analysis results revealed the presence of terpenoids, tannins, steroids, and phenols. Methanolic extract of plant had a maximum quantity of phenol (28.3 ± 2.0 mg) and flavonoids (25.2 ± 1.2 mg) than others. Similarly, the methanolic extract showed excellent antibacterial activity and exhibited the highest inhibitory effect against Pseudomonas aeruginosa (12 ± 0.5 mm), Salmonella typhi (10 ± 0.6 mm), Staphylococcus aureus (9 ± 1.0 mm), and Escherichia coli (7 ± 1.3 mm). The average minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were reported in the range of 3.2 to 6.9 µg/ml (for MIC) and 6.0 to 14.5 µg/ml (for MBC), respectively. The remarkable antioxidant activity was observed in chloroform and methanol extract on the DPPH radical scavenging activity with the lowest IC50 value of 197.245 μg/ml (chloroform) and 201.04 μg/ml (methanol) and compared with control (ascorbic acid 239.36 μg/ml). GC-MS analysis revealed the presence of six major bioactive compounds as follows; 2,Octylcyclopropene-1-Heptanol (42.81%), Hexadecanoic acid (26.63%), 1-methylene-2b Hydroxymethyl-3,3-Dimethyl-4b-(3Methylbut-2enyl)-C (9.3%), Phytol (7.5%), (1,3,3-Trimethyl-2-Hydroxymethyl-3,3-Dimethyl-4–3-Methylbut-2-Enyl)-C (7.2%). 3,7,11,15-Tetramethyl-2-Hexadecen-1-Ol (6.3%). The FT-IR spectrum reflected the presence of the twelve peaks at the range of 3746.38 cm-1 (O-H stretch alcohols), 3424.18 cm-1 (O-H stretch phenols), 2926.01 cm-1 (C-H stretch alkanes), 2857.55 cm-1 (C-H stretch alkanes), 2084.04 cm-1 (-C = C stretch alkynes), 1595.76 cm-1 (N-H bend primary amines), 1402.49 cm-1 (C-C stretch in ring aromatics) and others. This study suggests S. xanthocarpum as a potential candidature for having better antibacterial and antioxidant property and identified several bioactive compounds by GC-MS analysis.
with 2 ml of iodine solution. A dark blue or purple colour formation indicates the presence of carbohydrate.
Steroids, terpenoids, and alkaloids
Crude extract was mixed with 2 ml of chloroform, and concentrated H 2 SO 4 was added sidewise. A red colour produced in the lower chloroform layer indicates the presence of steroids. Crude extract was dissolved in 2 ml of chloroform and evapourated to dryness. To this, 2 ml of concentrated H 2 SO 4 was added and heated for about 2 min. A greyish colour indicates the presence of terpenoids. Crude extract was mixed with 2 ml of 1% HCl and heated gently. Mayer's and Wagner's reagents were added to the mixture. Turbidity of the resulting precipitate was taken as evidence for the presence of alkaloids.
Phenols, flavonoids, and tannins
Crude extract was mixed with 2 ml of 2% solution of FeCl 3 . A blue-green or black colour formation indicates the presence of phenols and tannins. Crude extract was mixed with 2 ml of 2% solution of NaOH. An intense yellow colour was formed which turned into colourless by the addition of few drops of diluted acid, and this indicates the presence of flavonoids.
Saponins and glycosides
Crude extract was mixed with 5 ml of distilled water in a test tube and was shaken vigorously. The formation of stable foam was taken as an indicator for the presence of saponins. Crude extract was mixed with 2 ml of chloroform and 2 ml of acetic acid. The mixture was cooled in ice, and then the concentrated H 2 SO 4 was carefully added. A colour change from violet to blue to green indicates the presence of steroidal nucleus (i.e., glycine portion of glycoside).
Quantitative phytochemical analysis
Determination of glycosides Glycosides of S. xanthocarpum plant extracts were quantitatively determined by the modified method of Solich et al. [16] 10% of each extracts were mixed with 10 ml freshly prepared Baljet's reagent (95 ml of 1% picric acid + 5 ml of 10% NaOH). After an hour, the mixture was diluted with 20 ml distilled water, and the absorbance was measured at 495 nm by Shimadzu UV/Vis spectrophotometer model 160A (Kyoto, Japan).
Determination of flavonoids
Flavonoids were quantitatively determined according to the method of Harborne. [17] The total flavonoid content was determined by the aluminium chloride colourimetric method. 0.5 ml aliquots of various extracts (1 mg/ml) were mixed with 1.5 ml of methanol, followed by the addition of 0.1 ml of 10% aluminum chloride, 0.1 ml of potassium acetate (1 M), and 2.8 ml of distilled water. The reaction mixture was kept at room temperature for 30 min. The absorbance of the reaction mixture was recorded at 415 nm. The calibration curve (0-8 μg/ml) was plotted using rutin as a standard.
Determination of protein
The S. xanthocarpum sample was extracted by stirring with 50 ml of 50% methanol (1:5 w/v) at 25°C for 24 h and centrifuged at 8,000 rpm for 10 min. 0.2 ml of extract was pipette out, and the volume was made into 1.0 ml with distilled water. 5.0 ml of alkaline copper reagent is added to all the tubes and allowed to stand for 10 min. Then, 0.5 ml of Folin's Ciocalteau reagent is added and incubated in dark condition for 30 min. The intensity of the colour was developed, and the absorbance was read with a spectrophotometer at 660 nm. [6] Determination of carbohydrates A total of 100 mg of different extract sample was hydrolysed in a boiling tube with 5 ml of 2.5 N HCl in a boiling water bath for a period of 3 h. It was cooled to room temperature, and solid sodium carbonate was added until agitation ceases. The contents were centrifuged, and the supernatant was made with 100 ml using distilled water. From this 0.2 ml of sample was pipette out and made up to 1 ml with distilled water. Then 1.0 ml of phenol reagent was added and followed by 5.0 ml of sulphuric acid. The tubes were kept at 25-30°C for 25 min. The absorbance was read with a spectrophotometer at 490 nm. [7] Determination of total steroids The estimation of total steroids in the different extracts of S. xanthocarpum was carried out by the modified method of Sabir et al. [18] Fifty grams of extracts was dissolved in 200 ml Aceto nitrile and incubated at 50-60°C for 2 h. It was filtered through Whatman No.1 filter paper, and 200 ml of methanol was added. Then, 3 ml of the extract was taken in a test tube with three replicates, and one blank. Then, 2 ml of Liberman-Burchard reagent was added to each tube. The reference standard solution was prepared by dissolving 10 gm of standard cholesterol in 10 ml chloroform. This was taken in five test tubes (1, 1.5, 2, 2.5, and 3.0 ml), and chloroform was used as blank. 2 ml of Liberman-Burchard reagent was added to each of them to develop the green colour, which is an indication of steroids. The final volume was made up to 10 ml by adding methanol and incubated in the dark for 15 min. The optical density of the standard solutions was determined with a spectrophotometer (Shimadzu) at 640 nm. The amount of steroids in the plant sample was determined by plotting the standard graph as mg/gm.
Determination of tannins
Tannin determination of sample was done according to the method of Van-Burden and Robinson. [19] 50 ml of distilled water was added to 500 mg of the sample taken in a 500 ml flask and kept in a shaker for 1 h. It was filtered into a 50 ml volumetric flask, and then 5 ml of the filtrate was pippette out into a test tube and mixed with 2 ml (10 fold diluted) of 0.1 M FeCl 3 in 0.1 N HCl and 0.008 M potassium ferrocyanide. Within 10 min, the absorbance of sample was measured with a spectrophotometer at 605 nm.
Determination of total phenolic content
The amount of total phenolic content of sample was determined as per the method of Velioglu et al. [20] using the Folin-Ciocalteu reagent. Aliquot of 0.1 ml of various extracts (4 mg/ml) was mixed with 0.75 ml of Folin-Ciocalteu reagent (10-fold diluted with dH 2 O). The mixture was kept at room temperature for 5 min, and 0.75 ml of 6% sodium carbonate was added. After 90 min of reaction, its absorbance was recorded at 725 nm. The standard calibration (0-25 μg/ml) curve was plotted using gallic acid. The total phenolics were expressed as mg gallic acid equivalent/gram dry weight. Negative control was prepared by adding 0.1 ml of DMSO instead of extracts.
Determination of saponins
Quantitative determination of saponin of samples was done according to Obadoni and Ochuko. [21] The samples were ground, and 10 g of each sample were put into a conical flask, and 100 cm 3 of 20% aqueous ethanol was added. The samples were heated over a hot water bath for 4 h with continuous stirring at 55°C. The mixture was filtered, and the residue was re-extracted with another 200 ml 20% ethanol. The combined extracts were reduced to 40 ml over a water bath at 90°C. The concentrate was transferred into a 250 ml of separating funnel, and 20 ml of diethyl ether was added and shaken vigorously and the aqueous layer was recovered. The purification processes were repeated, and 60 ml of n-butanol was added. The combined various extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evapouration, the samples were dried in the oven to a constant weight; the saponin content was calculated.
Determination of quinones 50 mg of the fine powder sample was soaked in 50 ml of distilled water for 16 h. This suspension was heated in a water bath (at 70°C) for 1 h. After the suspension was cooled, 50 ml of 50% methanol was added, and it was filtered. The clear solution was measured by spectrophotometer at a wavelength of 450 nm and compared with a standard solution containing 1 mg/100 ml of purpurin with a maximum absorption by a spectrophotometer at 450 nm.
Antibacterial activity
Bacterial strains: Multidrug resistant Escherichia coli and Staphylococcus aureus bacteria were previously isolated in our laboratory. [12] The strains were subcultured and used throughout the study. Two bacterial strains Salmonella typhi and Pseudomonas aeruginosa were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India.
Preparation of inoculums. The inoculum was prepared as described by Tereshuck et al. [22] from 24h-old cultures by picking numerous colonies and suspending with sterile saline (NaCl, 0.9 %) solution. Absorbance was read at 530 nm and adjusted with the saline solution to match to that of a 0.5 McFarland standard solution, corresponding to about 1.5 × 10 8 Colony Forming Units (CFU). The bacterial cultures were maintained at −20°C in glycerol stock and were sub-cultured in nutrient broth for 24 h at 30°C before it is used for experimental purpose.
Agar well diffusion method. Antibacterial activity of S. xanthocarpum extracts was carried out by the agar well diffusion assay discussed by Natarajan et al. [23] 25 ml of the molten agar (45°C) were poured into sterile petri dishes. The working cell suspensions were prepared, and 100 μl was evenly spread on the surface of the agar plates of Mueller-Hinton agar (Hi-Media, India). Once the plates had been aseptically dried, 5 mm wells were punched into the agar with a sterile Pasteur pipette. The residual extracts were dissolved in their extracting solvents to yield the final concentration -1 mg/ml -and sterilized by filtration (filter pore size 0.45 μm). A total of 100 µl (stock 1 mg/ml) of extracts was placed into the wells, and the plates were incubated at 37°C for 24 h. The respective wells 10% DMSO were used as negative control, and chloramphenicol (10 μg/ml) antibiotic was used as positive control. The diameter of growth inhibition zones around the well was noted. All the tests were performed in triplicates. [15] Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). The MICs of the plant extracts were determined in sterile 96-well microplates using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute, M07-A8, [24] as the lowest extract concentration suppresses the growth of microorganisms after 24 h of incubation at 37°C, whereas the MBC values defined as the lowest concentration of an antibacterial agent needed to inhibit the growth of a certain bacteria followed by subculturing using antibiotic-free media. In brief, extracts (100 µg/ml dissolved in 10% DMSO) and twofold serial dilution were prepared in a 96-well microplate. Chloramphenicol was used as positive control. The solution without extract served as a blank and negative control. Each microplate well included 40 µl of the growth medium, 50 µl of the diluted sample extracts, and 10 µl of the inoculums (10 6 CFU/ml). The wells containing no growth in the plate were taken as MBC.
DPPH free radical scavenging activity
The plant extracts were diluted with methanol to make 20, 40, 60, 80, and 100 μl/ml dilutions. Two milliliters of each dilution was mixed with 1 ml of DPPH solution (0.2 mM/ml in methanol) and mixed thoroughly. The mixture was incubated in dark at 20°C for 40 min. Absorbance was measured at 517 nm by UV-Visible spectrophotometer with methanol as blank. [25] Each experiment was performed in triplicates. The percentage scavenging of DPPH was calculated according to the following formula: AbsðcontrolÞ where Abs control is the absorbance of the control, and Abs sample is the absorbance of test.
Fourier transform-infrared (FT-IR) spectroscopy analysis
The FT-IR analysis of methanolic extract has been carried out as per the method of Jagmohan. [26] The powdered plant samples were mixed with potassium bromide (KBr pellet) and subjected to a pressure of about 5 × 10 6 Pa to produce a clear transparent disc (diameter 13 mm) and thickness (1 mm). IR spectra in frequency region 400-4000 cm −1 were recorded at room temperature on a PerkinElmer Fourier Transform Spectrometer equipped with an air-cooled deuterated triglycine sulphate (DTGs) detector. For each spectrum, 100 scans were co-added at a spectral resolution of 4 cm. The frequencies for all sharp bands were accurate to 0.01 cm. All the spectral values were expressed in percentage (%) transmittance. [27] Gas chromatography-mass spectrometry (GC-MS) analysis Statistical analysis All data were expressed as mean ± SD. Statistical analysis was performed using the SPSS 20.0 software.
Results
The phytochemical screening of S. xanthocarpum showed the presence of various types of chemical constituents. The flavonoid was present in all the extracts, whereas quinones was present in all the extracts except chloroform extract. Steroids and terpenoids were absent in all the extracts tested. Phenols and glycosides are occurred in acetone, ethyl acetate, hexane, and chloroform extract except methanol extract (Table 1). Preliminary phytochemical analysis is very important for the quantitative estimation of pharmacologically active natural compounds. The results of various quantitative phytochemical analyses of different solvent extracts of S. xanthocarpum are presented in Table 2. The level of carbohydrate was found higher in the methanol extract (7.2 mg/g) and lower in hexane extract (1.8 mg/g). The methanol extracts of this plant shown maximum amount of phenol (28.3 mg/g), flavonoids (25.2 mg/g), steroids (8.2 mg/g), quinones (7.3 mg/g), and terpenoids (6.3 mg/g) than other extracts. The solvent extracts of S. xanthocarpum were tested against five bacterial pathogens using agar well diffusion method. The results show methanol extract have significant antibacterial activity against P. aeruginosa (12 ± 0.5 mm) followed by S. typhi, S. aureus, E. coli, and C. diphtheriae. Ethyl acetate extracts exhibit moderate activity against Pseudomonas aeruginosa (8 ± 1.2 mm) and Staphylococcus aureus (7 ± 1.0 mm) having broad spectrum activity, and no inhibition was found in S. typhi. Chloroform, hexane, and acetone extract exhibit least antibacterial activity against S. aureus, P. aeruginosa, and C. diphtheriae ( Table 3
Jaberian et al. [32] analyzed the phytochemical nature of various medicinal plants, and their results were supported with the findings of current investigation. The methanolic extract of S. xanthocarpum exhibits the maximum number of phytochemicals than other extracts, which may be due to the polar nature of the solvents. This result was supported by Narender et al., [33] which found that the methanol used as leading solvent for extraction of variety of plant constituents than other solvents. Kalita et al. [34] stated that the flavonol compounds from medicinal plants exhibited better antioxidant property. The outcome of present study observed the maximum amount of total flavonoid (25.2 mg/mg) and phenol (28.3 ± 2.0 mg/mg) contents from the leaf of S. xanthocarpum.
Many studies suggest the role of phenolics and saponins obtained from plants acted as potent antibacterial agents against human pathogenic bacteria. [34,35] The present study clearly focused on crude extracts of S. xanthocarpum that were evaluated for antibacterial activity against selected bacterial cultures. The significant zone of inhibition was noticed in the crude methanol and ethyl acetate extracts against P. aeruginosa and S. aureus. Similarly, Nithya et al. [36] reported better inhibition zones (from the ethanol extracts of Thaaleesadhi chooranam) against E. coli, S. aureus, K. pneumoniae (around 12 mm). The highest antibacterial activity (14 mm) was observed in B. subtilis, and least activity was recorded in P. aeruginosa (8 ± 1.2 mm). Likewise, Aisha Ashraf et al. [37] reported the methanol and hexane extracts showed an excellent antimicrobial activity against A. niger. The minimum inhibitory action (5 mm) was observed by methanol extract against E. coli. The MIC and MBC values of selected bacteria were observed after 24 h incubation and resulted better values, i.e., 3.2 to 6.9 µg/ml (for MIC) and 6.0 to 14.5 µg/ml (for MBC). The organism like S. typhi and C. diptheriae showed higher values for MIC as well as MBC. Similarly, Mahfuzul Hoque et al. [38] reported S. aromaticum extract was found to be effective with a minimal inhibitory concentration (10 mg/ml) against B. cereus, S. aureus, E. coli, and P. aeruginosa. On the other hand, Mahboubi and Haghi [39] reported Mentha pulegium plant as an effective antibacterial activity with least MIC and MBC values.
The antioxidant activity of different extracts of S. xanthocarpum was determined using DPPH assay. It showed the highest activity was obtained in the methanolic extracts. Previously, Patil Dinanath et al. [40] reported the antioxidant activity of ethanol, chloroform, and ethyl acetate extracts of leaves and stems of S. xanthocarpum. Among them, ethanol extract of leaves and stem show better antioxidant activity. Likewise, Hoshyar et al. [41] proved the antioxidant activity of lemon exhibited the highest antioxidant activity in all three extractions. Similarly, Saumya et al. [42] reported Panax ginseng and Lagerstroemia speciosa, the percentage of inhibition was mainly dose-dependent manner, showing the IC 50 value of 3.18 μg/ml and 6.15 μg/ml, respectively, when compared to control (IC 50 value 3.35 μg/ml).
Conclusion
Solanum xanthocarpum contains many phytoconstituents such as flavonoids, carbohydrates, tannins, and phenols. Methanol extract of plant exhibited strong antibacterial effects against tested bacteria followed by other extracts due to the presence of high amount phenolics and flavonoids, and it was confirmed based on low minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) values. The same extracts was found to better antioxidant activity. Totally six major bioactive compounds and their functional groups have been identified by GC-MS and FT-IR spectral analysis, respectively. The outcome of the study is strongly recommends further isolation and structural elucidation of bioactive compounds from the plant and their potential against the different biological action. | v3-fos-license |
2019-03-11T17:23:28.142Z | 2019-02-28T00:00:00.000 | 73468595 | {
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} | pes2o/s2orc | Platelet-Rich Fibrin Extract: A Promising Fetal Bovine Serum Alternative in Explant Cultures of Human Periosteal Sheets for Regenerative Therapy
In 2004, we developed autologous periosteal sheets for the treatment of periodontal bone defects. This regenerative therapy has successfully regenerated periodontal bone and augmented alveolar ridge for implant placement. However, the necessity for 6-week culture is a limitation. Here, we examined the applicability of a human platelet-rich fibrin extract (PRFext) as an alternative to fetal bovine serum (FBS) for the explant culture of periosteal sheets in a novel culture medium (MSC-PCM) originally developed for maintaining mesenchymal stem cells. Small periosteum tissue segments were expanded in MSC-PCM + 2% PRFext for 4 weeks, and the resulting periosteal sheets were compared with those prepared by the conventional method using Medium199 + 10% FBS for their growth rate, cell multilayer formation, alkaline phosphatase (ALP) activity, and surface antigen expression (CD73, CD90, and CD105). Periosteal sheets grew faster in the novel culture medium than in the conventional medium. However, assessment of cell shape and ALP activity revealed that the periosteal cells growing in the novel medium were relatively immature. These findings suggest that the novel culture medium featuring PRFext offers advantages by shortening the culture period and excluding possible risks associated with xeno-factors without negatively altering the activity of periosteal sheets.
Introduction
Abundant growth factors and cytokines stored in platelet granules are released from activated platelets in response to tissue injury. These soluble factors are involved in wound healing and tissue repair [1]. In the 1990s, this essential role of platelets was exploited for regenerative therapy [2] and since then, therapies using platelet concentrates have been widely applied in various fields of regenerative medicine. In parallel with or even a little ahead of this therapeutic strategy, platelet lysates (PLs) have been used as a substitute for fetal bovine serum (FBS) [3] for in vitro cell expansion to reproducibly maintain cell proliferation [1]. Finding a possible alternative to FBS was strongly motivated by two major reasons: (1) limitation of the variability of FBS owing to the increased demands and decreased production ability; and (2) wide variability between batches that may affect end-product reproducibility, risks of pathogen contaminations, and ethical issues [1]. The quality of PLs also varied by source; however, shortage and risks of unexpected contamination could be avoided with the use of autologous platelets.
We have previously demonstrated a regenerative therapy with autologous periosteal sheets exhibiting osteogenic properties [4] for alveolar bone regeneration in more than 120 clinical cases [5][6][7] over the past 14 years on the basis of the evidence that osteogenicity, as well as osteoinductivity and osteoconductivity, are maintained in this grafting material [4,8]. Periosteal sheets are routinely expanded in vitro from small segments of alveolar periosteal tissues in the conventional medium supplemented with 10% FBS. Although no adverse events related to xeno-factors have been observed as a result of extensive washing with phosphate-buffered saline (PBS) prior to implantation, other aforementioned concerns, such as availability and efficacy of FBS, still pose difficulties. Furthermore, the requirement of a 6-week expansion period reduces the operational efficiency of cell-processing facilities, thereby increasing the economic burden. Therefore, we aimed to develop a xeno-free culture medium that may significantly shorten the period of expansion.
In a preceding study, we modified a chemically defined novel culture medium originally developed for the maintenance of mesenchymal stem cells suitable for human adult periosteal cells. This was accomplished by the addition of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and dexamethasone. Additionally, we adopted the extract of platelet-rich fibrin (PRFext) prepared from human peripheral blood samples to replace FBS. As the expansion of periosteal sheets necessitates only a limited amount of FBS replacement and as this supplement should be prepared in-house, we chose a more convenient way to obtain platelets and plasma instead of using the protocol of PL preparation. We confirmed that this novel complete medium facilitated the growth of periosteal sheets without causing genetic instability, as evident from karyotype testing. To test compatibility, we compared cell growth and fundamental characteristics of periosteal sheets prepared using the conventional culture medium (Medium199 + 10% FBS) and the newly modified stem cell medium supplemented with 2% PRFext. Figure 1 shows the onset of cell outgrowth, which indicates the days required for the migration of the first cell out of the original periosteum tissue segments. Some minor differences were reported depending on individual samples; however, no statistical difference was observed between groups. Cell outgrowth commonly occurred at 6-10 days of culture on average. Figure 2 shows the photomicrographs of the periosteal cells that migrated out from the isolated periosteum tissue segments. The cell density was maximum in the central region in cultures with MSC-PCM + 2% PRFext (C), while the lowest density was observed in the cultures with conventional Medium199 + 10% FBS (A). Differences in cell shape were observed in the peripheral region. The majority of periosteal cells showed a typical spindle shape in the conventional medium, while their shape was relatively branched in type, indicative of their immature phenotype [9][10][11]. These findings are consistent with the results observed with MesenPRO-RS medium [8]. Figure 3 shows the growth curves of periosteal sheets. Some individual differences were observed; however, overall these data indicate that MSC-PCM + 2% PRFext was the most effective of all media.
Growth of Periosteal Sheets
MSC-PCM + 4% FBS was equal or less effective than MSC-PCM + 2% PRFext, while the conventional medium delayed the growth of periosteal sheets. Figure 4 shows alkaline phosphatase (ALP) activity, a representative phenotypic marker of differentiated osteoblasts, in fixed periosteal sheets. Safranin-O staining indicated the size of individual samples. As the cell multilayer formation varied with different types of media, it is difficult to compare ALP activity among groups. Although cell growth in a horizontal plane is fundamentally different from cell growth in multiple layers, the observed effect was, to some extent, consistent with the growth rate results shown in Figure 3. By contrast, although calcium deposit formation largely relies on the nature of the original periosteum tissue segments, the conventional medium generally induced diffused mineralization, whereas MSC-PCM medium reduced it in limited regions. Figure 6 shows the distribution of PDGF-B, transforming growth factor beta 1 (TGFβ1), and collagen type I in the outgrown cell sheets. PDGF-B or antigenically similar proteins were not detected in any groups in Figure 6. The expression of TGFβ1 or similar proteins was slightly positive in the periosteal sheets expanded in the conventional medium. However, collagen type I was detected in all groups. As MSC-PCM + 2% PRFext produced the thickest cell multilayers, the volume of collagen type I matrix was the most abundant in the periosteal sheets expanded in this culture medium. Figure 7 shows the expression of the basic markers of mesenchymal stem cells, CD73, CD90, and CD105, in the cells growing in periosteal sheets. Comparison was performed only between two groups; namely, the conventional medium and MSC-PCM + 2% PRFext. Expression of CD105 was lower in the newly developed medium than that in the conventional medium; however, no statistical differences were observed. . X-axis: type of surface antigens. Statistical analysis was performed using the Mann-Whitney rank-sum test and no significant difference was observed between the two groups. N = 3 or 4 replicates.
Discussion
FBS is still considered a "magical" supplement for the successful cultivation of cells, although the associated disadvantages are well known. To improve the quality of the resulting cell-based products and their therapies, animal-derived factors should be completely eliminated from culture media. Several efforts have been directed toward the development of a chemically defined medium suitable for adherent cell cultures. In the initial phase of our project, we aimed to develop such a chemically defined medium or a medium free of animal components suitable for the cultivation of periosteal sheets. In comparison with single cell cultures, however, periosteal tissues require stronger adhesion systems that cannot be achieved by simply adding sufficient amounts of recombinant human adhesion molecules, such as fibronectin and vitronectin, as evident from our preliminary studies. Instead, such systems may be reproduced with the use of animal or human-derived sera. Therefore, we modified our aim to develop a xeno-free medium.
At the beginning of the second phase, we developed and patented a new expansion method using stocked human platelet-rich plasma (PRP) along with recombinant human bFGF to allow the growth of periosteal sheets [12]. This method provides a consistent source of fully confluent periosteal sheets in 100 mm dishes within 4 weeks. However, a thin fibrin membrane also forms, covering periosteal sheets that may cause easy detachment of periosteal sheets upon medium exchange. Thus, in the preliminary study, we attempted to evaluate alternative ways to utilize the factors from platelet concentrates.
In industry, it may be convenient and economical to use pooled allogeneic PRP, although complicated and costly extraction methods have to be introduced into the manufacturing process. For the preparation of small-scale homemade autologous PRP extracts, by contrast, the preparation protocol needs to be simple and cost-effective. The first choice is definitely platelet-rich fibrin (PRF) exudate or releasate. However, as various major adhesion molecules were found to be adsorbed on fibrin fibers in a preliminary experiment [Kawase et al., manuscript in submission], we homogenized the minced PRF preparations to release these adhesion molecules and used the obtained supernatant supplemented with small debris of fibrin fragments. This preparation protocol is fast, less labor-intensive, and produced better results during the initial adhesion and growth of periosteal sheets, even after reducing the content of PRFext to 2% (v/v).
The rapid growth induced by PRFext was not associated with the initial cell outgrowth, but was related to the acceleration of cell proliferation after outgrowth. As illustrated in Figure 8 and previously demonstrated [13], most periosteal cells are dead in the initial phase of culture, and the surviving cells actively replicate and migrate out to form periosteal sheets. Our results indicate that the added PRFext acted on cell outgrowth and subsequent cell proliferation, but not on cell turnover. The shortening of the cell turnover phase may allow further reduction in the period of periosteal sheet preparation to less than 3 weeks in the near future. As rapid proliferation needs to be balanced against genetic, phenotypic, and functional stability [1], we examined the compatibility of periosteal sheets prepared using the new culture medium in the validation stage of this study. Regarding genetic stability, the source of periosteal sheets, i.e., the cells from alveolar periosteum, is at a relatively late stage of differentiation compared to mesenchymal stem cells. In general, the genetic instability of cells correlates with their pluripotency and multipotency [14,15]; therefore, the majority of periosteal cells may be relatively genetically stable during expansion. In support of this speculation, we have previously demonstrated the least probability of cell transformation in X-ray-irradiated periosteal cells [16]. Furthermore, the qualitative analysis of a limited number of cells in karyotype testing (preliminary study) revealed no abnormality in the chromosomes from periosteal sheet samples at the end of the expansion period.
Regarding the rest of the criteria, the type of culture medium failed to have any significant influence on the expression of the conventional surface markers of mesenchymal stem cells, i.e., CD73, CD90, and CD105 [17]. ALP expression and calcium phosphate deposition were, to some extent, influenced by culture media. The addition of PRFext suppressed the spontaneous increase in ALP activity and consequent calcium deposit formation observed in the periosteal sheets expanded in Medium199 + 10% FBS. By contrast, MSC-PCM increased the accumulation of collagen around periosteal cells and consequently increased the thickness of periosteal sheets with an increase in growth rate. MSC-PCM induced maximum effects on sheet thickness in combination with PRFext.
Similar observations were recorded in a previous study using another stem cell medium, MesenPRO-RS medium supplemented with 2% FBS [8]. Although the ALP activity and the ability to form calcium deposits in vitro were lower, the periosteal sheets prepared with this medium showed potent osteogenesis similar to that achieved with the conventional medium upon subcutaneous implantation in animal models. Taken together with the evidence that collagen provides a platform for mineral deposition [18], the periosteal sheets prepared with MSC-PCM + 2% PRFext may possibly exhibit compatible osteogenesis.
The shortening of the preparation period is beneficial for both clinics serving this regenerative therapy and patients receiving this therapy, in terms of cost, operation efficiency, and treatment schedule. However, compatibility must be predefined and tested to ensure safety and efficacy of the resulting periosteal sheets [1] prior to clinical application. As expected, the present study demonstrates that the critical qualities of the periosteal sheet prepared with MSC-PCM + 2% PRFext are not negatively influenced during the process of expansion. The process of blood collection from patients can be estimated to be relatively low on the basis of predicted consumption as mentioned: for a medium size (2−3 tooth width) alveolar ridge augmentation, approximately 30 periosteal sheets are usually prepared. When 60 mm culture dishes are used, approximately 600 mL of the culture medium and approximately 12 mL PRFext are required for the 4-week culture. Since a 10 mL whole-blood sample, including 1 mL Acid Citrate Dextrose Formula-A (ACD-A), produces approximately 2.5 mL PRFext, approximately 45 mL peripheral blood should be collected as a sufficient starting volume prior to the explant culture. However, in case of smaller bone defects, such as periodontal bone defect, the volume of blood required for the culture can be reduced to between one-fifth and one-tenth.
In addition, this xeno-free medium minimizes the risk of unknown pathogen contamination. The newly developed MSC-PCM medium is exceptionally more expensive than the conventional Medium199, but the total cost may be markedly reduced by choosing MSC-PCM + 2% PRFext. Therefore, we proposed that this complete xeno-free medium may serve as a promising replacement medium for the conventional FBS-containing medium in the preparation of periosteal sheets.
Preparation of PRFext
Blood was collected from six healthy and non-smoking volunteers aged 24-44 years (three females and three males) using butterfly needles (21G 3/400; NIPRO, Osaka, Japan) and Vacutainer tubes (Japan Becton, Dickinson and Company, Tokyo, Japan). To prepare the PRFext, the blood samples were immediately (within approximately 2 min from blood collection) centrifuged by a Medifuge centrifugation system (Silfradent S. r. l., Santa Sofia, Italy) [19,20]. The red thrombus (the fraction of red blood cells) was eliminated with scissors and the resulting PRF preparations were minced using scissors, followed by homogenization with sterile BioMasher (Nippi, Tokyo, Japan), as illustrated in Figure 9 and as described previously [21]. The homogenized samples were centrifuged at maximum speed to exclude fibrin matrix fragments. The resulting supernatant was stored at −80 • C until use. Approximately 2.5 mL PRFext can be prepared from 10 mL whole-blood sample, including 1 mL ACD-A. The levels of PDGF-BB in the resulting samples usually ranged from 25 to 50 ng/mL [21].
Explant Culture of Periosteum Tissue Segments to Form Periosteal Sheets
Six patients aged 20-44 years (four females and two males) in need of wisdom tooth extraction participated in this study after providing written informed consent. Aliquots of periosteum tissues were aseptically dissected from the buccal side of the retromolar region in the mandible of healthy donors, washed thrice in Dulbecco's PBS without Ca 2+ and Mg 2+ , cut into small segments (~1 × 1 mm), and plated on 60 mm dishes. After incubation for 15-20 min under dry conditions in a CO 2 incubator, the conventional medium (Medium199 supplemented with 10% FBS), MSC-PCM medium (Kohjin Bio, Sakado, Japan) supplemented with 4% FBS, or MSC-PCM medium supplemented with 2% PRFext was added to cover the bottom surface of the dish. All media were commonly supplemented with 25 µg/mL of L-ascorbic acid, 100 U/mL of penicillin G, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B (Invitrogen, Carlsbad, CA, USA). The volume of media was increased in a stepwise manner as cell outgrowth proceeded.
Evaluation of Cell Outgrowth and Growth Rate
The onset of cell outgrowth, which indicates days required for the migration of the first cell out of the original periosteum tissue segments, was evaluated using an inverted microscope once every 3 days. Frequent examination of cell outgrowth can sometimes lead to detachment of periosteum tissue segments; hence, we did not perform a daily evaluation.
The growth rate was determined by measuring the lengths of the long (major) axis and short (minor) axis. Periosteal sheets were plated on a light box and measured by a caliper.
Histological Determination of ALP Activity
For ALP staining, periosteal sheets were fixed with 10% neutralized formalin on dishes and directly treated with an ALP staining kit (Muto Chemicals, Tokyo, Japan) for 4 h, followed by counterstaining with Safranin-O [4].
Histological and Immunohistochemical Examination for Calcium Deposition, Growth Factor Expression, and Collagen Accumulation
Periosteal sheets were gently detached using a cell scraper and immediately fixed with 10% formaldehyde in 0.1 M phosphate buffer, pH 7.4, overnight. Fixed samples were dehydrated using an ethanol series (70%−100%) and xylene and embedded in paraffin. The samples were sagittally sectioned at a thickness of 6 µm [4].
As previously described [20], the deparaffinized sections were subjected to antigen retrieval with Liberate Antibody Binding Solution (Polysciences, Inc., Warrington, PA, USA) and blocked with Block-Ace (Sumitomo Dainippon Pharma., Osaka, Japan) solution in 0.1% Tween-20-containing PBS. The sections were probed with a rabbit polyclonal anti-collagen type I antibody ( After being washed twice with PBS, the cells were analyzed by flow cytometry (Navios; Beckman Coulter, Miami, FL, USA). Data analysis and histogram overlay were performed using Navios Software (Beckman Coulter). For isotype controls, individual corresponding antibodies (all from BioLegend) were used [8].
Statistical Analysis
The data were expressed as mean ± standard deviation (SD). For multigroup comparisons, statistical analyses were performed to compare the mean values by Kruskal-Wallis one-way analysis of variance, followed by Steel-Dwass multiple comparison test (BellCurve for Excel version 3.00; Social Survey Research Information Co., Ltd., Tokyo, Japan). For two group comparisons, statistical differences were tested using the Mann-Whitney rank-sum test (SigmaPlot 12.5; Systat Software, Inc., San Jose, CA, USA). Differences with P values of less than 0.05 were considered statistically significant.
Conclusions
This preclinical study successfully validated the applicability of PRFext for FBS replacement in explant cultures of periosteum tissue segments to form periosteal sheets. These findings were established using the samples donated by healthy volunteers. For therapeutic use, however, autologous periosteum tissue and PRFext must be employed for the preparation of periosteal sheets. Thus, the efficacy of PRFext and the responsiveness of periosteum tissue may vary with individual samples, and more careful measures should be adopted while evaluating the quality of periosteal sheets prepared by this protocol. Funding: This study is financially supported by the budget provided by Kojin Bio, Co., Ltd. for the collaborative research investigation.
Acknowledgments: Kojin Bio, Co., Ltd. modified the stem cell medium suitable for the explant culture of human periosteum tissue in response to our requests and provided it by free of charge.
Conflicts of Interest:
A.I. who is an employee of Kohjin Bio, Co., Ltd. was involved in the collection, analyses and interpretation of data. Author T.K., M.N., K.O., T.U., Y.F., M.W. and K.N. state that there are no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Platelet-derived growth factor-B TGFβ1
Abbreviations
Transforming growth factor beta 1 EDTA Ethylenediaminetetraacetic acid | v3-fos-license |
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} | pes2o/s2orc | Praeruptorin B Mitigates the Metastatic Ability of Human Renal Carcinoma Cells through Targeting CTSC and CTSV Expression.
Renal cell carcinoma (RCC) is the most common adult kidney cancer, and accounts for 85% of all cases of kidney cancers worldwide. Praeruptorin B (Pra-B) is a bioactive constituent of Peucedanum praeruptorum Dunn and exhibits several pharmacological activities, including potent antitumor effects. However, the anti-RCC effects of Pra-B and their underlying mechanisms are unclear; therefore, we explored the effects of Pra-B on RCC cells in this study. We found that Pra-B nonsignificantly influenced the cell viability of human RCC cell lines 786-O and ACHN at a dose of less than 30 μM for 24 h treatment. Further study revealed that Pra-B potently inhibited the migration and invasion of 786-O and ACHN cells, as well as downregulated the mRNA and protein expression of cathepsin C (CTSC) and cathepsin V (CTSV) of 786-O and ACHN cells. Mechanistically, Pra-B also reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), p-mitogen-activated protein kinase kinase (MEK), and p-extracellular signal-regulated kinases (ERK) in RCC cells. In addition, Pra-B treatment inhibited the effect of EGF on the upregulation of EGFR–MEK–ERK, CTSC and CTSV expression, cellular migration, and invasion of 786-O cells. Our findings are the first to demonstrate that Pra-B can reduce the migration and invasion ability of human RCC cells through suppressing the EGFR-MEK-ERK signaling pathway and subsequently downregulating CTSC and CTSV. This evidence suggests that Pra-B can be developed as an effective antimetastatic agent for the treatment of RCC.
Introduction
Kidney cancer accounts for approximately 3% of adult malignancies, and yearly kidney cancer incidence rates are increasing in more developed regions of the world [1]. To date, most patients with kidney cancer have an excellent prognosis in the early stage; however, prior research has noted limited survival and poor outcomes in patients, which has been attributed to advanced or distant tumor metastasis [2]. At present, surgical intervention combined with chemotherapy is the standard therapy for patients with tumor metastasis; nonetheless, adverse side effects and drug resistance remain obstacles [3]. Therefore, the development of antimetastatic reagents for renal cell carcinoma (RCC) potentially improves current RCC treatment strategies.
Peucedanum praeruptorum DUNN (P. praeruptorum), a traditional Chinese medical herb, is well known for its pharmacological function in treating headaches, coughing, and vomiting. To date, many studies have indicated that angular-type pyranocoumarins and furanocoumarins are major constituents of dried roots of P. praeruptorum [4], and pharmacological studies have shown that these compounds may possess a wide variety of activities, such as anti-inflammatory [5], antiasthma [6], and neuroprotective [7]. Praeruptorins are major bioactive members of pyranocoumarin and can be divided into five species: A, B, C, D, and E. Praeruptorin A (Pra-A) is reported to exert a protective effect on osteoporosis through inhibiting the p38/AKT/c-Fos/NAFTc1 pathway [8]. Pra-C was observed to mitigate cardiac damage and have a clear effect on blood pressure in spontaneously hypertensive rats, suggesting its potential as a novel drug for the treatment and prevention of cardiovascular diseases [9]. One study reported that Pra-B inhibits sterol regulatory element-binding proteins (SREBPs) to improve hyperlipidemia and insulin resistance [10]. Moreover, Pra-A and Pra-C were indicated to possess cytotoxic activity and induce apoptosis against lymphocytic leukemia cells [7,11]. Another study demonstrated that praeruptorins enhanced the sensitivity of doxorubicin, paclitaxel, and vincristine in cancer cells [12], suggesting a potential anticancer effect. However, the effects and molecular mechanisms of the antitumor effect of Pra-B on RCC have thus far not been clarified.
The extracellular matrix (ECM) is a highly dynamic and continuous process during composition, reorganization, and degradation. It has the function of maintaining tissue homeostasis and is responsible for cell-cell interaction, cell migration, and cell proliferation. However, the dysregulation of ECM's dynamics process may lead to the development of different diseases [13]. ECM degradation by extracellular proteinases is a key step in tumor cell invasion and metastasis. Among them, the expression of matrix metalloproteinase (MMP) activity has been highly correlated with cancer cell metastasis and has thus been considered a target for anticancer drugs in the literature [14,15]. Cysteine cathepsins are proteases that are frequently secreted into the extracellular environment and during the activation of MMPs, which regulate the invasion of cancer cells [16]. Studies have implicated that overexpression of CTSC and CTSV expression in various different malignant tumors, such as breast ductal carcinoma, colorectal carcinomas, and pancreatic [17][18][19], and it was suggested to be associated with poor prognosis in HCC [20]. Moreover, Zhang et al. observed that CTSC mediated hepatoma tumor cell proliferation and metastasis by activation of the TNF-α/p38 MAPK pathway [21]. Keegan et al. demonstrated that TNF-α induced monocyte-endothelial cell and increased the CTSV activity through dependency on JNK signaling pathways in cardiovascular disease [22]. Although these studies have discovered CTSV and CTSC involved in tumor progression, the intracellular signaling cascades linking the Pra-B regulate the levels of CTSV and CTSC in RCC cells for further investigation.
In this study, we investigate the inhibitory effect of Pra-B on migration and invasion in RCC and further identify underlying molecular mechanisms for these effects. Our results demonstrated that Pra-B suppressed cellular motility through reducing the mRNA and protein expression of CTSC/CTSV and suppressing the EGFR-MEK-ERK signaling pathway. This suggested that Pra-B has potential as an antimetastatic agent in human RCC cells. Figure 1A illustrates the chemical structure of Pra-B. An MTT assay was used to examine the cell viability and cytotoxicity of various concentrations of Pra-B (0, 10, 20, 30, 40, and 50 µM) for 24 h, which led to the observation that treated with Pra-B doses higher than 40 µM, resulted in the reduction of cell viability in 786-O and ACHN cells, but doses lower than 30 µM did not induce cytotoxicity ( Figure 1C,D). However, Pra-B exhibited no cytotoxicity in human HK-2 cells ( Figure 1B). These results revealed that Pra-B is not toxic to human RCC cells at 30 µM; therefore, serial Pra-B concentrations in the range of 0-30 µM were used in subsequent experiments. Figure 1A illustrates the chemical structure of Pra-B. An MTT assay was used to examine the cell viability and cytotoxicity of various concentrations of Pra-B (0, 10, 20, 30, 40, and 50 μM) for 24 h, which led to the observation that treated with Pra-B doses higher than 40 μM, resulted in the reduction of cell viability in 786-O and ACHN cells, but doses lower than 30 μM did not induce cytotoxicity ( Figure 1C,D). However, Pra-B exhibited no cytotoxicity in human HK-2 cells ( Figure 1B). These results revealed that Pra-B is not toxic to human RCC cells at 30 μM; therefore, serial Pra-B concentrations in the range of 0-30 μM were used in subsequent experiments.
Pra-B Inhibited Cell Migration and Invasion in 786-O and ACHN Cells
To examine the effect of Pra-B on cellular migration and invasion ability in renal cell carcinoma, we treated 786-O and ACHN cells with various concentrations of Pra-B (0-30 μM) for 24 h. Our results revealed that an increasing dose of Pra-B significantly reduced 786-O and ACHN cell migration and invasion ability (Figure 2A). On the basis of quantitative assessment, the migrate cell numbers of cells treated with 20 and 30 μM of Pra-B was reduced by 42% and 79% in 786-O cells, and 60% and 82% in ACHN cells, respectively. In addition, such doses of Pra-B inhibited 58% and 80% of cell invasion in 786-O cells, and 62% and 86% in ACHN cells, respectively ( Figure 2B). These results demonstrated that Pra-B has an inhibitory effect on 786-O and ACHN cell migration and invasion capacities.
Pra-B Inhibited Cell Migration and Invasion in 786-O and ACHN Cells
To examine the effect of Pra-B on cellular migration and invasion ability in renal cell carcinoma, we treated 786-O and ACHN cells with various concentrations of Pra-B (0-30 µM) for 24 h. Our results revealed that an increasing dose of Pra-B significantly reduced 786-O and ACHN cell migration and invasion ability (Figure 2A). On the basis of quantitative assessment, the migrate cell numbers of cells treated with 20 and 30 µM of Pra-B was reduced by 42% and 79% in 786-O cells, and 60% and 82% in ACHN cells, respectively. In addition, such doses of Pra-B inhibited 58% and 80% of cell invasion in 786-O cells, and 62% and 86% in ACHN cells, respectively ( Figure 2B). These results demonstrated that Pra-B has an inhibitory effect on 786-O and ACHN cell migration and invasion capacities.
Pra-B Inhibits the Expression of CTSC and CTSV in 786-O Cells
Cathepsin-related proteins have been broadly implicated in cancer [23], and we even previously reported CTSC as being a target in the suppression of RCC migration and invasion [24]. To identify the effect of Pra-B on CTS-related mRNA expression, we treated 786-O cells with Pra-B (30 μM) for 24 h and used qRT-PCR analysis to screen the expression of CTS-related gene expression. The results indicated that the mRNA expression of CTSC and CTSV was significantly reduced in response to Pra-B treatment ( Figure 3A). Furthermore, Western blot analysis also demonstrated that the protein expression of CTSC and CTSV was reduced upon Pra-B treatment in 786-O and ACHN cells ( Figure 3B,C).
Pra-B Inhibits the Expression of CTSC and CTSV in 786-O Cells
Cathepsin-related proteins have been broadly implicated in cancer [23], and we even previously reported CTSC as being a target in the suppression of RCC migration and invasion [24]. To identify the effect of Pra-B on CTS-related mRNA expression, we treated 786-O cells with Pra-B (30 µM) for 24 h and used qRT-PCR analysis to screen the expression of CTS-related gene expression. The results indicated that the mRNA expression of CTSC and CTSV was significantly reduced in response to Pra-B treatment ( Figure 3A). Furthermore, Western blot analysis also demonstrated that the protein expression of CTSC and CTSV was reduced upon Pra-B treatment in 786-O and ACHN cells ( Figure 3B,C). and protein levels were subsequently analyzed using immunoblotting. The histogram represents the densitometric analysis of CTSC and CTSV protein expression. β-actin was used as the loading control. * p < 0.05, ** p < 0.01 relative to the control. Data are presented in terms of mean ± SD, as determined in at least three independent experiments.
Pra-B Suppressed Activation of the MEK-ERK Signaling Pathway.
Numerous studies have explored the MAP kinase and its molecular mechanisms involved in the regulation of tumor cell migration and invasion [25]. To determine which MAPKs pathway was involved in downregulating of migration and invasive ability of Pra-B, we treated 786-O and ACHN cells with Pra-B (0, 10, 20, and 30 μM) and performed Western blot analysis to observe the activation of signaling pathways. As illustrated in Figure 4A, Pra-B inhibited the phosphorylation h, and protein levels were subsequently analyzed using immunoblotting. The histogram represents the densitometric analysis of CTSC and CTSV protein expression. β-actin was used as the loading control. * p < 0.05, ** p < 0.01 relative to the control. Data are presented in terms of mean ± SD, as determined in at least three independent experiments.
Pra-B Suppressed Activation of the MEK-ERK Signaling Pathway
Numerous studies have explored the MAP kinase and its molecular mechanisms involved in the regulation of tumor cell migration and invasion [25]. To determine which MAPKs pathway was involved in downregulating of migration and invasive ability of Pra-B, we treated 786-O and ACHN cells with Pra-B (0, 10, 20, and 30 µM) and performed Western blot analysis to observe the activation of signaling pathways. As illustrated in Figure 4A, Pra-B inhibited the phosphorylation of ERK expression in 786-O and ACHN cells; however, the phosphorylation of JNK and p38 had no effects after Pra-B treatment.
Pra-B Attenuated EGF-Induced Migration and Invasion Ability Through the Activation of EGFR-MEK-ERK Signaling Pathway
To examine the effect of Pra-B on the phosphorylation of EGFR-MEK-ERK pathways of RCC cells, we analyzed the expression of p-EGFR and p-MEK, which are upstream of p-ERK, and the results indicated that their phosphorylation expression was significantly inhibited upon Pra-B treatment 786-O and ACHN cells, not influencing total EGFR/MEK/ERK expression ( Figure 4B). These results suggest that the EGFR-MEK-ERK activation signaling pathways are involved in the Pra-B-mediated inhibition of human RCC migration and invasion. To further determine the role of Pra-B in EGF-induced migration and invasion of 786-O cells, we found that Pra-B significantly inhibited EGF-induced cell migration of 786-O cells, compared with EGF-treated alone. Similar results were achieved with a Matrigel-based invasion assay ( Figure 5A). In addition, to further clarify whether p-EGFR, p-MEK, p-ERK, CTSC, and CTSV in Pra-B-treated RCC cells were involved, we performed a Western blot analysis. As shown in Figure 5B, the increments of p-EGFR, p-MEK,
Pra-B Attenuated EGF-Induced Migration and Invasion Ability through the Activation of EGFR-MEK-ERK Signaling Pathway
To examine the effect of Pra-B on the phosphorylation of EGFR-MEK-ERK pathways of RCC cells, we analyzed the expression of p-EGFR and p-MEK, which are upstream of p-ERK, and the results indicated that their phosphorylation expression was significantly inhibited upon Pra-B treatment 786-O and ACHN cells, not influencing total EGFR/MEK/ERK expression ( Figure 4B). These results suggest that the EGFR-MEK-ERK activation signaling pathways are involved in the Pra-B-mediated inhibition of human RCC migration and invasion. To further determine the role of Pra-B in EGF-induced migration and invasion of 786-O cells, we found that Pra-B significantly inhibited EGF-induced cell migration of 786-O cells, compared with EGF-treated alone. Similar results were achieved with a Matrigel-based invasion assay ( Figure 5A). In addition, to further clarify whether p-EGFR, p-MEK, p-ERK, CTSC, and CTSV in Pra-B-treated RCC cells were involved, we performed a Western blot analysis. As shown in Figure 5B, the increments of p-EGFR, p-MEK, p-ERK, CTSC, and CTSV expression through EGF induction were significantly decreased after Pra-B treatment at 20 and 30 µM. These findings indicate that the EGFR signaling pathway is involved in the inhibitory effect of Pra-B on EGF-induced EGFR-MEK-ERK phosphorylation as well as CTSC and CTSV expression in 786-O cells.
p-ERK, CTSC, and CTSV expression through EGF induction were significantly decreased after Pra-B treatment at 20 and 30 μM. These findings indicate that the EGFR signaling pathway is involved in the inhibitory effect of Pra-B on EGF-induced EGFR--MEK-ERK phosphorylation as well as CTSC and CTSV expression in 786-O cells. were harvested to detect the p-EGFR, t-EGFR, p-MEK, t-MEK, p-ERK, t-ERK, CTSC, and CTSV protein expression levels through immunoblotting. β-actin was used as the loading control. The expression of these proteins was detected by densitometry as an average relative ratio compared to β-actin from three different experiments. ** p < 0.01 relative to the control. # p < 0.05 relative to EGF-treated alone. Data are presented in terms of mean ± SD, as determined in at least three independent experiments. Scale bar = 50 μm.
Discussion
Several targeted drugs treating tumors by their histological and molecular subtype have been developed from natural products; these drugs have improved the treatment outcomes and distal metastasis of patients in the last decade [26,27]. Despite these notable advances, some unresolved issues remain, such as optimal drug selection for patients. Therefore, research on those bioactive agents that can be derived from natural products, especially their effectiveness for improving patient survival, is crucial. In this study, we demonstrated that a seselin-type coumarin, Pra-B, had no effect on cellular viability in normal human proximal tubule cells (HK2) and RCC cell lines (786-O and ACHN). However, Pra-B significantly inhibited RCC cells migration and invasion ability as well as downregulated CTSC and CTSV protein expression in a dose-dependent manner. Furthermore, Pra-B inhibited EGFR-MEK-ERK phosphorylation but had no effect on the JNK and p38 pathways. These results suggest that Pra-B can act as an antimetastatic agent through suppressing CTSC and CTSV expression as well as migration and invasion through downregulating the EGFR-MEK-ERK signaling cascade in RCC cells ( Figure 6). In our previous study, we noted Pra-A's antiproliferation and antimetastatic abilities in cervical cancer HeLa cells. In addition, we found that the effect of Pra-B was mediated by the PI3K/AKT/NF-κB signaling pathway in blocking cervical cancer cell metastasis [28]. Although Pra-B is a major bioactive Cells were harvested to detect the p-EGFR, t-EGFR, p-MEK, t-MEK, p-ERK, t-ERK, CTSC, and CTSV protein expression levels through immunoblotting. β-actin was used as the loading control. The expression of these proteins was detected by densitometry as an average relative ratio compared to β-actin from three different experiments. ** p < 0.01 relative to the control. # p < 0.05 relative to EGF-treated alone. Data are presented in terms of mean ± SD, as determined in at least three independent experiments. Scale bar = 50 µm.
Discussion
Several targeted drugs treating tumors by their histological and molecular subtype have been developed from natural products; these drugs have improved the treatment outcomes and distal metastasis of patients in the last decade [26,27]. Despite these notable advances, some unresolved issues remain, such as optimal drug selection for patients. Therefore, research on those bioactive agents that can be derived from natural products, especially their effectiveness for improving patient survival, is crucial. In this study, we demonstrated that a seselin-type coumarin, Pra-B, had no effect on cellular viability in normal human proximal tubule cells (HK2) and RCC cell lines (786-O and ACHN). However, Pra-B significantly inhibited RCC cells migration and invasion ability as well as downregulated CTSC and CTSV protein expression in a dose-dependent manner. Furthermore, Pra-B inhibited EGFR-MEK-ERK phosphorylation but had no effect on the JNK and p38 pathways. These results suggest that Pra-B can act as an antimetastatic agent through suppressing CTSC and CTSV expression as well as migration and invasion through downregulating the EGFR-MEK-ERK signaling cascade in RCC cells ( Figure 6). In our previous study, we noted Pra-A's antiproliferation and antimetastatic abilities in cervical cancer HeLa cells. In addition, we found that the effect of Pra-B was mediated by the PI3K/AKT/NF-κB signaling pathway in blocking cervical cancer cell metastasis [28].
Although Pra-B is a major bioactive compound of Peucedanum praeruptorum Dunn, its underlying mechanisms differ depending on the type of tumor cell it is interacting with.
compound of Peucedanum praeruptorum Dunn, its underlying mechanisms differ depending on the type of tumor cell it is interacting with.
Figure 6. Illustration of how Pra-B inhibits the migration and invasion of human RCC cells through suppressing EGFR-MEK-ERK activation depending on CTSC and CTSV expression.
Cancer cell metastasis is often accompanied by a series of ECM destruction leading to subsequent tumor cell migration and invasion [29]. In the context of carcinogenesis, cysteine cathepsins secreted into the extracellular environment contribute to tumor ECM degradation [30]. Recent studies have indicated that CTSD is involved in breast cancer invasion, and inducing CTSD may facilitate breast and gastric cancer cell migration [31,32]. In addition, studies have indicated that CTSB and CTSS mediate cancer progression and metastasis in lung cancer, renal cancer, and gastric cancer [33][34][35]. In our previous study, we discovered that CTSC is highly expressed in RCC cells and involved in inhibited the migratory and invasive ability in timosaponin AIII-treated RCC cells [24]. Our present results also demonstrate that the expression of CTSC and CTSV is inhibited by Pra-B as well as involved in the migration and invasion of RCC cells. Therefore, CTSC and CTSV may play crucial roles in the invasion of RCC cells and have a potential antimetastatic effect on RCC.
EGFR, also called ErbB1 or HER1, is a member of the ErbB family of receptors, which are transmembrane glycoproteins that stimulate their corresponding signaling pathway, including the KRAS-MEK1/2-ERK1/2 pathway, phosphoinositide 3-kinase (PI3K)-AKT kinase pathway, and STAT signaling pathway [36,37]. Evidence is accumulating for a critical role played by the EGFR-mediated signaling pathway in cell proliferation, survival, angiogenesis, and cancer metastasis [38]. A study observed the inhibition of CTSS-induced cancer-cell autophagy through the EGFR-MEK1/2-ERK1/2 cascade [39]. CTSD is highly expressed in colorectal tumors and positively correlated with the expression of EGFR [40]. Furthermore, the E3 ubiquitin ligase NEDD4 interacted with EGFR, which mediated lung cancer cell migration through activating CTSB [34]. The conclusions of the aforementioned studies suggest that the EGFR and CTS families closely regulate the carcinogenesis process. Numerous natural products have been identified as potential drugs based on their ability to target the EGFR signaling pathway. Butein, isolated from Rhus verniciflua, can inhibit EGFR phosphorylation, following the inhibition of the activation of its downstream signaling pathway [41]. An extract of Magnolia spp., Honokiol, has been demonstrated to be a phenolic compound with multiple activities, especially anticancer properties. Honokiol inhibited cancer cell migration and invasion through downregulating EGFR phosphorylation [42]. In conjunction with the aforementioned findings, our results demonstrated that through inhibiting the Cancer cell metastasis is often accompanied by a series of ECM destruction leading to subsequent tumor cell migration and invasion [29]. In the context of carcinogenesis, cysteine cathepsins secreted into the extracellular environment contribute to tumor ECM degradation [30]. Recent studies have indicated that CTSD is involved in breast cancer invasion, and inducing CTSD may facilitate breast and gastric cancer cell migration [31,32]. In addition, studies have indicated that CTSB and CTSS mediate cancer progression and metastasis in lung cancer, renal cancer, and gastric cancer [33][34][35]. In our previous study, we discovered that CTSC is highly expressed in RCC cells and involved in inhibited the migratory and invasive ability in timosaponin AIII-treated RCC cells [24]. Our present results also demonstrate that the expression of CTSC and CTSV is inhibited by Pra-B as well as involved in the migration and invasion of RCC cells. Therefore, CTSC and CTSV may play crucial roles in the invasion of RCC cells and have a potential antimetastatic effect on RCC.
EGFR, also called ErbB1 or HER1, is a member of the ErbB family of receptors, which are transmembrane glycoproteins that stimulate their corresponding signaling pathway, including the KRAS-MEK1/2-ERK1/2 pathway, phosphoinositide 3-kinase (PI3K)-AKT kinase pathway, and STAT signaling pathway [36,37]. Evidence is accumulating for a critical role played by the EGFR-mediated signaling pathway in cell proliferation, survival, angiogenesis, and cancer metastasis [38]. A study observed the inhibition of CTSS-induced cancer-cell autophagy through the EGFR-MEK1/2-ERK1/2 cascade [39]. CTSD is highly expressed in colorectal tumors and positively correlated with the expression of EGFR [40]. Furthermore, the E3 ubiquitin ligase NEDD4 interacted with EGFR, which mediated lung cancer cell migration through activating CTSB [34]. The conclusions of the aforementioned studies suggest that the EGFR and CTS families closely regulate the carcinogenesis process. Numerous natural products have been identified as potential drugs based on their ability to target the EGFR signaling pathway. Butein, isolated from Rhus verniciflua, can inhibit EGFR phosphorylation, following the inhibition of the activation of its downstream signaling pathway [41]. An extract of Magnolia spp., Honokiol, has been demonstrated to be a phenolic compound with multiple activities, especially anticancer properties. Honokiol inhibited cancer cell migration and invasion through downregulating EGFR phosphorylation [42]. In conjunction with the aforementioned findings, our results demonstrated that through inhibiting the EGFR-MEK-ERK signaling pathway, CTSC and CTSV expression are involved in Pra-B's suppression of EGF-induced migration and invasion in RCC cells.
Currently, tyrosine kinase inhibitors (TKIs) have been approved by the Food and Drug Administration for the treatment of RCC, and increasing evidence suggests that they are most effective and safe, with less toxicity [43]. It is well known that tyrosine kinase inhibitor (TKI)-targeted therapies such as sorafenib, pazopanib, or sunitinib that are combined with nature bioactive compounds or flavonoids may exert promoted antitumor or anti-invasive therapeutic effects and significantly decrease the systemic toxicity induced by combined targeted therapy; the results may be a possible lower dose [44]. However, we need to clarify two questions: First, what are the antitumor effects and molecular mechanisms of the combination of TKI-targeted therapy and Pra-B treatment of RCC cells? Second, are there any interactions or signaling pathways between the combination of TKI-targeted therapy and Pra-B? The answers to these questions should be studied in more focus and detail on the molecular mechanism of RCC in further research in the future.
Cell Viability
Cell viability was measured using MTT reagent. Cells were counted at 8 × 10 3 /100 µL and seeded in 96-well plates (Greiner Bio-one, Germany). After 24 h, the cells were cultured with 0.1% dimethyl sulfoxide (DMSO) or Pra-B (10, 20, 30, 40, and 50 µM) for 24 h. Subsequently, the medium was replaced with fresh medium containing MTT reagent (0.5 mg/mL) and incubated at 37 • C for 4 h. The product of formazan followed solubilization with 1 mL of isopropanol, and the color intensity was measured at 570 nm using a Multiskan MS ELISA reader (Labsystems, Helsinki, Finland).
Cell Migration and Invasion
Migration and invasion assays were performed per a previously described method [45]. In brief, cells were counted at 4 × 10 5 /3 mL and seeded in a 6-cm dish (Greiner Bio-one). After 24 h, the cells were treated first with Pra-B (10, 20, and 30 µM) for 24 h, and subsequently using 48-well modified Boyden chambers containing polycarbonate filter inserts (Millipore) with 8-µm pores in RPMI1640 or MEM medium. These filter inserts were coated with Matrigel (10 µL, BD Biosciences) prior to the invasion assay. Cells were counted at 2 × 10 4 /50 µL and placed in the upper part of the chamber, which contained serum-free RPMI1640 or MEM medium, before being incubated for 16 h. The migrated cells were counted using an inverted microscope (200×, Leica). Three sets of five microscopic fields were counted for each sample.
Statistical Analysis
Differences in experimental results were statistically analyzed using one-way analysis of variance in SPSS (version 10.0). Differences between the control or Pra-B-treated groups were analyzed by one-way ANOVA and Dunnett's post hoc test. Results were expressed in terms of mean ± standard deviation in triplicate, with a p-value < 0.05 or < 0.01 indicating statistical significance.
Conclusions
The present results suggest that the EGFR-MEK-ERK pathways may be key mediators of Pra-B's antimetastatic action through the inhibition of the expression of CTSC and CTSV. Ours is the first study to demonstrate the roles and possible mechanisms of EGFR, CTSC, and CTSV on the metastasis of human RCC. Our findings uncover the molecular mechanisms underlying the role of Pra-B, and CTSC/CTSV is considered a potential antimetastatic target against RCC cells. Funding: This research was funded and supported by grants from Changhua Christian Hospital, Changhua, Taiwan (108-CCH-IRP-013). | v3-fos-license |
2019-11-07T14:18:02.432Z | 2020-01-01T00:00:00.000 | 209484695 | {
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} | pes2o/s2orc | Ozone mediated depolymerization and solvolysis of technical lignins under ambient conditions in ethanol †
Technical lignins are highly available and inexpensive feedstocks derived from current large scale biomass utilizing industries. Their valorization represents a bottleneck in the development of biore fi neries, as the inherently complex lignin structure often su ff ers severe condensation during isolation, leading to their current application as low value fuel. Processes able to depolymerize technical lignins into value-added (intermediate) molecules are of great interest for the development of integrated, viable routes aiming at the full valorization of lignocellulosic biomass. Here, we report an e ff ective ozone mediated depolymerization of four technical lignins (Indulin-AT Kraft, ball-milled Indulin-AT Kraft, Alcell organosolv and Fabiola organosolv) in ethanol under ambient conditions without the need for catalysts. 52 – 87 wt% of these nearly ethanol insoluble lignins was broken down into soluble fragments upon ozone exposure. The average molecular weight of the soluble fragments was shown to have decreased by 40 – 75% compared to the parent materials. A range of (di)carboxylic acids and (di)ethyl esters was identi fi ed, accounting for up to 40 wt% of the ozonated lignin oils. These products are the result of phenol ring-opening reactions as well as oxidative cleavage of unsaturated linking motifs followed by partial esteri fi cation. Reactivity varied substantially among the lignin feedstocks. For instance, lower particle sizes and higher degradation of the native lignin structure were shown to be bene fi cial for the e ff ective action of the ozone. Our results show that a straightforward ozonation process under ambient conditions can depolymerize recalcitrant lignins into oxygenated fragments and low molecular weight products soluble in ethanol. These can potentially be used for the synthesis of high-value drop-in chemicals.
Introduction
Lignin is one of the three main fractions of lignocellulosic biomass, corresponding to up to 40 wt% and consists in its native form of a highly crosslinked and methoxylated phenylpropanoid network (Fig. 1A). 1 Compared to the carbohydrate fraction, lignin is largely unutilized and oen burned for low value energy generation. 2 The complex structure of this biopolymer suffers severe degradation during isolation in typical industrial processes, e.g. Kra for pulp and paper production. 3,4 This makes further valorization of these so-called technical lignins (produced at large scales, e.g. 50 million tons per year of Kra lignin 5 ) via depolymerization challenging. 6 Technical lignins are highly condensed and chemically heterogeneous, with substantially lower amounts of the labile C-O linkages present in abundance in native lignin 7,8 (Fig. 1B). Within the biorenery concept, which aims to achieve full valorization of biomass by converting all of its fractions into value-added chemicals, materials and fuels, lignin stands as a promising source of renewable carbon. 1,9 Therefore, the development of processes able to deconstruct the recalcitrant structure of technical lignins to yield value-added (intermediate) chemicals is of great interest.
Several strategies for the depolymerization of technical lignins have been suggested. For instance, catalytic hydrotreatment has been widely studied to hydrodeoxygenate and hydrocrack lignin to yield fuels and chemicals. A range of heterogeneous catalysts have been reported with and without the use of solvents, e.g. CoMo and NiMo supported catalysts typically used in petro-based reneries, [10][11][12] noble metal supported catalysts, [13][14][15][16][17][18] inexpensive iron ores 19,20 and catalysts based on transition metals such as Ni. [21][22][23][24][25][26][27] Despite the high monomer yields of up to 35 wt% observed (largely comprised of aromatics and alkylphenolics), costs related to hydrogen and catalysts are still impeditive, harsh reaction conditions are typically needed (i.e. temperatures > 350 C, high pressures) and signicant carbon losses to solids and the gas phase occur.
Oxidative depolymerization strategies have also been reported for lignin valorization, using oxygen, air and hydrogen peroxide as typical oxidants. Various catalytic systems have been investigated, in which homogeneous catalysts are oen employed, e.g. TEMPO 28,29 and polyoxometalates. [30][31][32] Heterogeneous catalysts (e.g. chalcopyrite, 33 metal-supported, 34 metal oxides, 35 metal composites, 36 hydrotalcites 37 ) and new approaches using biomimetic catalysts, 38,39 anchored TEMPO 40 and ionic liquids [41][42][43] have been explored as well. The applied conditions are usually milder than those employed in reductive strategies. Products derived from lignin oxidation include aromatic acids and aldehydes, phenolic building blocks and dicarboxylic acids (DCAs) with several potential applications as specialty and platform chemicals, fuels and additives. Overall, the use of catalysts adds extra complexity to lignin processing due to separation and reusability difficulties, fast deactivation, poisoning, high costs and irreversible morphologic changes in the case of heterogeneous catalysts. 44 Non-catalytic routes for the upgrading of technical lignins include pyrolysis [45][46][47] and solvolysis, [48][49][50][51][52] which despite the promising results, also need high temperatures to efficiently break down the structure and minimize repolymerization pathways. In addition, low monomer yields and high carbon losses to the gas, solid and aqueous phases are common.
Ozonation is a less explored process for lignin valorization, despite the high reactivity of ozone towards lignin-like structures at mild conditions without the need of catalysts. 53,54 Ozone can be easily generated in situ, either from oxygen or dry air, and technology is well-established and available at all scales. Furthermore, ozone has a short half-life, thus any residual ozone in the system quickly decomposes to O 2 , which facilitates safe operation. This provides an overall clean procedure that does not need further separation steps to remove reagents. 55 Previous studies showed that ozonated biomass solutions contain a range of aromatic aldehydes, quinones and carboxylic acids derived from the lignin fraction. 33,56,57 An investigation of alkali lignin ozonation reported that the thus produced esters are suitable for applications as fuel additives. 55 In another study, ozonated lignin was shown to be suitable for the production of vitrimer materials with potential use as recoverable adhesives. 58 Recent publications from our group 59,60 showed the potential of pyrolytic lignin (PL) ozonation for the production of biobased DCAs and methyl esters at mild conditions (i.e. 0 C and atmospheric pressure), having methanol as solvent. In addition, model compound studies also showed the high reactivity of ozone towards phenolic motifs and unsaturated C-C bonds. These results inspired us to move from low molecular weight PL's to more complex and degraded lignin feeds, which are known to contain high amounts of the said functionalities, 61 using even milder conditions (Fig. 2).
Thus, here we report the depolymerization and subsequent dissolution of four initially ethanol insoluble technical lignins by a simple ozone treatment. All experiments were performed under ambient conditions at relatively short reaction times of under two hours, using absolute ethanol as the solvent. The use of ethanol is advantageous over methanol due to its very low toxicity and biobased character. Two types of lignin feeds were chosen based on their high industrial availability and challenging structure, 5 i.e. Kra lignin (Indulin-AT, abbreviated here as KL) and Alcell organosolv lignin. 62 Our studies show that ozone is remarkably effective for the depolymerization and solubilization of these lignins. A ball-milled KL was added to the scope to evaluate the particle size inuence on the process. Furthermore, Fabiola lignin, which is an organosolv lignin obtained under milder conditions than the Alcell, 63 was also evaluated to get insights on the inuence of lignin condensation on the ozonation extent.
Chemicals
Indulin-AT (Kra lignin, KL) was supplied by MeadWestvaco Specialty Chemicals, USA. Indulin-AT is a puried form of pinederived KL and does not contain hemicellulose. The ball-milled KL was obtained by ball milling KL for 30 minutes at 1200 RPM (Retsch 400). The Alcell (ethanosolv from a mixture of hardwoods) and Fabiola (acetosolv from beech wood) organosolv lignins were supplied by ECN-TNO. The extraction procedure and associated conditions of the latter are described elsewhere. 63 All lignins were obtained in powder form and their particle sizes were determined by dynamic light scattering (DLS). Tetrahydrofuran (THF), toluene, ethanol, deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), pyridine, cyclohexanol, chromium(III) acetylacetonate and 2chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphopholane were purchased from Sigma-Aldrich. All chemicals in this study were used as received.
Ozonation experiments
The ozonation experiments were performed under ambient conditions. A 100 mL round bottom ask containing a magnetic stirrer was used. 2 g of lignin and 45 g of ethanol were added in each experiment. For the longer experiments of 120 minutes, 2 g of lignin and 60 g of ethanol were used. Ozone (diluted in oxygen) was bubbled in the mixture through a pipette. A ow of 4 L min À1 of oxygen was fed into the ozone generator (model LAB2B from Ozonia), producing 9.5 g of O 3 per h. Stirring was set to 1500 RPM. The reaction time varied from 20 to 120 min, while all other process parameters were kept constant. Aer each reaction, the oxidized mixture was ushed with air for around 2 minutes to remove residual ozone and ltered for the recovery of solids. Ethanol was removed by vacuum evaporation (2 h, 250 mbar and 40 C) to yield the nal lignin oil, which was weighted and analyzed in detail.
Since the evaluated lignins have very low solubility in ethanol and the solvent participates in the ozonation to some extent (vide infra), the amounts of dissolved lignin and mass incorporation were calculated based on the solids recovered aer each reaction (here called insoluble lignin). Hence, these solids were considered unreacted lignin and provided an indication of how much of the initial lignin was extracted to the solvent during ozonation (i.e. dissolved lignin). By knowing the amount of dissolved lignin, the mass incorporation could be quantied as well (eqn (1) and (2)). For a better comparison between the experiments, an incorporation ratio (IR) factor was dened (eqn (3)). The reader is referred to the ESI † for an example of calculations using the data obtained from a typical ozonation experiment.
CAUTION: Ozone is a highly reactive and toxic gas. Chronic exposure and exposure to high concentrations of ozone may cause respiratory difficulties and eye irritation. The apparatus described in this report should only be operated with proper ventilation in a fume hood. Generation of ozone requires a highvoltage discharge, therefore care is advised to isolate the highvoltage components of the apparatus from any nearby solvents.
Feed and lignin oil analyses
Prior to the ozonation experiments, the four lignin feeds were characterized by GPC (weight average molecular weight, M w ), HSQC NMR (linking motifs as well as the aromatic unit composition syringyl/guaiacyl/p-hydroxybenzene S/G/H ratio), 13 C-NMR (overall chemical makeup of carbons), 31 P-NMR (hydroxyl types and content), elemental analysis (C, H, N, S) and DLS (particle size). The chemical composition of the ozonated lignin oils was also assessed by a series of techniques; GPC (M w and molecular weight distribution), TGA (charring tendency and volatility), GC-MS (identication of thermally stable monomers), HPLC (carboxylic acids identication and quantication), HSQC NMR and 13 C-NMR (structural features), Karl Fischer analysis (water content) and elemental composition (C, H, N, S elemental analyzer). pH measurements of the ethanol and KL mixtures before and aer ozonation (diluted 50 wt% in water) were performed using a 848 Titrino plus apparatus from Metrohm.
GPC analyses of the feed and products were performed using an Agilent HPLC 1100 system equipped with a refractive index detector. Three columns in series of MIXED type E (length 300 mm, i.d. 7.5 mm) were used. Polystyrene standards were used for calibration. 0.05 g of the sample was dissolved in 4 mL of THF together with 2 drops of toluene as the external reference and ltered (lter pore size 0.45 mm) before injection.
Thermogravimetric analyses (TGA) were performed using a TGA 7 from PerkinElmer. The samples were heated under a nitrogen atmosphere (nitrogen ow of 50 mL min À1 ), with heating rate of 10 C min À1 and temperature ramp of 30-900 C.
Gas chromatography/mass spectrometry (GC-MS) analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a RTX-1701 capillary column (30 m  0.25 mm i.d. and 0.25 mm lm thickness) and a Quadrupole Hewlett-Packard 6890 MSD selective detector attached. Helium was used as carrier gas (ow rate of 2 mL min À1 ). The injector temperature was set to 280 C. The oven temperature was kept at 40 C for 5 minutes, then increased to 250 C at a rate of 3 C min À1 and held at 250 C for 5 minutes. All samples were diluted 10 times with THF.
The HPLC analytical device used for carboxylic acids iden-tication and quantication consisted of an Agilent 1200 pump, a Bio-Rad organic acids column Aminex HPX-87H, a Waters 410 differential refractive index detector and a UV detector. The mobile phase was 5 mM aqueous sulfuric acid at a ow rate of 0.55 mL min À1 . The HPLC column was operated at 60 C. Since the products were not fully soluble in water, a water extraction step (proportion of 1 : 10 of ozonated lignin and water) was needed, and the aqueous phase was further analyzed. Calibration curves of the targeted acids were determined experimentally to provide an accurate quantication and were based on a minimum of 4 data points with excellent linear tting (i.e. R 2 > 0.99).
Heteronuclear single quantum coherence (HSQC) NMR spectra were acquired on a Bruker NMR spectrometer (600 MHz) with the following parameters: 11 ppm sweep width in F2 ( 1 H), 220 ppm sweep width in F1 ( 13 C), 8 scans and a total acquisition time of around 1 h. Sample preparation involved the dissolution of the sample in DMSO-d 6 (15 wt%). Spectra were processed and analyzed using MestReNova soware, refer to the ESI † for integration details. 13 C-NMR spectra were acquired on a Bruker NMR spectrometer (600 MHz) using a 90 pulse and an inverse-gated decoupling sequence with relaxation delay of 5 seconds, sweep width of 220 ppm and 2048 scans, with a total acquisition time of 3.5 h and TMS as reference. Sample preparation involved the dissolution of the sample in DMSO-d 6 (15 wt%). Spectra were processed and analyzed using MestReNova soware, refer to the ESI † for integration details. Hydroxyl content analyses were performed using 31 P-NMR following a procedure described elsewhere, 7 using cyclohexanol as the internal standard and CDCl 3 as solvent. 31 P-NMR spectra were acquired on a Bruker NMR spectrometer (600 MHz) at 293 K using a standard 90 pulse, 256 scans and 5 s of relaxation delay. Spectra were processed and analyzed using MestReNova soware, refer to the ESI † for integration details.
The water content was determined by Karl Fischer titration using a Metrohm 702 SM Titrino titration device. About 0.01 g of sample was injected in an isolated glass chamber containing Hydranal (Karl Fischer solvent, Riedel de Haen). The titrations were carried out using the Karl Fischer titrant Composit 5K (Riedel de Haen). All analyses were performed at least 3 times and the average value is reported.
Elemental analysis (C, H, N, S) was performed using a Euro-Vector EA3400 Series CHN-O analyzer with acetanilide as the reference. The oxygen content was determined by difference. All analyses were carried out at least in duplicate and the average value is reported.
Dynamic light scattering (DLS) measurements were performed using a Brookhaven Zeta-PALS analyzer equipped with a 35 mW red diode laser (nominal wavelength of 660 nm), reading at a measurement angle of 90 . The measurements were carried out on aqueous colloidal solutions (1 wt% of lignin concentration) at 25 C. The mean effective diameter of each lignin sample (i.e. particle size) was obtained aer 10 measurements assuming that the particles are spherically shaped.
Results and discussion
Characterization of the lignin feeds Table 1 shows relevant properties of the four lignins used in the ozonation experiments, which were characterized in detail by GPC, NMR techniques, elemental analysis and DLS. These lignins can be separated in two subsets, i.e. a subset comprised of sowood derived G-based Kra lignins (KL and ball-milled KL) and a subset comprised of mixed S/G-based organosolv lignins (Alcell and Fabiola). Apart from the differences in monomer unit composition, KL and ball-milled KL also present relatively higher contents of sulphur and nitrogen as well as a lower M w . The rst subset provides a comparison regarding the average particle size (i.e. 1080 nm versus 460 nm). Besides the slightly lower M w of the ball-milled KL, no signicant differences were observed between the properties of KL and ball-milled KL, showing that the conditions applied in the ballmilling step prevented major structural changes. Furthermore, the low b-O-4 content of KL likely did not allow for further lignin degradation, as specically these linkages were shown to be prone to cleavage during ball-milling. 64,65 The second subset compares two lignins which mainly have a different level of condensation. Accordingly, the Fabiola lignin used in this study is less condensed than the Alcell lignin due to the milder conditions applied in the organosolv process. 63 This is conrmed by its higher oxygen content, higher amount of C-O linkages (mainly b-O-4, in line with the higher amount of aliphatic OH provided by 31 P-NMR) and lower amount of S condensed units shown by HSQC NMR analysis. Furthermore, 13 C-NMR results shows signicantly less aliphatic C-H motifs in the Fabiola lignin and relatively more methoxy groups attached to aromatic rings, also suggesting a structure that resembles more native lignin. In addition to that, the results from 31 P-NMR show a lower amount of phenolic OH groups (which are known to increase with lignin degradation 61,66,67 ) in this lignin.
Product yields and macromolecular properties
To demonstrate the reactivity of ozone towards these technical lignins, each was exposed to ozone for different times (20 min to up to 60 or 120 min, Fig. 3). All experiments were performed under ambient conditions by bubbling ozone through a suspension of the lignin in ethanol. The experiment reacting KL with ozone for 20 min was performed in triplicate to investigate reproducibility and showed good results (standard deviation of 0.7% on the measured weight of the different product fractions). Clear visual changes were observed during the experiments, indicating promising reactivity at these mild, non-catalytic reaction conditions. For instance, all the lignins initially present low solubility in ethanol (varying between 4-33 wt%, see blank runs without ozone in Fig. 3) which increased dramatically upon ozone exposure, and the ozonated solutions turned orange-red. The lignin oils aer ethanol removal show a clear difference from the starting dark brown solid lignins, being a low viscosity oil with an orange-red color (see Fig. S2 † for an example). Accordingly, the lighter color is related to the decrease of conjugated aromatic structures and thus indicates reactivity at unsaturations. 68 Fig . 3 also shows that the lignin oil yields signicantly surpass the amount of dissolved lignin aer ozonation. Such mass increase is a direct result of oxygen incorporation into the structure and also suggests that the solvent participates in some reaction pathways. For instance, solvent incorporation was previously observed when using methanol as the solvent. 59 In a similar fashion, ethanol may be incorporated via ester and acetal formation reactions of the acids and aldehydes/ketones respectively, which are all formed during ozonation (vide infra). Furthermore, ethanol might also serve as a capping agent that stabilizes highly reactive (oxidized) phenolic intermediates by O-alkylation of hydroxyl groups. 69,70 Average incorporation ratios of 2.0, 1.9, 1.8 and 2.1 were observed for KL, ball milled KL, Alcell and Fabiola lignin, respectively. This indicates that for a determined amount of lignin solubilized during ozonation, the amount of lignin oil aer ethanol removal will be approximately doubled.
Since signicant mass incorporation occurs, precise mass balances are obscured and both the yields of lignin oil and insoluble lignin have to be evaluated. For instance, the yields of lignin oil showed an overall increase with longer ozonation times, and results varied distinctly among the lignins (Fig. 3, O3 entries). For the KL, ball-milled KL and Alcell lignins, the high yields of lignin oil were accompanied by desirable low amounts of insoluble lignin aer ozonation. Alcell lignin showed the least solid residue (13 wt%) and thus highest dissolved lignin which might relate to its relatively higher initial solubility in ethanol, being itself an ethanol/water extracted lignin. As the lignins are nearly insoluble in ethanol, mass transfer limitations of ozone from the gas to the liquid phase and subsequent transfer to the solid lignin particles play an important role in this system and may determine the overall rate of the reactions. The use of ball-milled KL with a signicantly smaller particle size led to a slightly higher initial lignin solubility and indeed an overall increased solvation and less insoluble residual lignin compared to KL. This is likely due to better dispersion and a higher surface area, facilitating reactions to take place. It is expected that optimized set-ups with proper attention to mass transfer issues (e.g. intense mechanical stirring using overhead, self-inducing expellers) may lead to higher lignin oil yields at shorter reaction times.
For the Fabiola lignin, a plateau for the amounts of dissolved lignin was observed from 20 to 60 minutes of reaction time, in which relatively low yields of lignin oil and high amounts of insoluble lignin (i.e. around 50 wt%) were observed. This surprising result is likely related to the structural features of this lignin. For instance, the milder organosolv extraction procedure used for this lignin (compared to the Alcell process) results in a structure more similar to native lignin, 71,72 i.e. less condensed and with a higher amount of C-O bonds and methoxy groups (vide supra, Table 1). Literature shows that C-O linked lignins are usually easier to depolymerize, as C-O bonds are more labile when compared to C-C bonds. [73][74][75] Nonetheless, the apparently contradictory results here observed can be explained by specic reactivity trends of ozone. A recent publication from our group showed that the b-O-4 linking motifs are relatively resistant to ozone attack, and that the major inuence for lignin disruption during ozonation particularly comes from available phenolic hydroxy groups. 59 Such phenolic groups are mostly the results of structural modications of native lignin during processing. 61,66,67,76 Furthermore, the presence of C-C double bonds (i.e. stilbene linkages) as reported in Kra lignin 61,77,78 increases its reactivity towards ozone due to the high electronic density of such moieties. 79,80 Hence, in the case of an ozone treatment, condensed structures as in technical lignins (i.e. Kra and Alcell lignins) are benecial for their reactivity and subsequent dissolution in ethanol, leading to the higher lignin oil yields observed.
GPC analyses were performed to compare the molecular weight distribution of the lignin feeds and their ozonated lignin oils (Fig. 4). The results clearly show that the lignin fragments being solubilized during ozonation are substantially lower in molecular weight compared to the initial lignin feed, indicating that ozone simultaneously breaks down the structure and increases its solubility in ethanol. Furthermore, repolymerization pathways usually observed in oxidative processes due to the formation of radicals 30,81 seem to be suppressed as no molecular weight increase was observed at elongated reaction times. This positive observation is likely a result of the acidic reaction medium and solvent system used, as ethanol is reportedly an efficient radical scavenger able to quench reactive lignin fragments 70,82,83 under such conditions. Accordingly, while the mixture of ethanol and KL before reaction was slightly acidic (average pH of 6, standard error of 0.1), the lignin oil solution aer ozonation for 20 minutes had a substantially higher acidity (average pH of 2.3, standard error of 0.1). Lignin-solvent reactions may also play a role on inhibiting competitive repolymerization pathways. 48 For most cases, increasing the reaction time from 20 to 60 minutes led to an increase of the lower M w fractions due to the subsequent oxidation of the lignin fragments solubilized in ethanol, which is known to happen at extended ozonation times. 54,55,59 The amounts of these fractions were greatly increased at the longer reaction times of 120 minutes (KL and ball-milled KL), likely accompanied by carbon loss due to CO 2 formation from over-oxidation. 84 As set-up limitations hindered an accurate analyses of the gaseous products, full mass and carbon balances could not be determined. An overall decrease of 40-75% in the M w (based on the initial M w of each lignin) ratify the high activity of ozone towards lignin structures and that no substantial repolymerization of the solubilized ozonated fragments occurs.
TGA results are in line with GPC, showing a substantial increase in volatility due to depolymerization ( Fig. 5 and S3 †). Accordingly, the amounts of non-volatiles decreased by 60-80% (based on the initial TGA non-volatile residue of each lignin) with the ozone treatment, being of around 10 wt% aer ozonation. No substantial variation on the TGA residue was observed under the different reaction times applied. Furthermore, the temperature of maximum mass loss shied from around 350-400 C to values lower than 250 C. These results ratify the extensive depolymerization of lignin and show that the smaller fragments produced are also more volatile and thermally stable, since no substantial repolymerization was observed under the high temperatures applied during TGA. Such lowered charring tendency is of great interest for fuel and fuel additives applications. 55,85 Elemental analyses showed large differences in the composition of the lignin oils when compared to their former lignins. Results are displayed in a Van Krevelen plot, see Fig. 6. For instance, the O/C ratio increase is expected as more oxygen is incorporated into the lignin structure, and the H/C ratio increase is likely a result of ethanol incorporation via esterication reactions (which are accompanied by water formation). Despite possible water losses during ethanol removal, positive correlations could be observed when plotting the H/C molar ratios versus the water contents determined for the lignin oils (Fig. S4 †). Having the Alcell data as reference, an estimated value of 36 wt% of the total mass incorporation aer ozonation for 60 minutes could be attributed to the incorporation of ethanol via esters and acetals.
Lignin oil composition and structural transformations
Chromatographic analyses were performed on the lignin oils to identify and quantify low molecular weight products. GC-MS qualitative analyses show an extensive formation of (di)ethyl esters in the volatile and thermally stable fraction (Fig. 7). This conrms that ethanol is incorporated in the products mainly through esterication reactions of the (di)carboxylic acids produced, being these a result of ring-opening reactions largely favored by ozone. 54,55 Such observations are in line with elemental analyses results (vide supra) and with a previous study from our group, in which (di)methyl esters were identied aer exposing a solution of pyrolytic lignin and methanol to ozone. 59 Acetals and aromatic oxidation products were also identied, e.g. vanillic acid, ethyl vanillate and hydroquinone.
Through an extraction of the product oil with water and subsequent HPLC analyses of the obtained aqueous phase, a range of (di)carboxylic acids could be identied and quantied ( Fig. 8 and Table S3 †). Importantly, as the HPLC analyses were performed under hydrolysis conditions, it was not possible to quantify acids and esters separately. Overall, the yields of these organic acids increased with ozonation time and reached up to 40 wt% of the lignin oil. Minor amounts of formic and acetic acid arise from the oxidation of ethanol. This was proven by blank ozonation experiments with ethanol (reaction times of 20/40/60 minutes) that yielded 0.9/1.2/1.4 wt% and 1.6/ 2.2/2.8 wt% of formic and acetic acid, respectively. The decrease in detected acids at longer reaction times (i.e. 120 minutes for the ball-milled KL and 60 minutes for the Fabiola lignin) might be related to the over-oxidation of small (di)carboxylic acids to CO 2 . 84 In general, the chromatographic analyses show that the low molecular weight products produced during ozonation are largely a result of lignin conversion into (di)carboxylic acids, which are esteried to their respective (di)ethyl esters due to the presence of ethanol in an acidic environment. In order to determine the chemical composition of the whole lignin oil rather than solely the lower molecular weight fraction, NMR analysis was used to further elucidate global structural transformations during ozonation. Representative 13 C-NMR spectra and the integration results of the lignin feeds and their lignin oils aer 60 minutes of ozonation show substantial structural differences ( Fig. 9 and 10). Regardless of the lignin used, products were richer in aliphatic bonds aer ozonation. While aliphatic C-O bonded carbons are a direct result of oxidation and ring opening reactions, the increase in C-H bonded carbons is related to the incorporation of ethanol within the structure. Aromatic carbons showed an overall decrease that varied between 40-55% depending on the lignin, and new carbon signals for carbonyl groups could be observed, being expected due to the formation of (di)carboxylic acids and esters. The substantial decrease in methoxy groups attached to aromatics is also an indication of ring-opening reactions. Importantly, while the distribution of bonds in the lignin oils is similar in all cases, variations in the yields depending on the lignin substrate used must be recalled in order to select an adequate feed for ozonation. When considering such aspects, the ball-milled Kra and Alcell lignins are the most promising options, as a large fraction of them (i.e. 71-87 wt%) was solubilized by the ozone mediated depolymerization under the applied conditions (vide supra).
Qualitative results provided by HSQC NMR ratify the aromatic disruption, as well the disappearance of well-dened linkages typically found in lignin (i.e. b-b, b-5, b-O-4), 71 see Fig. 11 for a representative spectra of KL and its lignin oil (1 h ozonation) and Fig. S5-S7 † for the spectra of the lignin oils obtained from the other feeds. Overall, similar trends were observed that relate to the formation of ethyl esters, acetals and aliphatic chains containing carbonyl and hydroxyl groups. In addition, the aliphatic C-H region is less heterogeneous (as C-O linkages were formed) and contained just a few signals, which are mostly derived from ethanol incorporation. NMR and GC-MS analyses of a blank ozonation experiment with ethanol indicated the formation of some acids, esters and acetals (Fig. S8-S10 †) which can contribute to the observed signals and are in line with the results previously observed using methanol. 59 Fig . 12 illustrates the ndings of this work in a scheme. Overall, it was shown that technical lignins could be converted in high yields into ethanol-soluble fragments by an ozone mediated depolymerization operated under room conditions. The obtained lignin oils have signicant lower M w and a less aromatic character due to ring-opening reactions. A range of low molecular weight compounds was identied, particularly DCAs and their derived (di)ethyl esters. Both ethanol and ozone are incorporated to give esters and acetals as main functional groups in the product mixture. Various applications in the food and pharma industries are envisioned for the DCAs, 33 while esters can be used as high value fuels and additives. 55 Furthermore, there is great potential for obtaining lignin-based materials from ozonated fragments 58 and oxygenated aromatics. 86
Conclusions
The results presented here clearly show the potential of ozonation for breaking down recalcitrant technical lignins and promoting their solvation in ethanol, which is a recyclable and biobased solvent option with low toxicity. Accordingly, the Kra and organosolv lignins evaluated were converted under ambient conditions and without the need of catalysts. Lower particle sizes favored higher yields due to the increased contact areas and better dispersion of the lignin particles in the liquid medium. Furthermore, increased structural degradation of the native lignin structure prior to ozonation was shown to be benecial for the effective conversion of the lignin feed. This is likely due to the increased amounts of reactive phenolic motifs and C-C double bonds in comparison with native lignin. Both Kra and Alcell lignins were shown to be suitable feeds for the process, leading to high lignin oil yields containing up to 40 wt% of low molecular weight compounds. The depolymerized lignin fragments have a substantially lower M w (i.e. 40-75%) and much higher volatility compared with their former lignins. While ozonation favored phenol ringopening pathways that produce (di)carboxylic acids, ethanol was shown to suppress repolymerization pathways and incorporate within the lignin structure mostly as ethoxy groups in esters and acetals. Longer reaction times led to higher yields of small (di) carboxylic acids/esters, and likely the over-oxidation of some molecules to CO 2 . These results clearly show that ozonation is effective in converting recalcitrant technical lignins into ethanolsoluble fragments containing valuable monomers and oligomers with a more aliphatic character. Further in-depth studies of crucial process parameters (i.e. ozone input, residence time, stirring speed) may tune the product distribution towards the desired applications. Accordingly, the use of optimized set-ups can minimize mass transfer limitations, suppressing undesired secondary reactions in the liquid phase and providing a more efficient use of ozone. Within the biorenery concept, which aims to synergistically valorize all biomass fractions, lignin ozonation can serve both as a way to produce valuable oxygenated products (e.g. DCAs) and as a straightforward pretreatment to depolymerize, solubilize and make the lignin structure more accessible for further processing by strategies typically hampered by its recalcitrance (e.g. hydrotreatment). Due to the high ester content, H/C ratio and volatility, ozonation can be a starting point for converting technical lignins into oils suitable for applications as fuels and fuel additives.
Conflicts of interest
There are no conicts to declare. | v3-fos-license |
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