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} | pes2o/s2orc | Sphingolipid-Transporting Proteins as Cancer Therapeutic Targets
The understanding of the role of sphingolipid metabolism in cancer has tremendously increased in the past ten years. Many tumors are characterized by imbalances in sphingolipid metabolism. In many cases, disorders of sphingolipid metabolism are also likely to cause or at least promote cancer. In this review, sphingolipid transport proteins and the processes catalyzed by them are regarded as essential components of sphingolipid metabolism. There is much to suggest that these processes are often rate-limiting steps for metabolism of individual sphingolipid species and thus represent potential target structures for pharmaceutical anticancer research. Here, we summarize empirical and biochemical data on different proteins with key roles in sphingolipid transport and their potential role in cancer.
Introduction
Lipids are essentially defined by their insolubility in water. In particular, membrane lipids, such as sphingolipids, can be transported in biological systems only within one membrane or between two different membranes. Since many processes controlled by sphingolipids are separated spatially, the transport of these molecules is of particular importance. The most obvious example is the spatial separation of processes of de novo sphingolipid biosynthesis. In contrast to degradation of sphingolipids, which is mostly catalyzed by soluble enzymes, all enzymes involved in the biosynthesis of sphingolipids are membrane-bound. Starting with serine-plamitoyl transferase (SPT), ceramide is synthesized in four steps within the cytosolic leaflet of the ER membrane. The synthetic steps downstream of ceramide, leading to formation of sphingomyelin, glucosylceramide, and more complex glycosphingolipids (GSL) take place at different localizations of the Golgi apparatus (GA). Therefore, there is much to suggest that the transport of intermediates between the different membranes is an essential component of sphingolipid biosynthesis. In addition, previous studies have shown that the function and activity of individual sphingolipid species is dependent on their specific subcellular or extracellular locations. This suggests a closer examination of the underlying transport processes.
In classical medicinal chemistry, it is a recognized fact that, in particular, enzymes that catalyze the rate limiting steps are well-suited pharmacological targets. The characterization of enzymes and their conversion rates, however, is usually carried out in vitro in cell lysates or even with purified enzymes. It is therefore obvious that the importance of sphingolipid transport proteins as potential bottlenecks of sphingolipid metabolism has not yet been sufficiently appreciated. Given the importance of sphingolipid metabolism as a potential pharmacological target in cancer, in this review, we would like to highlight proteins that are specifically involved in sphingolipid transport even if, in view of their recent discovery, only limited epidemiological data is available.
Ceramide Transfer Protein (CERT)
The ceramide transport protein (CERT) is a protein that transports ceramides in a nonvesicular manner from endoplasmic reticulum to Golgi membranes, thereby influencing the metabolism, cellular concentration, and biological activity of this lipid [12]. When discovered, it was the first specific sphingolipid transport protein, making it a prototype for this type of transporter. Ceramide is a central component in sphingolipid metabolism and functions as a precursor for downstream sphingolipids and glycosphingolipids of higher complexity. Its concentration is controlled by more than 11 different enzymes [13]. It is synthesized in three separate pathways, which include sphingomyelin cleavage, de novo synthesis, and the salvage pathway [13]. The latter is a recycling pathway, fed by the degradation product sphingosine and by external sphingolipids. Ceramide has been implicated in several biological roles, particularly in the induction of apoptosis [13][14][15][16]. Ceramide levels are elevated following various triggers such as ultraviolet light, cytotoxic agents, ionizing radiation, or tumor necrosis factor alpha (TNF-α) [17]. In cancer, the lipid has been reported to be the main regulator of chemotherapy-induced cell death triggered by compounds, such as taxane, through induction of apoptosis [18]. Thus, several approaches to cancer chemotherapy are pharmacological manipulation of sphingolipid metabolism aiming to enhance cell ceramide as a proapoptotic molecule.
CERT was identified by functional rescue experiments, showing that it is responsible for trafficking of ceramide from endoplasmic reticulum to Golgi independent of vesicular transport, but dependent of ATP [12]. During de novo synthesis of the membrane lipid sphingomyelin, the most
Ceramide Transfer Protein (CERT)
The ceramide transport protein (CERT) is a protein that transports ceramides in a nonvesicular manner from endoplasmic reticulum to Golgi membranes, thereby influencing the metabolism, cellular concentration, and biological activity of this lipid [12]. When discovered, it was the first specific sphingolipid transport protein, making it a prototype for this type of transporter. Ceramide is a central component in sphingolipid metabolism and functions as a precursor for downstream sphingolipids and glycosphingolipids of higher complexity. Its concentration is controlled by more than 11 different enzymes [13]. It is synthesized in three separate pathways, which include sphingomyelin cleavage, de novo synthesis, and the salvage pathway [13]. The latter is a recycling pathway, fed by the degradation product sphingosine and by external sphingolipids. Ceramide has been implicated in several biological roles, particularly in the induction of apoptosis [13][14][15][16]. Ceramide levels are elevated following various triggers such as ultraviolet light, cytotoxic agents, ionizing radiation, or tumor necrosis factor alpha (TNF-α) [17]. In cancer, the lipid has been reported to be the main regulator of chemotherapy-induced cell death triggered by compounds, such as taxane, through induction of apoptosis [18]. Thus, several approaches to cancer chemotherapy are pharmacological manipulation of sphingolipid metabolism aiming to enhance cell ceramide as a proapoptotic molecule.
CERT was identified by functional rescue experiments, showing that it is responsible for trafficking of ceramide from endoplasmic reticulum to Golgi independent of vesicular transport, but dependent of ATP [12]. During de novo synthesis of the membrane lipid sphingomyelin, the most abundant sphingolipid, this intermembrane translocation is the rate-limiting step. CERT is derived from the COL4A3BP gene (as annotated), which encodes two alternatively spliced proteins, the 624 and 598 amino acid isoforms, mostly termed as CERTL and CERT. The shorter splicing isoform is identical to GPBPD∆26, which was originally termed Goodpasture antigen-binding protein (GPBP) [19]. CERT is ubiquitously present inside the cell. CERTL, its longer splice-variant is a nontypical serine/threonine kinase, which is partially secreted outside the cell, where it exists in solution or in a membrane-associated form [20]. Both isoforms are capable of transferring ceramide between cellular membranes [12,21]. The CERT protein is a soluble protein and comprises a set of characteristic domains and motifs: Starting from the amino terminus, a pleckstrin homology (PH) domain,~120 amino acids in length, mediates binding to phosphatidylinositol 4-phosphate (PI4P) and therefore directs the CERT to the PI4P enriched Golgi complex [12]. It has been shown that the transfer process itself is not ATP dependent, but it has been speculated that the ATP dependence in situ may be essential to keep PI4P in a phosphorylated form. The PH domain is followed by the so-called middle region,~250 amino acids in length, containing motifs that have been speculated to play a role in a potential homo-or hetero-oligomerization [19]. Furthermore, this region harbors the so-called FFAT motif, which docks the protein to the ER membrane via its interaction with the VAP protein (vesicle-associated membrane protein-associated protein) [22]. Finally, the C-terminus is formed by the START domain (steroidogenic acute regulatory protein), consisting of about 230 amino acids. The START domain is able and sufficient to bind and transfer ceramide in vitro, between donor and acceptor vesicles. The CERT protein is regulated by phosphorylation at multiple sites and phosphorylation generally attenuates the transfer process [23,24].
CERT is essential for embryogenesis. In CERT mutant embryos, sphingomyelin and plasma-membrane ceramides are reduced, while ceramide levels in ER and mitochondria are elevated, leading to severe mitochondrial dysfunction [25]. Interestingly, in the mutant embryos, the cell cycle was arrested, but without increased apoptosis, no growth arrest was reported in different cell lines with depleted CERT function. In contrast, CERT knock-out did not lead to ceramide accumulation in primary mouse embryonic fibroblasts (MEFs), neither in the ER nor in the mitochondria. However, higher concentrations of hexosylceramides have been described in these cells, suggesting a compensatory reaction of the cells to avoid high concentrations of cytotoxic ceramide in the ER. Nevertheless, substantial morphologic defects in the mitochondria and other organelles have been observed in the mutant MEFs.
CERT and Cancer
Swanton and colleagues showed that downregulation of CERT resulted in sensitization of tumor cells to various cytotoxic compounds by enhancing ER stress [26]. The authors used RNA interference (RNAi) screening in different tumor cell lines (MDA-MB-231/ breast cancer, HCT-116/colorectal cancer, and A549/non-small cell lung cancer) to evaluate all protein kinases and enzymes involved in ceramide metabolism, regarding their contribution to either resistance or sensitivity to chemotherapeutics such as paclitaxel. Paclitaxel belongs to the taxane group of anticancer drugs, which, through stabilization of microtubules, causes mitotic arrest and ultimately apoptosis. The authors identified a set of genes that confer resistance to paclitaxel, some of them being also implicated in mitotic spindle formation checkpoints. Knockdown of the related mRNAs potentiated the arrest of mitosis and induced initiation of endoplasmic reticulum stress, resulting in increased sensitivity to taxane. Among the different transcripts, COL4A3BP (CERT) triggered the most significant sensitization to taxane when it was knocked down. Indeed, CERT knock-down sensitized all types of cells to paclitaxel and additionally HCT-116 cells to 5-FU, MDA-MB-231 cells to doxorubicin, and A549 cells to cisplatin. Moreover, in drug-resistant cell lines as well as in ovarian cancer residual tumors after paclitaxel therapy, the authors identified an enhanced expression of CERT. Therefore, CERT could be a pharmacological target in cancers resistant to chemotherapy. Finally, HPA-12, a small-molecule inhibitor of CERT, was effective in elevating ER stress, supporting the hypothesis that ER stress is inversely correlated with CERT-mediated clearance of ceramide from the ER in the respective cell lines [26]. In another study, the same authors showed that among others COL4A3BP is a suitable marker for paclitaxel treatment outcome in therapy of triple-negative breast cancer [27]. Later, it turned out that ER stress induction is not the only contribution for CERT inhibition-mediated sensitization of cells to anticancer drugs, but also chromosomal instability (CIN) [28]. Furthermore, the sensitization of cells to chemotherapy following CERT depletion comes along with an increased expression of Lysosome-associated membrane protein 2 (LAMP2), which in turn leads to increased autophagosome-lysosome flux [28]. The authors provided evidence that the silencing of CERT resulted in decreased ceramide transport and thus increased levels of ceramides, even compared to paclitaxel-treated cells and the combination of CERT knock-down and paclitaxel treatment resulted in a synergistic elevation of the total ceramide levels.
Despite these results, the suitability of CERT as a potential pharmacological target molecule in cancer cells is not always given and the story is not as simple as it may seem. One study provided evidence that CERT expression in triple-negative breast cancer (TNBC) and metastatic prostate cancer was decreased [29]. CERT obviously contributes to cell survival or cell death in more than one manner and a lowering of CERT expression can also provide advantages to tumor cells. This may also match with a recent study, which revealed a novel function of CERT and CERTL in the classical innate immune response, suggesting CERTL's participation in apoptotic cell clearance [30]. CERT downregulation may be a way for the tumor cells to attenuate complement activation and thus evade the immune system.
CERT Inhibitors
The first and most prominent CERT inhibitor, HPA-12, was identified to inhibit ceramide trafficking prior to the identification of CERT [31]. It is structurally related to ceramide and shows potent cellular activity at concentrations between 0.1 to 2.5 µM (Figure 2). Since then, various syntheses and derivatives of HPA-12 have been published [30,32]. Very recently, the first not ceramide-related CERT inhibitor, HPCB-5, was identified by a virtual screening approach [33]. Its cellular potency is very similar to that of HPA-12. COL4A3BP is a suitable marker for paclitaxel treatment outcome in therapy of triple-negative breast cancer [27]. Later, it turned out that ER stress induction is not the only contribution for CERT inhibition-mediated sensitization of cells to anticancer drugs, but also chromosomal instability (CIN) [28]. Furthermore, the sensitization of cells to chemotherapy following CERT depletion comes along with an increased expression of Lysosome-associated membrane protein 2 (LAMP2), which in turn leads to increased autophagosome-lysosome flux [28]. The authors provided evidence that the silencing of CERT resulted in decreased ceramide transport and thus increased levels of ceramides, even compared to paclitaxel-treated cells and the combination of CERT knock-down and paclitaxel treatment resulted in a synergistic elevation of the total ceramide levels.
Despite these results, the suitability of CERT as a potential pharmacological target molecule in cancer cells is not always given and the story is not as simple as it may seem. One study provided evidence that CERT expression in triple-negative breast cancer (TNBC) and metastatic prostate cancer was decreased [29]. CERT obviously contributes to cell survival or cell death in more than one manner and a lowering of CERT expression can also provide advantages to tumor cells. This may also match with a recent study, which revealed a novel function of CERT and CERTL in the classical innate immune response, suggesting CERTL's participation in apoptotic cell clearance [30]. CERT downregulation may be a way for the tumor cells to attenuate complement activation and thus evade the immune system.
CERT Inhibitors
The first and most prominent CERT inhibitor, HPA-12, was identified to inhibit ceramide trafficking prior to the identification of CERT [31]. It is structurally related to ceramide and shows potent cellular activity at concentrations between 0.1 to 2.5 µM (Figure 2). Since then, various syntheses and derivatives of HPA-12 have been published [30,32]. Very recently, the first not ceramide-related CERT inhibitor, HPCB-5, was identified by a virtual screening approach [33]. Its cellular potency is very similar to that of HPA-12.
FAPP2
FAPP2 or human phosphatidyl-4-phosphate adapter protein 2 is a protein essential for trans-Golgi network (TGN) to plasma membrane vesicular transport of glucosylceramide [34]. The protein is encoded by the PLEKHA8 gene, giving rise to 519 amino acids. It comprises a pleckstrin homology
FAPP2
FAPP2 or human phosphatidyl-4-phosphate adapter protein 2 is a protein essential for trans-Golgi network (TGN) to plasma membrane vesicular transport of glucosylceramide [34]. The protein is encoded by the PLEKHA8 gene, giving rise to 519 amino acids. It comprises a pleckstrin homology (PH) domain with 93 residues with two specific binding sites for PI4P. The PH domain is linked by a stretch of~200 amino acids to the C-terminal glycolipid transfer protein homology (GLTPH) domain of 212 amino acids.
In accordance with the existence of the GLTPH domain, the protein is able to selectively transfer glucosylceramide (GlcCer) from cis Golgi to the TGN [34]. GlcCer is the precursor for the biosynthesis of more complex GSL, which takes place on the luminal sheet of late Golgi membranes. Recently, the structure of FAPP2 was resolved and provided insight into the binding mode [35]. The selectivity for the ceramide moiety was found to originate from highly conserved His and Asp residues of the recognition center that bind the amide group of ceramide. A group of four hydrophobic residues plays a sensor role in sphingolipid chain length determination. The recognition center, together with so-called ID loop elements, determine the sugar head-group specificity for substrate glycosphingolipids.
Indeed, knock down of FAPP2 leads to significantly reduced concentration of complex GSL. A more detailed study specified that FAPP2 is essential for the synthesis of glycosphingolipids of the globo-series, while the ganglioside GM3 and other gangliosides, which are also derived from GlcCer, but in earlier Golgi membranes like the cisternae, are synthesized independently of FAPP2 [36]. Still, many aspects of FAPP2 action and the underlying mechanisms await further clarification. Since the synthesis of the complex GSL is located at the luminal side of the Golgi, it is yet unknown how the trans-bilayer movement is facilitated. This could be mediated by a flippase enzyme, catalyzing the trans-bilayer flip-flop, but such a factor is still elusive [37]. An alternative model proposes the retrograde transport of GSL from Golgi to ER, where they can be wrapped into vesicles, which can then be transported anterograde and fuse with the Golgi [38]. However the latter model may not explain the differences seen for the synthesis of globosides and GM3. Moreover, it is still not finally clear whether the role of FAPP2 in TGN vesicle budding is a consequence of its limiting role in GSL synthesis. The fact that the Trp407Ala variant of FAPP2, which is devoid of GlcCer binding and glycolipid transfer activity, shows a diminished TGN to plasma membrane vesicular transport when expressed in FAPP2−/− cells, points in this direction [37]. On the other hand, FAPP2 has an independent ability to bend membranes and to form tubular structures in model membranes. This feature also seems to be essential for TGN vesicle budding. Another interesting hint for the importance of FAPP2 comes from its role in HCV infection [39]. A viral protein was shown to activate an ER-derived PI4-kinase, leading to elevated levels of PI4P at the HCV replication complex. As a result, FAPP2 is hijacked (via its PH domain) to transport GSL to the HCV replication complex. Knock-down of FAPP2, in contrast, led to reduced viral replication and infectivity.
FAPP2 in Cancer
The data for a role of FAPP2 in cancer is rather limited. An early study described induction of apoptosis in colon cancer carcinoma cells after incubation with ribozymes targeting FAPP2 in the presence of Fas agonistic antibody [40].
In a recent publication, FAPP2 was remarkably upregulated in colon cancer samples compared to adjacent tissues in 52 out of 90 patients. In 20 patients, FAPP2 was unchanged and in 18 patients, FAPP2 was downregulated [41]. In FAPP2 upregulated cancer tissues, disruption of FAPP2 by CRISPR/Cas9 led to attenuated cell growth and colony formation. The tumorgenicity of FAPP2 downregulated xenografts in a nude mice model was also decreased. Further mechanistic evaluation showed that FAPP2 affects Wnt/beta-catenin signaling [41]. However, further research will be needed to confirm the potential of FAPP2 as a target for cancer therapy.
FAPP2 Inhibitors
Inhibitors of FAPP2 have so far only been described in one patent [42]. The compound Phlorizin leads to a notable decrease of the FAPP2 transfer activity. TAK-875, a selective agonist of GPR40 (Free fatty acid receptor 1), was identified as a FAPP2 inhibitor (Figure 2). Since FAPP2 downregulation leads to a concomitant decrease in glycosphingolipids, such inhibitors may be promising tools for substrate deprivation therapies in glycosphingolipid storage diseases [43].
Glycolipid Transfer Protein (GLTP)
Glycolipid transfer protein (GLTP) is 24 kDa soluble cytosolic lipid binding protein with a putative role in nonvesicular glycolipid trafficking. In vitro it has been shown to catalyze intermembrane translocation of various glycolipids and glycosphingolipids (GSLs) [44,45]. GLTP's in vitro functional properties and its structural features are subject of excellent reviews [46]. The X-ray crystal structures of GLTP with and without bound substrates have been resolved and revealed yet unknown folding motifs. Due to its novel architecture, GLTP has been considered the prototype of a new family of lipid-binding proteins [46,47]. The protein adopts a previously unknown all α-helical structure, which is arranged in a 'sandwich motif' to form a single glycolipid-binding pocket [45]. GLTP binds both ceramide-derived glycosphingolipids as well as glycerol-based glycolipids. However, the mono-or oligosaccharide-part must be linked to the lipid backbone via a beta-glycosidic bond. The rate-limiting step in GLTP-mediated transfer reactions was found to be the formation of the GLTP-substrate complex and its release from the donor membrane, which is consistent with a shuttle/carrier mode of action [48]. GLTP has no PH domain and its in vivo target membranes are uncertain. The high affinity of GLTP for GlcCer on the one side, together with a low affinity for membranes on the other side, suggests that GLTP may also function as a dynamic reservoir for GlcCer which could deliver its cargo when and where it is needed. In vitro experiments showed that the uptake of glycolipids by GLTP is a function of the molar fraction of the lipid within the donor membrane. It is likely that also in vivo the donor and acceptor membranes will be defined according to this principle [49].
While orthologous forms of GLTP in plants and fungi play a role in programmed cell death, the function of the protein in mammals is largely unclear [50,51]. At least, the depletion or overexpression of GLTP failed to induce apoptosis in mammalian cell lines, in agreement with earlier GLTP overexpression data [52]. However, when human GLTP was overexpressed in HeLa or HEK-293 cells, significant alterations in cell shape were observed [53]. In contrast to the tubule-forming activity of FAPP2, this feature depends on the presence of a functional glycolipid binding site [54]. Overexpression of GLTP increases glucosylceramide (GlcCer) and globo series (Gb3) content, while RNAi-mediated knock-down of GLTP leads to reduced Gb3 concentration in HeLa cells [50].
In cells with elevated levels of GlcCer, the GLTP expression was upregulated, while a decrease in GlcCer concentration, caused by glucosylceramide synthase knock-down, resulted in significantly reduced GLTP expression levels. Therefore, GLTP has been suggested to act as a sensor for newly synthesized glucosylceramide (GlcCer) with a putative regulatory role in interorganelle glycosphingolipid redistribution [50,54].
GLTP and Cancer
GSLs and other glycolipids play important roles in adhesion processes at the cell surface [55] as well as in neurodegeneration and cell death. Especially in colon cancer, initiation and progression is strongly associated with altered levels in glycosphingolipids [56]. Due to its proposed role as molecular transporter of GlcCer and other glycosphingolipids to the plasma membrane [57], and due to its potency to induce changes in cell shape changes, GLTP appears to be involved in progression and malignancy of cancer.
A functional study on miR-196B, a microRNA upregulated in colon cancer, showed its ability to suppress FAS-mediated apoptosis [58]. Later, three direct targets of miR-196B were identified and validated: HOXA5, HOXB6, and GLTP. Indeed, a comparison with adjacent nontumorous tissues showed that GLTP protein expression was lowered in colorectal cancer tissues [59]. Moreover, very recently, the overexpression of human GLTP was shown to induce cell death via necroptosis in certain colon cancer cells [60]. GLTP overexpression resulted in cell cycle arrest and a dormant state for HCT-116 cells and normal colonic cells. These cells showed slightly elevated ceramide and unchanged S1P levels. In HT-29 cells, the same treatment resulted in a cell cycle arrest, which was accompanied by only moderately lowered ceramide levels together with a drastic reduction in S1P levels and finally a shift in the so-called 'sphingolipid-rheostat' leading to necroptotic cell death [60]. Moreover, it was found that the resistance of cells to undergo cell death upon GLTP overexpression [53] was associated with low expression levels of receptor-interacting protein kinase (RIPK-3) [60,61].
GLTP Inhibitors
To the best of our knowledge, no small molecule inhibitors of GLTP are known.
Ceramide-1-Phosphate Transport Protein (CPTP)
CPTP was originally termed GLTP1, due to its GLTP-related structure [51]. Later, it was found that instead of glycolipids the protein is specific for ceramide-1-phosphate and it was renamed to ceramide-1-phosphate transfer protein, CPTP [62]. The structural similarity of CPTP to GLTP was confirmed by X-ray structural analysis. CPTP contains a positively charged surface pocket. The C1P head group is recognized by positively charged amino acids and the K60A or R106L variants of the protein show virtually no C1P transfer activity [62]. The glycerol-lipid analog of C1P, phosphatidic acid, is not transferred, but the hydrophobic pocket suggests acceptance of a wide range of different chain lengths. The protein is mainly localized to the cytosol, but is also found associated with perinuclear membranes, the plasma membrane, and in the nucleus. Knock down of the CTPT by RNAi elevates intracellular levels of C1P of different chain lengths mainly in the TGN and leads to partial fragmentation of the Golgi apparatus and these effects were not rescued by overexpression of mutants devoid of transfer activity. It is likely that CPTP transfers C1P, after its synthesis in the TGN, to the plasma membrane and other compartments. However, the structural analysis of the protein did not reveal any domains for targeted recognition, such as a PH domain [62]. It is thus still unclear whether the protein can mediate a directional transfer of C1P. However, recently, it was shown that the protein and its Arabidopsis orthologue ACD11 are directly and specifically stimulated by phosphatidyl serine (PtdSer), which provides evidence for a head-group-specific interaction site on the protein's surface [63].
CPTP and Cancer
Ceramide-1-phosphate, C1P, has opposite activity to ceramide and acts as a mitogenic and prosurvival lipid [64]. Intracellularly produced C1P is also a mediator of inflammatory processes and via PLA2 stimulation and increased production of eicosanoids, thereby contributing to chronic inflammation. In addition, extracellular C1P has been reported to stimulate motility of cells, including pancreatic cancer stem cells [65]. In their initial study, Brown and colleagues showed that siRNA-mediated downregulation of CPTP increased cellular C1P, while-obviously as a result of concomitantly decreased plasma membrane C1P-the phospholipase A2 (PLA2)-mediated release of arachidonic acid and downstream eicosanoids is stimulated. Along this line, the Arabidopsis orthologue of CPTP, ACD11 (accelerated cell death 11) was shown to elevate prodeath sphingolipids when mutated [66].
Therefore, CPTP is regarded as a potential biomarker in cancer [67]. A more recent study showed that CPTP is a regulator of autophagy and inflammasome activation [68]. A knockdown of CPTP induced autophagy in epithelial cells by up to ten-fold and activated Caspase1 and elevation of cytokines.
Recently, it was shown that CPTP is a direct target of miR-328 [69]. Upregulation or delivery of this miRNA and thus downregulation of CPTP sensitizes non-small cell lung cancer cells to radiotherapy or colorectal cancer cells to chemotherapy. Also, a recent meeting abstract suggests that CPTP (and GLTP) regulates neoplastic progression of colon and breast cancer cells [70]. It seems that CPTP expression levels are often altered in various cancer biopsies, but a conclusive study in this direction is still elusive and more solid evidence for CPTP being a promising anticancer target is needed.
CPTP Inhibitors
There are currently no known small molecule inhibitors of CPTP.
Sphingosine-1-Phosphate Transporters
Sphingosine-1-phosphate is a potent bioactive molecule involved in signaling events. It is produced inside cells from sphingosine by sphingosine kinase 1 or 2 (SphK1/2). After its transport to the outer leaflet of the plasma membrane, it can act in an autocrine or paracrine manner on a number of specific S1P receptors (S1PR1-5), which are G-protein coupled receptors of the plasma membrane. By binding to these receptors, S1P can trigger a plethora of downstream biological effects that are "prosurvival" such as cell migration. S1P signaling via S1PRs is involved in many pathologies, including autoimmune diseases and cancer [71,72]. For this so-called "inside-out signaling", there must be defined mechanisms of transport of S1P outside the cells. Indeed, it is a well-established fact that blood and body fluids display high concentrations of S1P, in contrast to low levels in tissues. Besides the ABC transporters, which have been reported to play some role in S1P evasion of cells, there have recently been two distinct specific S1P transporters described, SPNS2 [73] and Mfsd2b [74]. Results from knock-out mice suggest that each of the transporters accounts for roughly 50% of plasma S1P levels. In contrast, mice with genetic knock-outs for different ABC transporters did not show altered S1P levels. While Mfsd2b is highly abundant in the plasma membranes of red blood cells, platelets, and in spleen and bone marrow [74], SPNS2 is expressed mainly in epithelial cells and in other cells and tissues, except bone marrow and erythrocytes and platelets [73]. Based on the current knowledge, each transporter is discussed individually.
Spinster Homolog 2 (SPNS2)
Spinster homolog 2 is a member of a large superfamily of non-ATP-dependent organic ion transporters. SPNS2 was discovered as a S1P transporter from Zebrafish studies [75]. A mutation in this gene caused a block in the migration of cardiac precursor cells and a split heart phenotype, which mimicked the phenotype of an S1P-receptor 2 (S1PR2) knock-out. In the following, it was shown that overexpression of SPNS2 increased plasma S1P and dihydrosphinganine-1-P levels, but not levels of sphingosine. Downregulation conversely led to decreases in the plasma levels of S1P. In contrast to zebrafish, knock-out mice are viable and appear normal, except organ-specific defects like deafness or vascular and retinal neurologic defects probably due to defects in the migration of neurons during retina development. These defects were observed in global S1P knock-outs, but not if the knock-out was specific for different tissues. This clearly shows that SPNS2 has a dual role in regulating systemic but also local and tissue specific S1P levels. As mentioned above, the global SPNS2 knock-out reduced the plasma S1P to about 60% and to the same extent as endothelial cell specific knock-out. Interestingly, SPNS2 systemic or endothelial cell-specific knock-out lead to lymphopenia [76], in contrast to the Mfsd2b knock-out, which results in a similar decrease in plasma S1P. This indicates that S1P produced from endothelial cells has a specific role in regulation of lymphocyte trafficking [77].
SPNS2 and Cancer
Due to the power of S1P as a signaling molecule, the role of S1P transporters in cancer is worthy of in-depth investigation [78]. In a first study, SPNS2 was downregulated in a small cohort of human lung tumors and overexpression of SPNS2 in non-small cell lung carcinoma cells led to increased S1P secretion, decreased cell migration, and induction of apoptosis [79]. SPNS2 knock-down also led to decreased EGF-mediated invasion of HeLa cells [80]. Interestingly, in this study, which showed that the mitotic transition was dependent on SPNS2 and the S1PRs, the effects were not blocked by an anti-S1P antibody, suggesting that there might be endogenous secretion pathways for SPNS2 as well.
Apart from these results for SPNS2 in tumor cells, the most striking result highlights the importance of healthy tissue SPNS2 for metastasis. In a recent study, more than 800 randomly selected knock-out mice were tested for their susceptibility to metastatic colonization of the lung. Among all mice, those having the SPNS2 gene knock-out showed the lowest metastatic burden [81]. Interestingly, the low level of metastasis was not due to an inability of cancer cells to invade the lung, but instead due to an unfavorable environment for metastatic colonization in the lung. Despite the lymphopenia seen in the SPNS2 knock-out mice, the level of natural killer cells in the lungs was increased, together with the T cell activities of CD4+ and CD8+ T cells, which all contribute to reduced metastasis. These results strongly suggest targeting of SPNS2 as a strategy to reduce metastasis after surgical resection of tumors.
SPNS2 Inhibitors
To our knowledge, there are no known small molecule inhibitors of SPNS2.
Mfsd2b
Since the knock-out of SPNS2 resulted only in a reduction of plasma S1P to about 60%, it was clear that there must be another source for plasma S1P [78]. Very recently, Mfsd2b was identified as an ATP-dependent S1P transporter in erythrocytes and platelets [74,82]. Knock-down of this transporter reduced plasma S1P to about 50%. Therefore, it is now believed that SPNS2 from endothelial cells and Mfsd2b from red blood cells and platelets contribute to most if not all plasma S1P in equal portions, respectively.
Mfsd2b
Up to now, there is no direct data for the recently discovered Mfsd2b in cancer. However, the known role of S1P in cancer suggests that an investigation of this topic would be highly worthwhile. It is known that many tumors overexpress sphingosine kinase 1, but trials with PF-543, a potent inhibitor of this enzyme, were unsuccessful. In contrast, experimental therapy with Sphingomab, an S1P antibody which can "soak" plasma S1P, showed promising results in a mouse model of renal cell carcinoma. The antibody also shows suppression of lung metastasis, which matches the results from SPNS2 knock-out leading to decreased systemic or plasma S1P levels.
Mfsd2b Inhibitors
Mfsd2b was found to be expressed in the erythroid cell line MEDEP-E14. In these cells, Mfsd2b was inhibited by very high amounts of glyburide ( Figure 2). The amount of extracellular NBD-labeled S1P was reduced to 50% compared to untreated cells in the presence of 500 µM glyburide [82].
Conclusions
Sphingolipid transfer proteins and sphingolipid transporters are recently characterized key components for sphingolipid metabolism and function. They are components of a complicated and finely balanced network of metabolic processes that can only take place at specific sites within cells or even organelles. These proteins thus provide the cell with an additional level of regulation that can possibly also be addressed by pharmacological intervention. A simplified overview of this network with the transport proteins discussed here is shown in Figure 3. At present, many aspects of intracellular sphingolipid transport are still unclear. For example, there is clear evidence that members of the ABC transporter family of proteins such as MDR1 or p-glycoprotein also play an important role as flippases for GlcCer and sphingomyelin, but the actual in vivo mechanisms await further elucidation [83,84].
Although not much is known about individual proteins and their roles in cancer, the preliminary results obtained so far are highly promising. However, it must be emphasized once again that most-if not all-data come from preclinical studies. Therefore, future systematic clinical trials are required. A similar situation also exists for small molecule inhibitors, which are often barely potent and selective, if they exist at all. We therefore hope that this review article helps to highlight this promising group of proteins and to stimulate further investigations. We believe that the data so far, together with the undisputed importance of sphingolipids in cancer, justify this. | v3-fos-license |
2018-04-03T00:05:53.766Z | 2016-10-10T00:00:00.000 | 7904032 | {
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} | pes2o/s2orc | Optimization of Physiochemical Parameters during Bioremediation of Synthetic Dye by Marasmius cladophyllus UMAS MS8 Using Statistical Approach
In many industrial areas such as in food, pharmaceutical, cosmetic, printing, and textile, the use of synthetic dyes has been integral with products such as azo dye, anthrax, and dyestuffs. As such, these industries produce a lot of waste by-products that could contaminate the environment. Bioremediation, therefore, has become an important emerging technology due to its cost-sustainable, effective, natural approach to cleaning up contaminated groundwater and soil via the use of microorganisms. The use of microorganisms in bioremediation requires the optimisation of parameters used in cultivating the organism. Thus the aim of the work was to assess the degradation of Remazol Brilliant Blue R (RBBR) dye on soil using Plackett-Burman design by the basidiomycete, M. cladophyllus UMAS MS8. Biodegradation analyses were carried out on a soil spiked with RBBR and supplemented with rice husk as the fungus growth enhancer. A two-level Plackett-Burman design was used to screen the medium components for the effects on the decolourization of RBBR. For the analysis, eleven variables were selected and from these four parameters, dye concentration, yeast extract concentration, inoculum size, and incubation time, were found to be most effective to degrade RBBR with up to 91% RBBR removal in soil after 15 days.
Introduction
Synthetic dyes are chemicals that are important alternatives in many industries. Since 1856, synthetic dyes have been used as they are economical to produce and create brighter, more colour-fast, and easy applications. However, most industrial dye processes involve the dye solution to be released to the surrounding water and soils. It has been reported that about 10-15% of all dyestuff is directly lost to wastewater [1] because of inadequate treatment of wastewater and poor waste management. Thus, this will lead to dye contamination in soil and water bodies [2]. Azo, anthraquinone, and phthalocyanine are the most common groups of synthetic dyes used [3]. Remazol Brilliant Blue R (RBBR) is a compound that is normally used to analyse azo dye degradation in laboratory condition [4]. RBBR, or Reactive 19, is an anthraquinonebased dye [5] which is an important dye in textile industries. It also represents a class of toxic and recalcitrant organopollutants that has identical structure to some polycyclic aromatic hydrocarbons (PAH) [6]. RBBR, an anthraquinone-based dye, is not easily degraded even by bacteria due to the presence of the aromatic structure [7]. The chemical structure and specification of the RBBR dye are shown in Table 1 [5].
Among industrial effluents, wastewater from textile and dyestuff industries is one of the most difficult to be treated. This is because industrial textile dyes have been purposely designed to be highly resistant to washing, chemical agents, 2 The Scientific World Journal solvents, and environmental factors such as action by sunlight. The synthetic and complex aromatic molecular structures of synthetic dyes also make them more stable and difficult to be degraded by microbial attack [8].
Conventional wastewater treatment plants using activated sludge treatment are unable to sufficiently treat dye containing wastewater with up to 90% of reactive textile dyes still remaining after the treatment [9]. In another study done by Shaul et al. (1991) [10], 11 out of 18 azo dyes studied were found to pass through activated sludge process substantially untreated while another 4 azo dyes were just adsorbed onto the waste activated sludge. Only 3 dyes (Acid Orange 7, Acid Orange 8, and Acid Red 88) were biodegraded by the activated sludge process.
These environmental pollutants are the contaminants that enter the environment and cause adverse changes. The pollutants can be cleaned up but at a high cost to the people. Furthermore, there are several limitations in the use of physicochemical methods such as adsorption, coagulation, precipitation, filtration, and oxidation [11]. This is because those methods are not environmental friendly and cost competitive compared to bioremediation [12]. Bioremediation on the other hand, by using fungi or bacteria, is an alternative way of cleaning up pollutants [11].
White rot fungi are important and have been used to discover organisms that can degrade synthetic compounds. For example, lignin biodegradation of white rot fungi involves the degradation of aromatic xenobiotics, heterocyclic aromatic hydrocarbons, synthetic high polymers, chlorinated aromatic compounds, and various dyes which are all environmentally persistent pollutants [13]. In the paper and pulp industry, degradation of lignin is needed to eliminate lignin from the wastewater [14]. For the degradation of lignin, white rot fungi produce extracellular enzymes, namely, lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac), which are involved to effectively depolymerize lignin producing carbon dioxide and water [14,15]. Apart from that, white rot fungi have a high lignin degradation ability due to their strong oxidative activity and low substrate specificity of their ligninolytic enzymes [14]. Previous studies showed RBBR biodegradation by various fungi such as Phanerochaete chrysosporium and Irpex lacteus, [4,16]. Irpex lacteus is one of white rot fungi that have been reported to be one of the first bioremediation agents of soil [16].
Here we report on the bioremediation of RBBR by M. cladophyllus UMAS MS8, in soil and under aerobic conditions. Eleven parameters in the bioremediation process were tested with the aim to determine the optimal parameters. Statistical approaches utilizing Plackett-Burman design were applied to optimize the concentration of dye, concentration of yeast, and incubation time as variables that affected the bioremediation rate.
Microorganism Preparation and Culture Maintenance.
Marasmius cladophyllus UMAS MS8 was used in this study and obtained from Molecular Biology Laboratory, Universiti Malaysia Sarawak (UNIMAS). The fungus was grown on malt extract agar (MEA) for a period of 7 to 10 days at 29 ∘ C. Malt extract agar (MEA) was prepared by dissolving 33.6 gL −1 of malt extract in distilled water, with pH 5.6, and autoclaved for 1 h at 121 ∘ C. Stock fungus culture was maintained in 20% (v/v) glycerol stored at −20 ∘ C, in sterile distilled water and on plates of malt extract agar (MEA) medium added with 0.05% (w/v) chloramphenicol.
Soil and Substrate Preparation.
Top soil sample was used and oven-dried for 4 days at 70 ∘ C before being used in the experiment. The soil was sieved through 2.0 mm mesh sieve and the physicochemical properties were partially characterized. Results showed that the soil used was clay textured with pH of 4.73 (1 : 5 H 2 O) and contained organic carbon of 70.133 g kg −1 (w/w) glucose equivalent, a total nitrogen of 80.06 mg L −1 , and bioavailable P of 1.96 mg kg −1 . These indicated a ratio of C : N : P of 35.8 : 40.8 : 1 which was lower than the commonly used C : N : P ratio of 100 : 10 : 1.
The Scientific World Journal 3 The sterile soil was prepared by autoclaving three times continuously at 24-hour intervals for 55 min at 121 ∘ C.
Rice husk that was used as growth enhancer was airdried and large particles were removed. Sterile rice husk was prepared by autoclaving for 1 h at 121 ∘ C and the moisture content was determined by drying 3 g of rice husk in an oven for three days at 70 ∘ C. After three days, rice husk was weighted again until a constant mass was obtained. The total water loss of rice husk also was determined using the same equation as for the soil % of Moisture Content = (Weight of the air-dried RH (g)) − (weight of oven-dried RH (g)) Weight of the air-dried RH (g) × 100. (1)
Screening of RBBR Dye Degradation on Agar Medium and Bioremediation Experiments on Soil. Marasmius cladophyllus
UMAS M8 was screened for RBBR decolourization capability on malt extract agar (MEA) supplemented with 0.02%, 0.06%, and 0.1% (w/v) RBBR dye. The agar plates were inoculated with 1.0 cm 2 (in diameter) of mycelium plug from a 7-day-old fungal mycelium. The fungal isolate together with the control plate was prepared in triplicate and incubated for 3 to 15 days in the dark at room temperature [17]. Bioremediation experiments were performed in 50 mL of Erlenmeyer flasks with 6 g of dry soil that was homogenously spiked with 0.1% (w/v) RBBR solution. A 30% (w/w) rice husk was used and added onto the top of the soil. The spiked soil was individually treated with 1.0 cm 2 of fungal mycelial plug with 70% (w/v) water moisture content. Noninoculated flasks with the respective dye concentrations were used as controls. Each culture condition was prepared in triplicate and incubated in the dark at room temperature under static condition. Other parameters were standardized using the experimental design layout.
Screening of Important Nutrient Components Using
Plackett-Burman Design. Plackett-Burman design was used for screening the important medium components with respect to their main effects but not their interaction effects on RBBR decolourization. Plackett-Burman design provides an efficient and rapid method to screen and select ingredients with maximum number of variables [17,18]. A 12run Plackett-Burman design was applied to this study to evaluate eleven variables at two levels: maximum (+1) and minimum (−1). The design and levels of each variable are shown in Table 2 and all trials were performed in triplicate. The medium was formulated as per the design and the response was calculated as the rate of dye decolourization and expressed in percentage. Table 3 represented the list of ingredients and concentrations chosen of 12 screening experiments.
The main effect of each variable was determined with the following equation: where is variable main effect, + and − are the RBBR degradation percentages in trials where the independent variable ( ) was present in high and low concentrations, and is the number of trials divided by 2.
Experimental error was estimated by calculating the variance between the two dummy variables using following equation: where eff is the variance of the effect, is the effect for the dummy variable, and is the number of dummy variables used in the experiment.
The standard error (SE) of the effect was the square root of eff and the significance ( value) of the effect of each variable on phenol degradation was measured by Student's -test as follows: where ( ) is the effect of variable .
Residual RBBR Extraction and Quantification.
Residual RBBR extraction was carried out according to the procedure as described by Novotný et al. [16] with some modification, by using a multisolvent system (chloroform, methanol, distilled water: 1 : 1 : 1, v/v). Each of the solvents was added separately, followed by mixing and vigorous shaking of the soil samples. Then the flasks contents were sonicated for 15 min and filtered through filter paper. Chloroform was separated from the filtrate with the help of micropipette. The filtrate was centrifuged for 10 min at 10,000 rpm, then placed in an open glass petri dish, and allowed to evaporate at 100 ∘ C for 6-8 h. The residue was redissolved in 7 mL of ddh 2 O and centrifuged for 10 min at 10,000 rpm. The control, soil without the RBBR, underwent similar procedure. Residual RBBR dye was quantified by measuring the absorbance at 595 nm [16]. Change in colour was observed which provided the rate of biodegradation of dye [19]. Percentage of degraded RBBR dye was calculated using (5) as follows: Percentage of decolourisation (%) = Ac − As Ac × 100, where Ac is the absorbance at the maximum absorption wavelength of dye in the control flask at time, , and As is the absorbance at the maximum absorption wavelength of dye in the sample flask at time, [17,20].
4
The Scientific World Journal
Screening of Degradation Assay in Agar.
Marasmius cladophyllus UMAS MS8 was used to decolourize RBBR and evaluate the dye decolourization characteristics. This work was carried out on solid agar medium and observation showed that the fungus grew on MEA supplemented with RBBR dye. MEA supplemented with 0.1% (w/v) of RBBR showed complete decolourization with formation of halo on agar plate after 9 days of incubation with 1.0 cm 2 of mycelial plug ( Figure 1). Other results were also recorded and shown in Table 4. The fungal isolate together with the control plate was prepared in triplicate and incubated for a period of 3 to 15 days at 28 ∘ C. (Table 6) were calculated and showed that , , , , and had positive effects on RBBR dye degradation, whereas , , , , and had negative effects. This positive effect indicated that these parameters will increase the RBBR dye degradation by increasing their concentration from low to high level. The variable with confidence level of above 95% is considered as significant parameters. Values of greater than 0.1000 indicated that the model is not significant. However, in this case, the model -value of 22.44 implies that the model was significant since its value is largely greater than 0.1000. There is only a 0.08% chance that an -value this large could occur due to noise. It was clear that variables , , , and were the significant factors, while , with confidence levels below 95%, was considered as insignificant and thus was not selected.
Screening Using Plackett-Burman
The significant effects (factors and interactions) having value less than 0.05 were selected as it is statistically different from zero at the 95% confidence level. On the other hand, the insignificant effects (factors and interactions) having value higher than 0.05 were then excluded. Since the values of Prob > less than 0.0500 indicated that the model terms are significant, thus in this case , , , and are the significant model terms. However, for this present study, only three variables (dye concentration, yeast concentration, and incubation time) were chosen since they were having the least number of values. Moreover, for a better response surface method, it is advisable to have only three parameters.
The significance of the model examined by the determination coefficient ( 2 ) showed that more than 0.9492% of variance was attributable to the variables 29.03% of the total variance that cannot be explained by the model. The adjusted coefficient ( 2 adjusted) and predicted coefficient ( 2 predicted) were calculated to ensure the quality of fit of the polynomial model for RBBR bioremediation [24]. The 2 Pred of 0.7969 was in agreement with the 2 Adj of 0.90694. This indicated that the overall mean was a better predictor of the response than the current model. Adequate precision, which measures the signal to noise ratio, showed a value of 14.508 (>4 value is desirable) indicated an adequate signal. Thus, this model can be used to navigate the design space. The Pareto chart ( Figure 2) offers a convenient way to view the results obtained by Plackett-Burman design and the order of significance of the variable affecting RBBR degradation. The vertical line in the chart defines the 95% confidence level.
The experimental values of RBBR degradation and theoretical values as predicted by Plackett-Burman design model equation showed a close agreement for all the medium components (Table 8). Errors represent the deviation value Table 2.
between predicted and actual values and were calculated based on (6) as follows:
Conclusion
Based on Plackett-Burman design method, the work found that 4 (dye concentration, yeast extract concentration, inoculum size, and incubation time) out of the 11 factors investigated were the most effective factors on RBBR biodegradation in soil. However, only three variables (dye concentration, yeast extract concentration, and incubation time) were selected for further analysis since they have the least values. Therefore, the Plackett-Burman design used in this work provided an efficient and rapid method for screening and selecting ingredients with a minimal number of experiments. | v3-fos-license |
2020-02-29T14:05:06.946Z | 2020-02-25T00:00:00.000 | 211555035 | {
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} | pes2o/s2orc | Anticancer Potential of Raddeanin A, a Natural Triterpenoid Isolated from Anemone raddeana Regel.
Natural compounds extracted from plants have gained immense importance in the fight against cancer cells due to their lesser toxicity and potential therapeutic effects. Raddeanin A (RA), an oleanane type triterpenoid is a major compound isolated from Anemone raddeana Regel. As an anticancer agent, RA induces apoptosis, cell cycle arrest, inhibits invasion, migration and angiogenesis in malignant cell lines as well as in preclinical models. In this systemic review, the pharmacological effects of RA and its underlying molecular mechanisms were carefully analyzed and potential molecular targets have been highlighted. The apoptotic potential of RA can be mediated through the modulation of Bcl-2, Bax, caspase-3, caspase-8, caspase-9, cytochrome c and poly-ADP ribose polymerase (PARP) cleavage. PI3K/Akt signaling pathway serves as the major molecular target affected by RA. Furthermore, RA can block cell proliferation through inhibition of canonical Wnt/β-catenin signaling pathway in colorectal cancer cells. RA can also alter the activation of NF-κB and STAT3 signaling pathways to suppress invasion and metastasis. RA has also exhibited promising anticancer potential against drug resistant cancer cells and can enhance the anticancer effects of several chemotherapeutic agents. Overall, RA may function as a promising compound in combating cancer, although further in-depth study is required under clinical settings to validate its efficacy in cancer patients.
Introduction
Cancer is a disease which arises through uncontrolled cell division leading to the formation of a tumor, which metastasizes to other body parts through the lymphatic and circulatory systems [1]. According to latest reports, around 18.1 million people were affected worldwide from different types of cancer in 2018. Up to 9.1 million cancer deaths were reported in 2018 and an increase up to 20.3 million is expected in 2026 [2]. Asia had the highest cancer death rate (57.3%), followed by Europe and America at 20.3% and 14.4%, respectively. Most of the deaths were attributed to lung cancer (2.1 million) followed by breast cancer (2.1 million) and colorectal cancer (1.8 million) [3]. The mortality rate of cancer can be reduced by understanding the etiology of cancer through advancement of diagnosis techniques, prevention strategies and treatment [4]. Cancer therapies including surgery, radiotherapy, chemotherapy and molecular targeted therapy have stabilized cancer prevalence to some extent [5], radiotherapy, chemotherapy and molecular targeted therapy have stabilized cancer prevalence to some extent [5], but unfortunately, these therapeutic strategies have been found to be less effective due to late diagnosis, lack of selective therapeutics, high toxicity, and ability of cancer cells to develop resistance against available treatments [6]. Therefore, several attempts have been made to develop novel treatments in fighting cancer and to improve patient survival.
Along with various available treatments, the use of natural plant products as medicines provides a new horizon for the treatment of various types of diseases [7][8][9][10][11]. The source of about 60% of the available drugs are natural raw materials from plants [12][13][14][15][16][17]. Natural drugs being used in clinics such as paclitaxel, vinblastine and camptothecin have gained a considerable amount of importance due to their reduced side effects [18]. Saponins, particularly triterpenoid saponins, have been used in Traditional Chinese Medicine (TCM) to cure cancer malignancies, for instance, they target the metastatic, invasive and angiogenic potential of cancer cells [19][20][21]. Saponins are also reported to reduce the resistance of neoplastic cells against chemotherapeutic agents, which is one of the protective mechanisms of cancer cells [22,23]. The progression of cancer cells is primarily mediated through the activation of multiple signaling pathways including the deregulation of MAPK, JAK/STAT and PI3K/Akt pathways which support carcinogenesis [24][25][26][27].
Anemone raddeana Regel is a medicinal plant, recognized by its vernacular name as "Liangtoujian" in China. The distribution of this plant is not only limited to China, but throughout the globe, particularly in Russia, Korea, and Japan [28,29]. The rhizome of Anemone raddeana Regel is used in Chinese conventional therapies to cure rheumatism, arthritis, neuralgia, paralysis and other diseases [30][31][32]. The anemone herb contains oleanane triterpenoid saponins accompanied by lactones, alkaloids, saccharide, triterpenoids and fats [33,34]. In the past few years, various bioactive saponins compounds have been extracted from this plant, including RA, which has gained importance due to its anti-inflammatory, analgesic, and antitumor activities [33]. RA (C47H76O10) is an oleanane class triterpenoid saponin, isolated from the roots of Anemone raddeana, possessing a 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside as the sugar moiety ( Figure 1) [31]. The anticancer potential of RA is linked with the presence of sugar moieties and its carboxyl group (-COOH) at the C-28 position (Figure 1). The free carboxyl group plays an important role in the cytotoxic potential of RA. In addition, the presence of rhamnopyranose, glucopyranose and arabinopyranosyl groups can also act as important moieties to enhance its therapeutic potential. A study reported that the modification at C-28 through esterification can lead to the reduction of cytotoxic potential, thus further confirming the role of this carbon moiety in the biological activities A study reported that the modification at C-28 through esterification can lead to the reduction of cytotoxic potential, thus further confirming the role of this carbon moiety in the biological activities reported for RA [31]. Various studies has shown that RA possesses cytotoxic potential through inhibition of proliferation, invasion and induction of apoptosis in multiple human carcinogenic cells including breast cancer, hepatocellular carcinoma, gastric cancer, and non-small cell lung carcinoma cells [35][36][37][38]. In addition, RA has also shown inhibitory properties at low concentrations against histone deacetylases (HDACs), further suggesting its cytotoxicity against cancer cells [31]. Likewise, on account of its reported pharmacological safety, RA can be used in combination with other anti-cancer drugs to enhance the sensitivity against resistant tumor cells. In this review article, the cytotoxic and therapeutic potential of RA has been comprehensively analyzed.
Molecules 2020, 25, 1035 3 of 21 reported for RA [31]. Various studies has shown that RA possesses cytotoxic potential through inhibition of proliferation, invasion and induction of apoptosis in multiple human carcinogenic cells including breast cancer, hepatocellular carcinoma, gastric cancer, and non-small cell lung carcinoma cells [35][36][37][38]. In addition, RA has also shown inhibitory properties at low concentrations against histone deacetylases (HDACs), further suggesting its cytotoxicity against cancer cells [31]. Likewise, on account of its reported pharmacological safety, RA can be used in combination with other anticancer drugs to enhance the sensitivity against resistant tumor cells. In this review article, the cytotoxic and therapeutic potential of RA has been comprehensively analyzed.
RA treatment effectively inhibited the initiation and proliferation of colorectal cancer cells through the suppression of the canonical Wnt/β-catenin signaling pathway. The inhibition was conducted by inactivating the Wnt co-receptor LRP6 and phosphorylating GSK-3β, an activator of downstream target genes c-Myc and Cyclin D1 [45,73,74]. c-Myc activation links with the activation of pleiotropic transcription factor, cell cycle progression, proliferation, and metabolism. Furthermore, treatment with RA suppressed invasion and osteosarcomas through the modulation of NF-κB signaling pathways by targeting IκBα phosphorylation to attenuate transcriptional activity of the NF-κB signaling pathway. These mechanisms lead to the downregulation of downstream matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) proteins [37,52]. These proteins are associated with the invasive and migratory potential of cancer cells [37]. Another finding reveals that RA initiated apoptosis and inhibited metastasis in osteosarcoma cells by stimulating ROS levels to trigger JNK activation. RA also suppressed MAPK and ERK pathways involved in resistance of cell growth, metabolism, autophagy [75], induction of apoptosis [76,77], and invasion [37]. In addition, RA has been reported to modulate STAT3 transcription factors involved in the expression of a large number of genes which plays a role in many physiological processes such as development, differentiation, metabolism, immunity and cancer progression in osteosarcoma [78]. RA was found as a potential antiangiogenic candidate through the modulation of VEGF mediated phosphorylation of VEGFR2 and downstream protein kinases FAK, PLCγ1, JAK2, Src and Akt in colorectal cancer tumors [79].
Pharmacokinetics Studies
Due to the rapid dispersion and removal, RA exhibits a low bioavailability and concentration in rat plasma. This may be due to the high molecular mass (897.1 D) of RA and poor membrane permeability as a result of the hydrophilic sugars in the RA structure. Moreover, this compound suffers from a short half-life and less systematic exposure due to the vulnerability of fast and large scale biliary excretion through active transport, as RA belongs to the saponins family [80]. A simple, fast and sensitive high-performance liquid chromatography electro spray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was employed to characterize the presence of RA in rat plasma. In vivo studies were conducted using an RA concentration of 2 mg/kg administered orally and intravenously to S.D rats. With 2.04−6.52% precision, 70% of RA was recovered without any clear matrix effect. The analysis indicated that due to poor lipid solubility, RA depicted less systemic absorption and thereby less bioavailability of 0.295% in plasma samples. This is identical to nearly all saponins reported previously in literature [81].
Another study was conducted using 0.75 mg/kg intravenous and intraperitoneal administrations of RA in Sprague Dawley (S.D.) rats. The blood sample was investigated at different time intervals which extended over 24 h for RA and glycyrrhetinic acid (internal standard) and analyzed under negative electrospray ionization in multiple reaction monitoring (MRM) mode [28]. The study indicated that the absolute recovery of RA was greater than 90.3% with a retention time of 2.1 min. The apparent distribution volume of RA was 0.11 L/kg less than total body volume 0.67 L/kg reflecting that RA widely disperses in blood compartments as compared to extravascular tissues. A recent in vivo study was carried out on mice by an LC-MS/MS system [82]. All mice were fed a single oral dose of 1.5 mg/kg and a blood sample was taken at different time intervals. The plasma of samples was stored at −80 • C for analysis. The study reveals that maximum concentration of RA was absorbed quickly at time duration of 0.33 h. They found low bioavailability with highest concentration of 12.3 µg/L. This dose of RA was the same as found in previous studies. Furthermore, RA exhibited rapid removal with a half-life of 3.5 h and it couldn't be detected in the plasma after 6h. The pharmacokinetic study of RA and its tissue distribution was observed in mice. After oral administration, among gastro-intestinal organs, the highest concentration of RA was detected in the stomach, followed by the colon and caecum. However, after 4 h of oral administration, RA could not be detected in any gastrointestinal organ [83]. The rapid dispersion and removal of RA was in accordance with the low bioavailability and low concentrations in rat plasma [28]. These studies depicted the low bioavailability of RA in plasma, although proper routes of administration are yet to be determined.
Role of RA in Cancer Prevention and Treatment
The therapeutic potential of RA against various cancers, including prostate, breast, gastric, colon, hepatic, cholangiocarcinoma, osteosarcoma, and glioblastoma has been extensively reported. Studies indicated that RA triggers apoptosis, inhibits proliferation, angiogenesis, and proved to be anti-metastatic in different cancers (Table 1). Hence, the mechanism of action of RA against different malignancies are described below.
Breast Cancer
Breast cancer is a clinically complicated condition with a high incidence and mortality rate in developed countries [84][85][86][87][88]. According to the vicious cycle of bone metastasis, bone cells can interact with breast cancer cells in which tumor cells release pro-osteoclastic factors to induce osteoclastogenesis, while the bone matrix releases pro-tumorigenic growth factors. This in turn can augment tumor expansion [89,90]. To impose osteoclast differentiation and consequently bone resorption, breast cancer cells release inflammatory cytokines such as nuclear factor-κB (NF-κB) ligand (RANKL), which leads to the activation of downstream signaling pathways such as MAPK, NF-κB, and SRC/AKT [70][71][72]. The studies of the therapeutic effect of RA specifically showed the inhibition of SRC/AKT pathway. In vitro, RA effectively prevented the RANKL associated osteoclastogenesis on bone marrow-derived macrophages (BMMs) and osteoblast differentiation [36]. Consistent with this result, treatment of RA at different concentrations considerably inhibited the Ti-particle-induced osteolysis by downregulating SRC/AKT signaling pathway in in vivo mice calvarial model. Hence, RA inhibited RANKL regulated SRC expression, and ultimately downregulated the AKT pathway which leads to the inhibition of osteoclastogenesis [36]. To get insight into the RA mechanism and the pathway, researchers found that RA treatment can cause inhibition of AKT phosphorylation, which was later rescued by using AKT activator SC-79. Hence these findings proved that RA specifically directed PI3K/Akt pathway, without interfering with MAPK and NF-κB signaling pathways. In vivo, Ra has been reported to restore osteolysis and decreased trabecular separation in the osteolysis female mouse model.
Another article reported by Guan et al., indicated that RA treatment induces cellular apoptosis and inhibits invasion and angiogenesis through the modulation of P13K/AKT/mTOR signaling pathway [64,65]. Importantly, this inhibition specifically reduced the multidrug resistance, which rendered various antitumor therapies as ineffective in breast cancer. Moreover, application of RA on breast cancer cell lines (MDA-MB-231, MCF-7 and T47D) induced autophagy and cytotoxicity through modulation of Akt-mTOR-eEF-2K signaling pathway, as evidenced by an elevated level of autophagy marker LC3, which is also considered as one of the hallmarks of cancer [67]. Upon pre-treatment with chloroquine at 20 µmol/L (an autophagy inhibitor), RA considerably enhanced cytotoxicity and induced apoptosis via intrinsic pathways through downregulating anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1) and upregulating caspase-3 and PARP expression. The morphological changes induced by apoptosis were also noticed by nuclear shrinkage, chromatin condensation, and fragmentation. Hence, it is suggested that through the modulation of AKT and autophagy, RA can reinforce the substantial level of apoptosis in breast cancer cells.
Cholangiocarcinoma
Cholangiocarcinoma (CCA) is a rare biliary adenocarcinoma characterized by aggressive metastatic and invasive tumors with poor outcomes [91,92]. Due to late diagnosis and vigorous growth, effective treatment is not available against CCA yet. To date, surgical resection remains the only therapeutic approach, but most cases result in death due to late diagnosis [93]. In addition, the resistance of cancerous cells towards available chemotherapeutics is one of the leading challenges [94]. 5-Fluorouracil (5-FU) is currently used in clinics as a chemotherapeutic agent, but due to resistance against CCA cells, the efficacy of this compound has been remarkably reduced Antitumor effect of 5-FU is enhanced by rosemary extract in both drug sensitive and resistant colon cancer cells [94]. The administration of RA promoted apoptosis in four cholangiocarcinoma cell lines. Amidst all, RA specifically impaired migration and hindered colony formation in LIPF155C and RBE cell lines. RA through its antitumor effect, sensitized bile duct cancer cells toward 5-FU and further mediated apoptosis in a 5-FU-resistant cell line. The Wee1 belongs to a protein kinase family involved in cell proliferation by halting cell cycle arrest in tumor cells [95,96]. RA increased apoptosis and impaired cellular functions via the activation of the Wee1 dependent signaling mechanism in RBE cell lines. The combined treatment of RA along with 5-FU (35µM) synergistically downregulated the expression of Bcl-2, cyclooxygenase-2, and Wee1 and upregulated Bax. In addition, RA mediated cell cycle arrest by modulating cell cycle-related protein cyclin E/D1. Through this process, the cyclin family forms an orchestrated series of molecular complexes to regulate cell cycle progression. In response to DNA damage, cyclin D-Cdk4/6 and cyclin E-Cdk2 in particular regulates G1-S transition [95,97]. Therefore, a high level of E/D1 cyclin protein can act as pro-apoptotic factors, possibly involved in the sensitization of cancerous cells toward radiation [98]. Hence, the synergistic effect of RA and 5-FU could be a potential therapeutic approach for cholangiocarcinoma.
Colorectal Cancer
Colorectal cancer (CRC) is a highly prevalent cancer globally and the third-leading cause of death after lung and breast cancer [99,100]. Cancer cells have the ability to induce angiogenesis, invasion, and metastasis to invade other parts of the body [101]. Ying and co-workers highlighted in their research that RA inhibited the progression of angiogenesis and metastasis of colorectal tumor [79]. RA successfully inhibited HUVEC proliferation and orchestrated the process of angiogenesis, including endothelial cell proliferation, motility, and capillary-like tube formation without affecting HCT-15 healthy endothelial cells, specifying its activity only against tumor endothelial cell. In the chick embryo chorioallantoic membrane (CAM) model, RA treatment effectively blocked blood vessel formation in a dose-dependent manner. The study was further extended to zebrafish model, where RA treatment was shown to disrupt nearly 68% of intersegmental vessel (ISVs) formation along with the deformed morphology of zebrafish. In HCT-15 xenograft mice models, RA dose-dependently showed substantial antiangiogenic potential through a remarkable decrease in micro vessel density (MVD) along with reduced tumor growth and weight. The above mentioned antiangiogenic effect of RA was mediated through the phosphorylation of VEGF-induced vascular endothelial growth factor 2 and the inhibition of downstream kinases and signaling pathways including focal adhesion kinase (FAK), JAK2, PLCγ1, Src, and Akt inhibition, involved in survival, migration and proliferation of EC. This mechanism was further hypothesized by molecular docking simulation, based on which RA pentacyclic triterpene moiety docked at the ATP-binding pocket of VEGFR2 kinase domain occupied with six amino acids and facilitate the formation of VEGFR2-RA complex to inhibit downstream molecular pathways.
PI3K/Akt signaling pathway constitutively expressed in malignant cells and activate several downstream proteins to regulate cell proliferation, cell metabolism, cell survival, and angiogenesis [62][63][64][65]. Activated PI3K/Akt pathway indirectly activates mTOR pathway which subsequently triggers activation of genes involved in apoptosis and cell cycle progression [8]. Chunqin et al. discovered that RA treatment dose-dependently stimulated apoptosis, G0/G1 cell cycle arrest and blocked cell cycle proliferation in colorectal HCT-116 cell line possibly through inhibiting the PI3K/AKT signaling pathway [102]. In the HCT116-xenograft mouse model, RA significantly reduced the tumor growth, whereas apoptotic cells were also seen in tumor tissue [102]. RA treatment significantly decreased the protein level of cyclin D1, cyclin E, p-PI3K, and p-AKT, suggested the strong anti-tumor potential of RA in vivo HCT116 cells induced xenograft mice.
Yu and co-workers discovered the anti-proliferative and apoptotic effects of RA in colorectal LOVO and SW480 cell lines [103]. The study was further extended to in vivo xenograft mouse model, where RA had significantly inhibited the tumor growth through modulation of Wnt/β-catenin signaling via downregulation of p-LRP6, upregulation of AKT inactivation, inhibition of β-catenin and removal of GSK-3β inhibition. In addition, RA prevented tumor through modulation of NF-κB signaling pathway, inhibited phosphorylation of IκB-α which led to an induction of the mitochondrial apoptotic pathway.
Glioblastoma
Glioblastoma multiforme (GBM) is an incurable primary brain tumor with a low long-term survival rate in affected patients. According to a recent report, the estimated incidence rate of GBM is around 5.62 per 100,000 people and the rate is growing [104,105]. Despite standard treatment, the aberrant metastatic potential of brain tumor cells, and incomplete surgical resection has declined the survival rate of less than one year after diagnosis [106]. Peng and coworkers have suggested the therapeutic potential of RA in reducing cell viability of four GBM cell lines (G112, T98, U87, and U251) compared with control cells [107]. RA treatment in these cell lines effectively reduced the level of MMP-2 and MMP-9, linked to the invasive and migratory potential of cancer cells. Treatment with RA induced apoptosis in glioma cells through increased ROS production, which lead to the activation of Jun N-terminal kinase (JNK) signaling pathway, which mediated high Bax/Bcl-2 ratio and subsequently caspase-3 and PARP upregulation. The activation of ROS/JNK signaling pathway was further verified by targeting cells with antioxidant NAC (N-Acetyl-L-cysteine) and caspase inhibitor (z-VAD-fmk), as a result reduced apoptotic rate, p-JNK and caspase-3 level authenticated the activation of this pathway in colorectal cells (T98 and U251). Interestingly, appearance of apoptotic cells after z-VAD-fmk treatment highlighted the occurrence of other possible mechanism in inducing apoptosis. Therefore, RA induced cell death also triggered through another mechanism along with apoptotic pathway. Furthermore, treatment of RA induced autophagy in glioma cells, since it is known to either support or inhibit apoptotic signaling. However, in this study, the application of autophagy inhibitors class III PI3K and 3-MA, exacerbated the RA mediated apoptosis in U251 glioma cell as noticed with caspase-3 overexpression. In U251-harbouring xenografts nude mice model, RA treatment exhibited curative effect with a significant drop in tumor size and induced apoptosis as noted by elevated level of caspase-3, LC3-I to LC3-II conversion and p-JNK.
Gastric Cancer
Gastric cancer (GC) is the third most extensively frequent cancer in males due to the increased resistance of gastric cancer cells toward clinically used chemotherapeutic agents [3,105,108]. The gastric cancer patient typically suffers relapse after surgery, which reduces the survival rate to less than five years [109][110][111][112][113][114]. Gang et al. discovered that RA treatment activated apoptosis and invasion in three dissimilar differentiation stage gastric cancer (GC) cells (BGC-823, MKN-28, and SGC-7901) [115]. Amongst all, RA remarkably reduced proliferation, adhesion, invasion, and migration in BGC-823 cells. However, a study reported by Hao et al., [37] also suggested the anti-proliferative potential of RA against SGC-7901 cells in a concentration dependent manner. Administration of RA on these cells hinder proliferation and induced apoptosis via mitochondrial apoptotic signaling cascade led to a drop in Bcl-2, Bcl-xL, survivin expression, whereas simultaneously upregulated the pro-apoptotic Bax, caspase-3, caspase-8, caspase-9 expression in addition to activation of PARP cleavage. Besides, the RA also attenuated the invasive, migratory, and angiogenic potential of tumor cells by inhibiting MMP-2, MMP-9, MMP-14, and Roc proteins. On the contrary, the E-cadherin (E-cad) expression and reversion inducing cysteine-rich protein with Kazal motifs (RECK) was significantly upregulated, which negatively associated with MMPs, therefore supported the notion that RA specifically prevented angiogenesis via MMPs inhibition. Furthermore, treatment of RA induced apoptosis and autophagy through modulation of p38/MAPK pathway as indicated by high level of p-p38 and ERK level in GC cells [75,76]. Moreover, LC3I to LC3II conversion along with phosphorylation of p-mTOR notably depicted the existence of autophagy in these cells, which probably protect cancerous cells from apoptosis and reduce the inhibitory potential of target compound [115]. Therefore, employing an autophagy inhibitor could be the best choice in RA-mediated apoptosis against gastric cancer cells.
Hepatocellular Carcinoma (HCC)
Hepatocellular carcinoma (HCC) is the aggressive malignancy of the liver and the second leading cause of mortality in the world [3]. Cisplatin is a well-established alkylating compound used regularly in human hepatocellular carcinoma (HCC) chemotherapies and radiotherapy [116], though accompanied by remarkable cytotoxicity [117,118]. Clinically, it is used in combination with other drugs to reduce toxicity. Cisplatin performs its function by repressing the tumor cellular DNA repair process [119]. RA, coupled with cisplatin, reduced its toxicity and showed a remarkable synergistic effect against tumor on QGY-7703 cells based on combination index (CI) values less than 0.8. Anaerobic condition in the tumor microenvironment enhanced tumor cell growth and metastasis due to which intracellular ROS production is used to evaluate the underlying proliferation or metastasis of HCCs [120]. RA, in combination with cisplatin, increases the ROS level and also facilitates the oxygen metabolism in HCC cells, including HepG2 and SMMC-7721. In addition, RA enhanced cisplatin effect through increasing sensitivity of resistant cells, which further activated their apoptosis. RA significantly inhibited proliferation by inducing S phase cell cycle arrest, whereas cisplatin induced cell cycle arrest at the G0/G1 stage [121]. The mRNA expression level of apoptotic genes was upregulated for p53 and Bax, whereas simultaneously led to a reduction of Bcl-2 and Survivin proteins [38]. Hence, RA treatment together with cisplatin, could serve as a potential therapeutic target in reducing toxicity of cancerous cells toward commercially available chemotherapeutic agent.
Lung Cancer
Lung cancer is the heterogeneous disease of lungs and the leading cause of global cancer mortality [3]. Among all lung cancers, 80% of cases belong to non-small cell lung cancer (NSCLC) with gradual increasing incidence and mortality rate [122]. Treatment of RA remarkably inhibited cell proliferation and blocked cell cycle progression of NSCLC H460 cells. RA significantly reduced proliferation of H460 in a concentration-dependent manner. RA modulated the Akt mediated G2/M phase arrest, down-regulated Bcl-2 and cleaved PARP expression, leading to reduced H460 cell proliferation apoptosis induction. These findings suggested the strong therapeutic potential of RA in combating cancer cells and increasing the survival of patients affected by NSCLC [35].
Osteosarcoma
Osteosarcoma is a highly aggressive bone tumor, associated with poor survival. Treatment with RA was shown to exhibit an anti-tumor effect via JNK mediated mitochondrial apoptosis pathway and inhibited metastasis on human osteosarcoma cell lines [123]. RA downregulated Bcl-2/Bax ratio, upregulated cleaved caspase-3 and PARP, as crucial components of apoptosis. RA increased cell apoptotic activity by ROS/JNK phosphorylation and downregulation of NF-κB transcriptional activity through a low level of p-IκBα and p65 [123]. After pre-treatment with RA, inhibition of p65 results in the sensitization of osteosarcoma cells. In addition, RA represses invasion and migration by downregulating MMP-2/9 level mediated with NF-κB pathway [123]. An in vivo study following exposure of RA in ROS xenograft models also confirmed the anti-cancer effects of RA on human osteosarcoma. Zhuoying and coworkers [124] suggested that RA treatment induced apoptosis and abrogated proliferation in osteosarcoma cells through modulation of the JNK/c-Jun and STAT3 signaling pathways. An in vivo study on tibial xenograft tumor model, administration of apoptosis in OS cells, which lead to the reduction of tumor size. Furthermore, another study indicated that RA had shown an antitumor effect in both drug-resistant and non-resistant OS cells [78]. Treatment with RA mediated apoptosis, blocked cell proliferation, as well as restricted colony formation by mediating interleukin-6 (IL-6) induced JAK2/STAT3 pathway. By contrast, RA treatment was shown to increase the levels of MDR1 and STAT3 in resistant OS cells, whereas high expression of these protein is related to the chemoresistance. The expression of MDR1 protein in both drug-sensitive and resistant OS cells, along with downregulation of STAT3 by using siRNA, increased the sensitivity of these cells toward doxorubicin treatment. The combine treatment of RA and doxorubicin increased the doxorubicin uptake by cell, result in increasing toxicity, ablated efflux, and reduced MDR1 expression in drug resistant cells by modulating STAT3 phosphorylation. Consistent with these results, RA reduced tumor growth by promoting apoptosis in doxorubicin-resistant OS tibia orthotopic model. Therefore, RA serves as a potential therapeutic for doxorubicin resistance treatment in OS.
Prostate Cancer
In men, prostate cancer is the most common type of malignancy and does not have therapeutic options in the advanced state [125,126]. The castration resistance after androgen deprivation therapy is the ultimate cause of death in castration-resistant prostate cancer (CRPC). The upregulation of full-length androgen receptor (AR-FL) and splice variants (AR-Vs, AR-V7, ARv567es, and AR-V9) are most likely involved in poor prognosis and castration resistance. It is, therefore, an urgent need to develop a drug that acts as a reactivation mechanism in combating CRPC. A recent report showed that RA has effectively attenuated the transcription of AR-FL and splice variants AR-Vs. Docetaxel is a first-line chemotherapy drug that has been implicated in CRPC and mediates its effect through stabilizing microtubules; however, AR-V7 is localized in the nucleus and is independent of the microtubule. Therefore, it remains insensitive toward docetaxel inhibition [127,128]. In CRPC cells, RA was reported to selectively target both the full-length AR-FL and splice variant AR-V mRNA expression and increase the growth inhibitory efficacy of docetaxel synergistically in both time-and dose dependent manners. RA inhibition is entirely dependent on AR other than androgen, as AR-null cells are left unaffected by RA treatment. This inhibition is of utmost importance because none of the drugs available in the market can directly target the full length and splice variants of the AR. However, compounds other than RA have been shown to decrease the levels of AR-FL and AR-Vs pre-clinically [129][130][131]. Taken together, the study provided a rationale for RA and its combination treatment against CRPC.
Chemosensitizing Properties of RA
Surgery, radiotherapy and chemotherapy are conventional clinical treatments for cancer [132]. However, more and more tumors have become resistant to chemotherapy in recent years, which has become a major obstacle to cancer treatment [111,113,114,[133][134][135]. 5-FU is the most common chemotherapeutic compound for cancer treatment [69]. RA sensitized cholangiocarcinoma cell lines to 5-FU treatment and ameliorated 5-FU resistance in bile duct cancer cells through activating multiple cell cycle and apoptosis-related factors, such as COX-2, Bax, Bcl-2, and cyclins E/D1 [136]. RA may also have the potential to enhance the growth inhibitory efficacy of docetaxel, the first-line chemotherapy for prostate cancer [137]. The growth and survival of prostate cancer cells rely on androgen receptor (AR) [136]. Splice variants of AR (AR-V) expression have been proposed to be a mechanism of docetaxel resistance. RA enhanced the growth inhibitory efficacy of docetaxel through suppressing both full-length (AR-FL) and AR-V expression and activities. Peng et al., reported that the activation of STAT3/NFIL3 signaling axis results chemotherapeutic resistance. In addition, RA reversed STAT3/NFIL3 signaling axis-mediated chemotherapy resistance in drug-resistance choriocarcinoma cell lines such as JEG-3/MTX (methotrexate-resistant-JEG-3 cells), JEG-3/5-FU-resistant-JEG-3 cells), and JEG-3/VP16 (etoposide-resistant-JEG-3 cells) [138].
Limitations and Future Prospects
The diverse pharmacological effects of RA have been analyzed in this review, indicating the therapeutic potential of RA against numerous cancer cell lines. Evidence has suggested that RA shows anticancer potential both in vitro and in vivo animal models. However, in vivo studies are confined to some cancers such as breast, colorectal, prostate and osteosarcoma. In addition, based on previous pharmacokinetic data, low bioavailability of RA in the systemic circulation is a major concern, therefore, there is a need to explore in depth mechanisms in order to increase the compound's bioavailability and to retain the metabolites for an optimal effect. Furthermore, despite having a large number of combination studies with other chemotherapeutic drugs, no clinical study is reported as yet. Hence, in an attempt to get further insights, available data can be employed in clinical settings.
Conclusions
This review provides a comprehensive detail about the diverse anticancer potential of RA in both in vitro and in vivo studies. The effect of RA is mainly exerted through the induction of apoptosis, cell cycle arrest and the inhibition of cell proliferation along with modulating cell signaling mechanisms in breast, cholangiocarcinoma, colorectal, liver, lung, prostate and osteosarcoma. Amidst various signaling pathways, PI3K/AKT has been the most significantly modulated by RA in different cancers. Furthermore, RA has induced synergistic effects in combination with other chemotherapeutic drug and increases sensitivity of tumor cells to apoptosis without posing toxic effect. Therefore, RA can be used as a novel anticancer agent against those malignancies that have developed resistance to chemotherapy. In preclinical studies, RA significantly reduced tumor growth, tumor size and metastasis. However, the effective concentrations against tumor cells varies depending on the type of cell and in vivo model system. Hence, clinical trials are required to establish the effectiveness of RA in clinical settings. The detailed study concluded that RA can be used as a promising anticancer compound.
Conflicts of Interest:
The authors declare no conflict of interest. | v3-fos-license |
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} | pes2o/s2orc | The Effect of Homogenization , CaCl 2 Addition and Pasteurization on White Cheese and Whey Composition
In this study it was aimed to determine the effect of different homogenization pressure, pasteurization process and CaCl2 addition on White cheese and whey composition. Pasteurization process increased ash and calcium content of cheeses, on the contrary to these, dry matter, ash, ash in dry matter and calcium content of whey decreased depending on pasteurization. The pH degree was lower in raw milk cheeses at draining stage and this caused more calcium loss from cheese curd of raw samples. Adjusted yield of cheeses increased in homogenized, pasteurized and CaCl2 added samples. While fat content of cheeses were higher in homogenized cheese samples; dry matter, pH and Ca were lower in whey belonging to homogenized milk cheese samples. Consequently, it can be said that pasteurization, homogenization and CaCl2 addition processes had positive effects on yield and composition of White cheeses.
Introduction
Homogenization and pasteurization of cheese-milk and CaCl 2 addition cause major physical and chemical changes in cheese.Homogenization of milk creates smaller globules with a greater total fat-water interfacial surface area.The surface active proteins, especially caseins, either as semi-intact micelles or as micellar fragments, cover the newly formed surface of the fat globules, and this protects the fat from coalescing (Everett andAuty 2008, Sharma andDalgleish 1993).As the pressure of the homogenization increased, the size of the fat globules in the milk decreased, and the caseins micelles required to spread even more widely over the surface of the fat (Dalgleish et al. 1996).In the absence of heating, the serum proteins seem to play a relatively minor role in this process (Dalgleish and Sharma 1993).But it has been shown that β-lactoglobulin and α-lactalbumin both interact with the κ-casein associated with the fat globules when whole milk is heated (Dalgleish and Blanks 1991;Houlihan et al. 1992).Experimentally, it is demonstrable that heating causes the incorporation of large amounts of serum protein into the fat globule fraction and occurrence is form as it were a second membrane layer around the fat globule (Dalgleish and Sharma 1993).Summarizing, heat treatment of milk results in a complex mixture of native whey proteins, whey protein aggregates and casein micelles covered with whey protein.Both heating and homogenization of milk leaded to physical and chemical changes in the milk and milk products, especially cheese, for example, the rennet coagulation of milk is considerably changed (Sharma and Dalgleish 1993), they are a lower drainage of whey from the rennet curd, increase the coagulum strength (Ghosh et al. 1994), increase the whiteness, moisture retention (Everett and Auty 2008;Tunick et al. 1993), they provide higher cheese yield because of lower fat losses in whey, lower free oil in cheese due to the increased degree of fat emulsification and increased rate of lipid hydrolysis for certain types of cheese, e.g., Blue cheese (Madadlou et al. 2007;Solorza and Bell 1998a;McMahon et al. 1997).
Heating of homogenized milk changed both physical and chemical properties in the milk fat globule membrane and serum proteins due to their interaction (Sharma andDalgleish 1993, 1994;Dalgleish andSharma 1993, Van Boekel andWalstra 1989).Both fore-warming and homogenization also affect the renneting and heat stability properties of milk (Sharma and Dalgleish 1994).
The effects of calcium addition to milk and pH are not independent events.Ca 2+ also reduces pH due to chelation of HPO 3 2-and H 2 PO 3 -ions, thus the increase in milk gel firmness and the reduced time for clotting to occur may be due to a decrease in pH rather than an increase in soluble calcium.A reduction of 0.01 units of pH per mM of Ca 2+ addition, up to 5 mM, has been reported by Ramet et al. (1981).Increasing the amount of total (ionic plus insoluble) calcium in cheese enhances casein-casein interactions, thereby reducing meltability and increasing cheese firmness (Everett and Auty 2008;Solorza and Bell 1998b).
White cheese is produced using raw milk, traditionally.However, in last two decades, pasteurization and CaCl 2 addition are becoming standard processes in White cheese making, but, homogenization is a rarely used application.The aim of this study was to evaluate the effect of homogenization and homogenization of cheese milk and CaCl 2 addition on cheese compositional parameters.
Material and Methods
Raw milk supplied from a Dairy Plant of Agriculture Faculty of Yüzüncü Yıl University.Lyophilised culture of Lactococcus lactis subsp.lactis and Lactococcus lactis subsp.cremoris were purchased from Peyma (Chr.Hansen's Co., İstanbul, Turkey).All chemicals were analytical grade (Sigma Chemical Co.St. Louis, MO, USA; Merck, Darmstadt, Germany).
Cheese production was carried out in Dairy Pilot Plant of Food Engineering Department of Yuzuncu Yil University.
White Cheese and Whey
For White cheese making, bovine milk was heated to 50 °C and then divided into three batches.The first batch was kept in unhomogenized form, the second batch was homogenized at 7.5 MPa (Giusti Corp., Wellingborough, Northants, UK) and the third batch was homogenized at 15 MPa.Then each batch was divided into two parts.The first part of milk was pasteurized (65 °C for 30 min) and the second part was not.After that each milk part was divided into two equal parts and CaCl 2 was added into a part.Thus 12 cheese milk batches were obtained totally (Table 1).
Pasteurized cheeses were prepared according to industrial method.After cooling (35 °C), CaCl 2 (150 ppm) was added into the pasteurized milk.Lyophilized cultures sub-cultured twice in sterilized reconstituted skim milk individually and a mixture (1/1, v/v) of these bacteria were inoculated to each treatment (20 g/L) (only for pasteurized samples).After 30 min, rennet extract (70 ppm, 1/10 000 strenght, Pınar Inc., Istanbul-Turkey) was added to treatment, which was incubated at 30 °C to develop the curd.After coagulation time (approximately 90 min) the curd was cut into 1 cm 3 with a curd knife, transferred to cheese cloth and allowed to stand for 3 h under pressure (10 kg weight) for draining the whey.After the removal of the whey, cheese mass was divided into blocks of about 7x7x7 cm with a knife.
Chemical Analyses
Total solids, fat, total nitrogen, titratable acidity and pH of the cheeses were determined according to AOAC methods (1995).Protein content of cheeses was calculated multiplying the total nitrogen content by 6.38.Moisture, non-fat dry matter (NFDM), moisture in NFDM, fat in DM, protein in DM and ash in DM used for Principal Component Analysis (PCA) were obtained by calculation.Cheese yields were converted to adjusted yield basing on their average dry matter content (Metzger and Mistry 1994), Ca was determined by atomic absorption spectrometry at 422.7 nm (Thermo Solaar AAS Spectrometry, Type M6 MK2, UK).P was determined by UV-Vis spectrophotometer at 400 nm (PGeneral T80 double beam UV-VIS Spectrophotometer, China) (AOAC 1995).For quantification these elements, samples were obtained by dry ashing at 550°C in a furnace and then solubilized in nitric acid (IDF 1992).
Statistical analysis
Experiment was performed on three replicates for White cheese and whey samples.Each analysis was done in duplicate following analytical procedure as defined above.All data were subjected to an analysis of variance (ANOVA) and separated by Tukey's Multiple Range Test perform using the SAS statistical software (SAS 2005).To gain insight into the structure of the data set, Principal Components Analysis (PCA) was performed.PCA is a well-known mathematical transformation of the raw data; it is an exploratory technique that indicates relationships among variables (Piggot and Sharman 1986).
White Cheese
The effect of different homogenization pressure, heat treatment and CaCl 2 addition into cheese-milk on composition and yield of White cheese were given in Compositional properties as ash, pH, acidity, Ca, adjusted yield (P<0.001), fat (P<0.01) and protein (P<0.05) of the White cheeses were found to be affected significantly by heating of cheese-milk.While ash, protein, pH, Ca and P were higher in the cheese made from raw milk; acidity, fat and adjusted yield of the pasteurized cheese were higher (Table 2).In pasteurized cheese, reduction in the numbers of naturally present bacteria in milk, addition starter lactic acid bacteria and their high acidifying activity possibly led to a faster development of acidity.The pH of pasteurized and pressure-treated milk cheeses was found lower than in cheeses made from raw milk (Buffa et al. 2005).Calcium ions released as dissociated form at low pH values and passed to whey, thus Ca, P and ash ratio decreased depending on pH decrease.
The heating of milk may be used to increase cheese yield, by binding the whey protein to the micelles (Dalgleish and Sharma 1993).Similar results were obtained in homogenized samples and CaCl 2 added samples.While fat content and yield of white cheese were higher (P<0.001) in homogenized samples.Homogenization of milk affects the casein network, thereby altering its basic structure and leads to lower curd firmness during rennet coagulation, curd shattering during cutting and increased fine losses to whey (Nair et al. 2000).Depending on the homogenization pressure, fat contents of the cheeses increased, conversely fat contents of whey decreased.As known, homogenization causes better fat recovery in cheese curd (Metzger and Mistry 1994;Walstra et al. 1999).
Addition of CaCl 2 to the milk was also observed to affect the adjusted yield (P<0.01), ash and Ca (P<0.05) (Table 2).Depending on the cheese manufacturing protocol (firmness of gel at cutting, cut programme), the addition of CaCl 2 may also increase the level of milk fat recovered to cheese, cheese moisture and cheese yield.The increase is likely to be due to the more rapid curd-firming rate, which would increase rigidity of the gel/curd during the early stages of syneresis, thereby limiting the ability of the matrix to rearrange and express whey (O'Callaghan and Guinee 2010).
Principal component analysis (PCA) was applied to compositional variables that were affected by pasteurization, homogenization pressure and addition of CaCl 2 .Results from the PCA belonging to White cheese showed that principal components (PC) 1 and 2 described about 72.39 % of the total variation of sample: 49.33 % PC1 and 23.06 % PC2 (Fig. 1).While PC1 was heavily loaded on homogenization, pasteurization, NFDM, moisture in NFDM, fat, fat in DM, protein, ash, pH, titratable acidity, Ca, P and adjusted yield, the second factor (PC2) was loaded on DM, moisture and CaCl 2 .There were positive significant correlations between homogenization and moisture in NFDM, fat, fat in DM, adjusted yield and negative significant correlations between homogenization and NFDM, protein and protein in DM.On the other hand, correlations between pasteurization and moisture in NFDM, fat, fat in DM, titratable acidity and adjusted yield were found significantly positive; and correlations between pasteurization and ash, ash in DM, NFDM, pH, P and Ca were determined significantly negative.CaCl 2 addition only correlated with DM of cheese, significantly.
Compositional variables such as dry matter, pH, Ca, P, nonfat dry matter, ash dry matter and protein, showed positive loadings with PC1, whereas moisture, titratable acidity, fat, pasteurization and protein dry matter showed negative loading with this factor (Fig. 1).The second factor (PC2) was high positive loadings with pH, Ca, ash, protein, P and non-fat dry matter and it showed negative loading with homogenization, pasteurization, CaCl 2 fat, dry matter, adjusted yield and titratable acidity.Results also showed that there is negative significant relationship among homogenization, heat treatment, moisture, pH, Ca, P, ash and protein (Fig. 1) Fig. 1.Principal component analysis of the effect compositional variables that were affected By pasteurization, homogenization pressure and addition of CaCl 2 in White cheese.
Whey
The composition of whey was given in Table 3.Generally, the dry matter, fat, pH (P<0.001) and protein (P<0.05)contents of whey obtained from pasteurized cheese were significantly lower.Positive relationship was determined between titratable acidity and Ca, P and ash.The observed increases in the levels of ionic calcium in the whey were probably due to the conversion of the colloidal calcium phosphate to ionic form.
Dry matter, pH and fat contents of whey decreased with homogenization pressure of milk compared with that of the control (P<0.001),possibly because of the loss of fat clumps in nonhomogenized sample during draining.In other studies, homogenization was reported to reduce the fat content of whey (Metzger andMistry 1994, Nair et al. 2000).The protein in cheese whey was also lower for homogenized samples compared to control sample.The lower fat and protein losses with homogenization could be attributed to the reduction in fat globule size and the modified fat globule membrane by adsorption of proteins onto fat globule surface.
Lower fat and protein content of homogenized whey samples reflected on the total solids content as well.Whey from control had higher total solids compared to that from homogenized treatments.According to the PCA analysis results showed that principal components 1 and 2 described about 69.35 % of the total variation of sample: 48.37 % for PC1 and 20.98 % for PC2.There was negative significant relationship among homogenization and some other compositional variables such as protein, fat, dry matter, Ca etc. (Fig. 3).As expected, parameters of whey were oppositely placed in the figure when compared White cheese parameters.
Conclusion
Homogenization, pasteurization and added CaCl 2 are common processes in dairy industry that can alter product characteristics.
Pasteurization process decreased pH, ash, calcium and phosphorus content of cheeses, on the contrary, these parameters of whey increased depending on pasteurization.Because of the addition of starter cultures into pasteurized milk could speed up acidification, decreased through soluble form of calcium and as a result, ash is reduced.
Homogenization of the cheese milk had a positive effect on fat retention and cheese yield.The addition of calcium into the milk enhanced the retention of this mineral in the cheeses.Therefore, in the industrial production method use of homogenization, pasteurization and CaCl 2 addition should be preferred for obtaining White cheeses at a standard quality.
Fig. 2 .
Fig. 2. Principal component analysis of the effect compositional variables that were affected by pasteurization, homogenization pressure and addition of CaCl 2 in whey
Table 1 .
Definition of cheese-milk types used in the experiment
Table 2 Table 2 .
The effect of different homogenization pressure, heat treatment and CaCl 2 addition into cheese-milk on composition and yield of White cheese a, b, c Means in each column with different superscripts were significantly different (P<0.05);P = pasteurized milk, R= raw milk
Table 3 .
The effect of different homogenization pressure, heat treatment and CaCl 2 to cheese-milk on composition of whey | v3-fos-license |
2016-05-04T20:20:58.661Z | 2013-07-30T00:00:00.000 | 14215880 | {
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} | pes2o/s2orc | Factors Controlling the Redox Potential of ZnCe6 in an Engineered Bacterioferritin Photochemical ‘Reaction Centre’
Photosystem II (PSII) of photosynthesis has the unique ability to photochemically oxidize water. Recently an engineered bacterioferritin photochemical ‘reaction centre’ (BFR-RC) using a zinc chlorin pigment (ZnCe6) in place of its native heme has been shown to photo-oxidize bound manganese ions through a tyrosine residue, thus mimicking two of the key reactions on the electron donor side of PSII. To understand the mechanism of tyrosine oxidation in BFR-RCs, and explore the possibility of water oxidation in such a system we have built an atomic-level model of the BFR-RC using ONIOM methodology. We studied the influence of axial ligands and carboxyl groups on the oxidation potential of ZnCe6 using DFT theory, and finally calculated the shift of the redox potential of ZnCe6 in the BFR-RC protein using the multi-conformational molecular mechanics–Poisson-Boltzmann approach. According to our calculations, the redox potential for the first oxidation of ZnCe6 in the BRF-RC protein is only 0.57 V, too low to oxidize tyrosine. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. We discuss the possibilities for modifying the BFR-RC to achieve this goal.
Introduction
The water splitting reaction of photosynthesis has been the most influential biologically catalyzed reaction on earth. Acquiring the ability to use water as a source of electrons about 2.5 billion years ago allowed oxygenic photosynthesis to power a massive increase in the diversity and numbers of aerobic life forms. Photosystem II (PSII) is the pigment-protein complex embedded in the thylakoid membranes of plant chloroplasts and cyanobacteria that catalyses the light induced oxidation of water and reduction of plastoquinone in oxygenic photosynthesis [1,2]. This catalytic function is performed by light driven electron transfer (ET) reactions through redox cofactors of the PSII reaction center. Oxidation of water by PSII requires several essential cofactors: a photoactive strongly oxidizing pigment (P680), a redox-active tyrosine (Y Z ) and the oxygen-evolving complex (OEC) containing four high-valence manganese ions bound by m-oxo bridges and a calcium ion [2,3]. Electronic excitation of P680 and subsequent electron transfer to the primary pheophytin electron acceptor forms the strongly oxidizing P680 N+ cation radical (E m ,1.12 V) which then oxidizes Y Z . Subsequently, Y Z oxidizes the OEC where water oxidation occurs after the accumulation of four oxidation equivalents in the Mn 4 CaO 5 cluster according to the S-cycle proposed by Joliot and Kok [4,5]. The coupling of water oxidation to photochemistry in PSII was a crucial milestone in the evolution of life allowing for the essentially unlimited conversion of sunlight energy to chemical potential energy which now powers most life on earth. The successful mimicking of this reaction in an artificial system could form the basis of a clean alternative energy source. Recently, bacterioferritin (cytochrome b1, BFR) was used as a protein scaffold for constructing a linear electron pathway that mimics some of the electron transfer components within PSII [6].
Ferritins are the principal iron storage proteins in most living organisms [7][8][9]. The protein BFR is a robust iron storage bacterial protein that forms a homodimer, with each subunit (,18.5 kDa) being composed of an antiparallel, four-helical bundle [10][11][12] (Fig. 1). The homodimers self-assemble into a dodecamer that forms a spherically shaped protein shell surrounding an internal cavity encapsulating an iron core composed of ferric hydroxyphosphate micelles [10]. Each BFR subunit binds 2 iron ions (Fe 2+ ) and contains seven tyrosine residues [10,12]. The BFR dimer binds one b-type heme at the interface between subunits [11]. In each subunit three tyrosines are located close to both metal ions and the heme (Fig. 1). Thus, a protein scaffold of BFR is suitable for constructing a photoactive reaction center mimicking the electron transfer reactions of PSII. A prototype of such a 'reaction center' (BFR-RC) has been created by replacing the heme with a photoactive zinc-chlorin-e 6 (ZnCe 6 ) pigment and the iron ions with Mn(II) ions [6]. It has been found that the bound ZnCe 6 species are capable of initiating electron transfer upon illumination, oxidizing a tyrosine residue and the bound manganese Mn(II) ions [6].
The mechanism of the light activated electron transfer observed in BFR-RC, however, remains poorly understood. It is not clear whether photooxidation of tyrosine and the bound manganese are sequential or independent events. One of the central questions key to understanding ET pathways, overall efficiency and limitations of BFR-RC is the unknown value of the oxidation potential of the ZnCe 6 cation radical in BFR-RC. The E m of ZnCe 6 provides the driving force for oxidation of cofactors and its value will determine which mechanisms of tyrosine oxidation are possible and whether this BFR-RC may eventually be able to oxidize water. To our knowledge the oxidation potential of ZnCe 6 has not been measured experimentally in either solvent or protein. The present article investigates the oxidation and reduction potentials of ZnCe 6 in solution and in BFR-RC with a combination of experimental and computational methods. Our focus is on understanding factors influencing the redox potential of ZnCe 6 within BFR-RC with an aim to identify possible means of controlling it.
Synthesis of ZnCe 6
ZnCe 6 was prepared by metallating chlorin e 6 with Zn(OAc) 2 by standard methods [13]. Typically, 40 mg (0.067 mmol) of chlorin e 6 and 37 mg (0.20 mmol) of Zn(OAc) 2 were stirred at room temperature in a CHCl 3 /CH 3 OH (8/4 ml) solvent mixture. The reaction was monitored by absorption spectroscopy. A red shift of the Q Y absorption band from 661 to 640 nm accompanied incorporation of zinc. We also confirmed formation of ZnCe 6 by observing the disappearance of the free base pigment protons by NMR spectroscopy. After completion of metallation the solvents were evaporated and the residue was washed with water and cooled methanol to get spectroscopic grade pure compound. The yield of reaction was 39 mg (89%).
Electrochemical measurements
Differential pulse voltammetric measurements were performed in pyridine and DMSO containing 0.1 M tetrabutylammoniumhexafluorophosphate (TBAPF 6 ) on a BAS Epsilon electrochemical analyzer (working: Pt, auxiliary electrodes: Pt wire; reference electrode: Ag). The pulse width, period and amplitude used were 50 ms, 200 ms and 50 mV, respectively. Sample concentration was 1 mM and ,100 fold excess of N-methylimidazole was added to prevent aggregation between ZnCe 6 molecules. Resultant solutions were purged with nitrogen gas for 10 min prior to the scan. The ferrocene/ferrocenium couple was used to calibrate the redox potential values. All experiments were performed at 296 K. The use of water as solvent for electrochemical measurements was avoided to eliminate reactions of cations and di-cations with water and any changes in E m which could arise from ionization of ZnCe 6 carboxylic acid groups.
Spectroelectrochemical measurements were performed at a platinum mesh electrode in a thin layer spectroelectrochemical cell (0.5 mm). Potentials were applied and monitored using the same potentiostat as for the differential pulse voltammetric measurements. Absorption spectra were recorded with an Ocean Optics USB650 Red Tide spectrometer.
Calculation of the shifts to the redox potential of ZnCe 6 due to coordination to axial ligands, ring substituent groups, and dielectric constant To calculate the shift to the redox potential due to axial ligation for the ZnCe 6 + /ZnCe 6 couple, we performed computations using density functional theory (DFT). The B3-LYP functional with the LanL2TZ+ basis set for the Zn atom and 6-311G+** for C, H, N and O were used. Gas-phase zero-point energies, thermal corrections, and entropic corrections were calculated using standard formulas for the statistical thermodynamics of an ideal gas using optimized geometries and scaled by 0.9613 B3-LYP/6-31G*/LanL2TZ+ frequencies [14]. Solvation energies of the studied species in the various solvents were calculated using the solvation model SM8 [15] at the B3-LYP/6-31G* level of theory. In all solvation energy calculations the LanL2TZ+ basis set was used for Zn.
Modeling BFR-RC structure
The X-ray bacterioferritin structure, (PDB ID: 3E1M), from E. coli with a resolution of 2.7 Å was used as a starting point for calculations. The structure was modified to match an engineered BFR-RC described in [6,16]. Two surface exposed histidines (H46, H112) were mutated to arginines, the heme was replaced with photoactive ZnCe 6 and iron ions were replaced with Mn(II) ions.
In BFR protein the heme binds in a symmetrical hydrophobic pocket located on a twofold axis between symmetry related subunits. As the structure of ZnCe 6 is similar to the structure of btype heme (ZnCe 6 has formyl, acetyl and propyl groups at positions 13, 15 and 17 and saturated ring IV while heme has propyl groups at 13 and 17 and all rings unsaturated) we placed ZnCe 6 in the same position and orientation as heme in the BFR. After initial placement of ZnCe 6 in its binding pocket, conformations of the three carboxylic groups of ZnCe 6 were optimized using a Monte-Carlo search as implemented in MCCE. The highest occupancy conformers were then used as a starting structure for a complete energy minimization of the whole BFR-RC with constrained backbone atoms which was then followed by a short 10-ps unconstrained molecular dynamics run. AMBER version 10 [17] was used for molecular mechanics computations. The ''pairwise'' generalized Born implicit solvent model [18] was used in these simulations. Initially we used standard ionization states for all aminoacids, and all 3 carboxylic groups of ZnCe 6 were protonated. The final RMSD of the protein backbone between the original PDB ID: 3E1M structure and BRF-RC was 0.15 Å . Next we performed ONIOM QM/MM geometry optimization of the whole protein with ZnCe 6 and its methionine ligands treated quantum mechanically at the B3LYP/6-31g* level using Gaussian 09 [19]. In QM/MM optimized structures both methionine ligands were almost equidistant from Zn. The equilibrium distances between Zn and S were found to be 2.80/2.85 Å . The QM/MM optimized structures of neutral and radical ZnCe 6 with its 2 methionine ligands were used to refine atom-centered point charges obtained from the initial model. The optimized structure of the BFR-RC with bound ZnCe 6 is available in PDB format in supporting Model S1.
Derivation of the atomic partial charges for ZnCe 6 The atomic partial charges for ZnCe 6 were obtained using the two stage RESP formalism [20], with a weighting factor of 0.0005/0.001 from a wavefunction computed at the HF/6-31G* level for H, N, C, O, and HF/LanL2TZ+ for Zn. Schematic diagram of ZnCe 6 is shown in Fig. S1, atomic point charges for ZnCe 6 are available in Table S1. QM calculations were done with the Gaussian09 package [19].
Computation of protonation pattern and shift of the redox potential of ZnCe 6 in BFR-RC MCCE2.4 [21] was used to predict the ionization state of each protein residue and cofactor as a function of E h and pH. The algorithm performs Monte-Carlo sampling of multiple aminoacid side chain geometric and ionization conformations, where conformer energies include electrostatic and van der Waals terms. Before sampling, rotamers of aminoacid side chains were generated using 60 degree increments for each rotatable bond, while conformers of carboxylic acid groups of ZnCe 6 were generated in 14 degrees increments. Pairs of conformers with clashes not exceeding 5 kcal/mol had their positions optimized. Finally, a genetic algorithm was used to optimize side chain conformers [22]. Electrostatic conformer-conformer pairwise interactions and reaction field energy for each conformer were computed by solving the linearized Poisson-Boltzmann equation using the Delphi program [23]. The dielectric constant was set to 4 inside the protein and 80 in the solvent. PARSE [24] radii and charges were used in Poisson-Boltzmann calculations for all elements except Zn. For Zn, a radius of 1.47 Å was used [25]. To obtain the E m of ZnCe 6 in BRF-RC the shift of the redox potential in the protein was added to the E m of the reference model system.
In calculations of the shift of E m in a protein environment several factors were considered: (i) the desolvation energy difference arising from moving the cofactor from water into protein , where the protein volume is simply considered as dielectric medium DDG desolv , (ii) the electrostatic and VDW nonelectrostatic interaction of the cofactor with the protein backbone DG bkbn , and (iii) the mean field pairwise interaction between the cofactor and side chains of residues in the protein in the distribution derived by Monte Carlo sampling DG mfe res . The difference in each energy term is between the oxidized and the reduced cofactor. Details of the calculations of these factors are described in [26].
In continuum electrostatics calculations ZnCe 6 and side chains of its methionine axial ligands were represented as a single residue with an oxidized or reduced conformer. The backbone atoms of the axial ligands remained as part of the protein backbone. QM treatment of the complex of ZnCe 6 ' (ZnCe 6 without carboxylic groups) with 2 methionine axial ligands yielded E m(sol) of 0.58 mV (see Results section 3.3) which was used as the reference for MCCE calculations. The three carboxylic groups of ZnCe 6 were treated as independently ionizable groups.
Results
Oxidation and reduction potentials of ZnCe 6 in solution ZnCe 6 has low solubility in most organic solvents. We were able to measure its redox potential in only two solvents (pyridine and DMSO). Although we observed oxidation waves in both solvents, only with N-methylimidazole were two clear waves visible. Voltammograms measured in DMSO without N-methylimidazole showed multiple redox waves (data not shown), suggesting that under these conditions several different aggregation states of ZnCe 6 co-existed in the sample. Aggregation occured most likely due to the coordination of Zn by Lewis base atoms of the substituent carboxyl groups. These aggregates can be broken down by addition of stronger Lewis bases such as N-methylimidazole. Indeed, after addition of N-methylimidazole to the ZnCe 6 /DMSO solution, two distinctive redox couples appeared in the oxidative scan. The major oxidation peaks were centered at 0.54 and 1.01 V vs. standard hydrogen electrode (SHE) (Fig. 2A).
In addition a smaller and broader feature was observed at 0.8 V. The spectral changes obtained during oxidation of ZnCe 6 at 0.54 V are shown in Fig. 2B. The Q Y band of the neutral compound at 640 nm decreased in intensity while a new absorption band grew at around 800 nm. These spectral features can clearly be assigned to a chlorin p cation radical. The difference between the first and the second oxidation of methylimidazolecoordinated zinc porphyrins has been reported to be in the range of 0.46-0.66 V [27]. In our voltammogram the peak centered at 1.01 V, matching this difference, was assigned to a two electron oxidation of ZnCe 6 . There was one redox couple in the reductive scan at 21.26 V tentatively assigned to the formation of the ZnCe 6 anion radical (Fig. 2).
Effect of axial ligands and carboxyl groups on the oxidation potential of ZnCe 6 in solution
To calculate the E m of ZnCe 6 in BFR we needed a reference E m corresponding to ZnCe 6 ligated by methionine without carboxyl groups in water. To obtain this value from the measured E m of ZnCe 6 in DMSO we computed shifts to the E m due to different solvents, axial ligands and carboxyl groups.
Axial ligands are known to induce changes in the electrochemistry of metalloporphyrins and metallochlorins [27,28]. For example, it was found that upon imidazole ligation the first oxidation potential of zinc porphyrins shifts negatively by 150 mV in CH 2 Cl 2 [27]. In contrast the second oxidation potential of complexed zinc porphyrins shifted positively by 50-270 mV when compared with the uncomplexed zinc porphyrins depending on the nature of the substituents [27].
Our DFT calculations showed that N-methylimidazole shifts the first oxidation potential of ZnCe 6 in water negatively by 70 mV, while the coordination to 2 methionines had a smaller negative shift of only 20 mV ( Table 1). This trend is similar to the results of previous computational work on chlorophyll a [28].
A second factor affecting the oxidation potential is the nature of ring substituent groups [29,30]. In general, electron withdrawing groups shift the oxidation potential up while electron rich groups shift it down. Protonated carboxyl groups, being electron withdrawing, are expected to up-shift the redox potential. This effect is pH dependent as ionization of carboxyl groups stabilizes the cation radical, and hence will down-shift the redox potential.
To estimate the shift of the redox potential of ZnCe 6 in BFR-RC protein we treated the carboxyl substituent groups and Znchlorin as separate units, affecting each other via classical electrostatic and VDW interactions. This approach allowed us to sample efficiently multiple conformational and ionization states of carboxylic acids. A similar approach was previously successfully used for calculation of heme redox potentials in different proteins [31]. These calculations require knowledge of the reference redox potential of Zn-chlorin without carboxyl groups in water, while experimentally we measured ZnCe 6 with three substituent carboxylic groups in DMSO. Therefore, to obtain the reference redox potential of Zn-chlorin we estimated differences in redox potential due to solvent and the presence of carboxyl groups computationally.
Our DFT calculations showed that the redox potential of both coordinated and non-coordinated ZnCe 6 changes insignificantly between DMSO and water. This result is consistent with previous experimental and theoretical studies of chlorophyll a [32] in solvents with different dielectric constant. However, the attachment of protonated carboxyl groups to Zn-chlorin in different coordination states leads to an increase of the redox potential by 0.13-0.14 V in water.
To estimate the accuracy of our DFT calculations we determined the E m s of 10 different zinc chlorins and compared them to experimental values [30]. All computed values were systematically lower than the experimental values by 0.1 V. After correction for this systematic shift, the computed E m s were within 0.03 V of the experimental values (Fig. S2). Overall, the reference oxidation potential corresponding to the first oxidation of bismethionine coordinated ZnCe 6 in water without carboxylic groups was estimated to be 0.58 V vs. SHE.
Oxidation potential of ZnCe 6 in BFR-RC
In BFR-RC ZnCe 6 is partially buried in the protein. Our Monte-Carlo simulations estimate that desolvation due to embedding into the protein's lower dielectric increases the E m of ZnCe 6 by 0.07 V. Another modest increase of the E m arises from interaction with dipoles of the protein backbone (0.05 V). In contrast the side chains of aminoacids (including the ZnCe 6 carboxylic groups) decrease the redox potential ( Table 2). The ionization state of the carboxyl groups is expected to affect the ZnCe 6 redox potential. While neutral carboxyl groups increase E m by 0.14 V in water (Table 1), ionized groups will stabilize the cationic form and shift the E m down. In addition the redox state of ZnCe 6 is bound to affect the pKas of its carboxyl groups. Oxidized ZnCe 6 will tend to lower the pKas and increase ionization of the carboxyl groups. This would tend to further lower the ZnCe 6 oxidation potential.
The case when all carboxyl groups are protonated before oxidation and remain protonated after oxidation provides an upper bound for the ZnCe 6 redox potential. With pKa values determined in the previous section, the charges of carboxylic groups with neutral ZnCe 6 at pH 6 are: 20.25, 20.56, 20.88 for formyl, acyl and propyl respectively when all carboxylic groups were titrated together. The probability of fully protonated ZnCe 6 (the product of occupancies of all 3 protonated groups) in this case is fairly low (4.4%). However, performing a titration of ZnCe 6 while the propyl group is fully charged decreases the redox potential by only 0.02 V compared to an all-neutral titration. The charges of carboxyl groups with the cation radical of ZnCe 6 at pH 6 increase to 20.93, 20.25, 20.93 for formyl, acyl and propyl respectively. The probability of a fully protonated cationic form of ZnCe 6 is very low. If all carboxylic groups are protonated in both cationic and neutral forms, the ZnCe 6 has an E m of 0.67 V ( Table 2). This result gives an idea of how high the E m of ZnCe 6 in BFR-RC could be if the carboxyl groups were to be replaced by neutral groups. In another extreme, when all carboxyl groups are ionized, ZnCe 6 has the lowest E m of 0.48 V ( Table 2). Of course the actual value of E m is somewhere between 0.48 and 0.67 mV. In agreement with upper and lower bound estimates we obtained an E m of 0.57 V at pH 6 in calculations when the ionization of titratable groups was sampled simultaneously with the redox titration of ZnCe 6 . Our calculations also showed that in the pH interval from 4 to 8, the E m decreases by 0.21 V (Fig. 3).
Contributions to the E m shift from individual aminoacids in the BFR-RC are listed in Table 3. Seven aminoacids, located close to ZnCe 6 , have the largest contributions to the E m shift (Fig. 4). Four of these aminoacids form two pairs of salt bridges between two monomers, while one pair (Asp50-Lys53) forms a salt bridge within a single protein subunit. Although each of these charged aminoacids would have a large effect on E m if considered separately, their participation in salt bridges neutralizes their effective charge greatly, thus decreasing their ability to shift the E m . Finally a large fraction of the decrease comes from Asn23. This aminoacid decreases E m by 20.02 V in both monomers of the homodimer resulting in total shift of 20.04 V. The two manganese clusters contribute 20.02 mV each, and the longrange electrostatic contributions from the rest of the protein aminoacids decrease the E m by 20.02 mV. Oxidation potentials of tyrosines in BFR-RC Three tyrosines in BRF-RC are located near ZnCe 6 and the dimetal center. Tyr25, 45 and 58 are found at 3.8/10.6, 13.7/4.5 and 5.8/10.4 Å from the di-metal center/ZnCe 6 respectively. The four other tyrosines are located at the periphery of the protein far from both cofactors. After performing Monte-Carlo sampling of protonated neutral and protonated cationic radical species to determine the redox potential of each tyrosine in BFR-RC we found that Tyr25 has the lowest oxidation potential.
The decrease of Tyr25's oxidation potential arises from the high polarity of its immediate environment which stabilizes the Tyr25 cation radical. The protein environment of Tyr25 is shown in Fig. 5. The polarity of the Tyr25 environment is reflected by increase in its pKa of ,10 units. Most of this shift is due to Asp90 and Glu47. The presence of these two negatively charged amino acids in the vicinity of Tyr25 destabilizes the deprotonated form of Tyr25, while the positively charged radical is favoured.
Our Monte-Carlo simulations indicated that at pH 8 the Tyr25 oxidation potential is decreased in BFR-RC by 160 mV relative to the tyrosine reference potential of 1380 mV in solution. The oxidation potentials of all other tyrosines in BFR-RC are increased by .250 mV. The largest contributions to the E m shift of Tyr25 are from Asp90 (2300 mV), the di-metal center with its ligands (2108 mV), Glu47 (250 mV), Glu44 (30 mV) and Asp50 (240 mV). Glu44, Glu47 and Asp90 are located approximately equidistantly from Tyr25, but only Asp90 has a large effect on its oxidation potential. This difference is because only Asp90 is fully ionized. Occupancy of the charged species of Glu47 is 0.2, and Glu44 which has the lowest influence on the shift of Tyr25 E m is neutral. The occupancy of the neutral species of both Glu47 and Glu44 is high because both of them are located in a hydrophobic environment (Leu40, Leu87, Leu134, Trp133). Despite the decreased oxidation potential of Tyr25 in BFR-RC (1220 mV) it is still too high for a cation of ZnCe 6 to oxidize.
Discussion
The E m of ZnCe 6 in DMSO is 0.54 V and our calculations predict that it remains not far from this value when ZnCe 6 is embedded in BFR-RC. This E m value is significantly lower than was anticipated [6], and well below that required to oxidize water.
It also raises the question of how ZnCe 6 oxidizes tyrosine in the BFR-RC, as in all known cases the redox potential of tyrosine in proteins is near 1 V [33][34][35]. However, light-induced tyrosine oxidation has been observed in BFR-RC [6]. How can tyrosine be oxidized by ZnCe 6 ?
In PSII there are three possible mechanisms for the oxidation of tyrosine in the redox reaction between P680 and Mn-cluster (reviewed in [36]): Where species of tyrosine are denoted as: YH, protonated neutral; YH z , protonated cationic radical; Y { , deprotonated anion; Y, deprotonated neutral radical. These standard potentials were measured for N-acetyl-L -tyrosinamide in aqueous solutions [36,37]. In aqueous solution, the pathway of tyrosine oxidation depends predominantly on pH. In a protein, these reactions can also be controlled by hydrogen bonds or electrostatic interactions of tyrosine with its local environment. Our computations suggest that none of the above reactions could be driven by photooxidation of ZnCe 6 . Reaction 1 requires the redox active tyrosine to be ionized. Tyrosine ionization can only occur if positive charges near the tyrosine significantly lower its pKa. None of the tyrosines in BFR-RC satisfy this requirement.
Reaction 2 requires a somewhat higher oxidation potential as compared to reaction 1. In solution Tyr is oxidized by this mechanism at +970 mV. Reaction 2 is proton-coupled and requires a suitable proton acceptor e.g. a nearby His, able to bind the proton released by tyrosine. None of the tyrosines in BFR-RC have suitable His proton acceptors nearby. However, we cannot exclude that water hydroxyls in specific hydrogen bond networks may accept protons from tyrosine.
In solution reaction 3 occurs at +1380 mV, in PSII the calculated E m for this reaction is raised to +1576 mV [35]. We estimated that Tyr25 has the lowest oxidation potential of all tyrosines in BFR-RC, its E m is around 1220 mV. Our calculations indicate that the single oxidized ZnCe 6 with oxidation potential of 570 mV at pH 6 would not be capable of tyrosine oxidation. Considering that a tyrosine cation radical EPR signal in BFR-RC has been observed after a prolonged exposure to saturating light or several saturating laser flashes [16,38] we suggest that a photogenerated ZnCe 6 di-cation [39] may be responsible for oxidizing tyrosine. The ZnCe 6 di-cation, having a solution value of 1022 mV, would have a sufficiently high E m to oxidize tyrosine in BFR-RC after taking protein effects into account.
It is clear from our results that the relatively low oxidation potential of ZnCe 6 limits its use in the construction of a BFR-RC that would eventually be capable of oxidizing water. What factors could help increase the oxidation potential of ZnCe 6 ? Oxidation potentials of reaction center chlorophylls in photosynthetic organisms span a wide range from only 500 mV for P700 in PSI [40] and P870 in purple bacteria [41] to 1,100-1,200 mV for P680 in PSII [42,43]. Several major factors give rise to the differences in E m [44]. Most of the difference in E m between reaction centers of PSI and PSII originates from the protein atomic charges and charges of cofactors. Together they up-shift The Em of ZnCe 6 in Engineered 'Reaction Centre' PLOS ONE | www.plosone.org the E m of P D1 in PSII by 325 mV, but, in contrast, shift the E m of P A in PSI down by 2125 mV [44].
One of the major factors raising the E m of P680 in PSII is the unique Mn 4 Ca cluster, bearing a large positive charge. This cluster alone shifts the E m of P D1 in PSII up by 214 mV [44]. The BFR-RC has two di-manganese centers composed of Mn(II) ions. Each of the di-manganese centers shifts the E m of ZnCe 6 up by 85 mV. However, this effect in BFR-RC is compensated by the charged ligands to the manganese which down-shift the E m by 290 mV. The combined effect of all aminoacid sidechains was found to shift the E m of BFR-RC down by about 292 mV, not as much as the sidechains of PSI lacking the Mn 4 Ca cluster. It is likely, however, that this down-shift would become smaller and maybe even turn into an up-shift with photooxidation of Mn(II) ions and formation of m-oxo bridges.
Another major factor contributing to the increase of the E m in P680 in PSII is its position close to the luminal edge of two transmembrane a-helixes (helixes d D1/D2 providing axial His ligands to P D1/D2 ). The protein backbone dipole of helix d shifts the E m of P D1 in PSII by about 95 mV. The corresponding helix in PSI (helix j) shifts the E m of P A down by 28 mV [44]. The organization of helices coordinating ZnCe 6 in the BRF-RC is more similar to PSI than to PSII which is reflected by similar contributions from the backbones (40 mV).
The choice of ZnCe 6 for the role of photoactive pigment is attractive because of its availability and solubility in water, however, our work demonstrated that this pigment is not capable of providing sufficient oxidative power for the water splitting reaction. One of the problems is the detrimental effect of ionized carboxyl groups on the redox potential. Our calculations indicate that a simple replacement of these groups with neutral groups would increase the E m of ZnCe 6 by 130 mV. Even higher potentials may be achieved by replacement of the acidic groups with basic groups.
Axial ligands to Zn are another factor known to affect redox potentials. For example, it has been shown that Met-Met coordination increases the potential of heme by about 200 mV compared to His-Met ligated heme [45]. In this aspect BFR-RC already has the best axial ligand, and replacing Met with His in BRF-RC would likely shift the potential down.
A large fraction of the E m decrease observed in BFR-RC comes from interaction with the two Asp23 residues. Interestingly, this polar aminoacid is located in the hydrophobic region of the ZnCe 6 binding pocket. In addition, this aminoacid is conserved among ferritins from several organisms (PDB ID: 2FKZ, 3E1M, 3IS8, 3FVB) suggesting that it serves to regulate heme potential. Replacing it with an aliphatic residue is expected to eliminate this negative effect. Another potential modification is replacement of Asp50. In native BFR Asp50 forms a salt bridge with Lys53. Replacement of Asp50 with a polar or even basic aminoacid would break this salt bridge without affecting the interaction between the two monomers. This would facilitate the interaction of Lys53 with the carboxylic acids of ZnCe 6 potentially neutralizing the effect of their negative charge.
In summary we have determined a number of protein structural factors contributing to the redox potential of ZnCe 6 in BFR-RC, and by emulating some of the features of PSII it may be possible to raise the potential by several hundred millivolts, but not likely all the way up to the 1.2 V required to oxidize water. | v3-fos-license |
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} | pes2o/s2orc | Puri fi cation and characterization of xylanases from Trichoderma inhamatum
Background: Two xylanases, Xyl I and Xyl II, were puri fi ed from the crude extracellular extract of a Trichoderma inhamatum strain cultivated in liquid medium with oat spelts xylan. Results: Themolecular masses ofthe puri fi ed enzymes estimated by SDS-PAGE and gel fi ltration were, respectively, 19and14kDaforXylIand21and14.6kDaforXylII.Theenzymesareglycoproteinswithoptimumactivityat50°C enzymes.Thexylanases 462.2 protein (Xyl I) 10.7, 4.0 mg·mL 4553.7 1972.7 -1 (Xyl II) on oat spelts and birchwood xylan, respectively. The hydrolysis of oat spelts xylan released xylobiose, xylotriose, xylotetrose and larger xylooligosaccharides. Conclusions: The enzymes present potential for application in industrial processes that require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries.
Introduction
Xylans are a diverse and complex group of polysaccharides with the common feature of a backbone of β-(1 → 4)-linked xylose residues [1], in which side-chains are attached at the C2 and C3 positions of D-xylosyl. These substituents can normally be acetyl, 4-0-methyl-D-glucuronosyl or L-arabinosyl groups [2,3]. Xylan is the predominant hemicellulose found in plant cell walls strongly associated to cellulose microfibrils and the strength of this interaction is inversely related to the degree of substitution of the main chain by side-groups [4].
The conversion of xylan into useful products represents a significant part of the effort to achieve economical viability of the lignocellulose biomass processing and to develop different ways to produce chemicals and renewable energy as well. Owing to its complex structure, the complete degradation of xylan requires the joint of several hydrolytic enzymes acting in synergy that is known as a xylanolytic system. Endo-β-1,4-xylanases (β-1,4-D-xylan xylanohydrolases, EC 3.2.1.8) are the most important xylanolytic enzymes, cleaving internal glycoside bonds in xylan backbone, reducing the degree of polymerization of the polymer [5,6]. The cleavage carried out by these enzymes is not aleatory, i.e. side chain decorations in xylan are recognized by xylanases, and the degree of substitution in the polymer influences the product of hydrolysis. This difference in substrate specificity among different xylanases has important implications in the deconstruction of xylan [3].
Interest in xylanolytic enzymes has increased in last decades due to their industrial applications in the food, feed, and pharmaceutical industries and for sustainable production of fuels and chemicals. Besides, they can be applied in some processes in which cellulolytic activity must be absent, to preserve the vegetal fibers, in the pulp and paper industries, and in the processing of flax, hemp and jute in the textile industries [7,8,9].
Fungi are commonly used as source of xylanases and their xylanolytic systems have been widely studied [6,10]. Trichoderma spp. xylanases are among the most known enzymes, therefore, this fungal genus is suited for further examination of function and application of these enzymes [11]. Trichoderma reesei Rut C-30 is the most well-known Trichoderma strain producing several xylanases and cellulases with different biochemical properties and specificities for substrates, as predicted by genome sequence [12], and also many enzyme preparations obtained from the large-scale cultivation of this fungus have been commercialized. Recently, rational and efficient systems for the production of cocktails containing different balances between xylanolytic and cellulolytic enzymes have been investigated for the different application of these enzymes [13]. Xylanases from other Trichoderma species have also been studied as those from Trichoderma harzianum, Trichoderma lignorum, Trichoderma longibrachiatum, Trichoderma koningii, Trichoderma pseudokoningii and Trichoderma viride [14,15]. Other xylanases, including that from a psychrotrophic Trichoderma strain have been purified and characterized [16], and also xylanase encoding genes from other Trichoderma species have been isolated, cloned and expressed in Escherichia coli [17], Saccharomyces cerevisiae [18] and Pichia pastoris [19].
When a new efficient xylanase-producing microorganism is isolated, it is essential to purify and characterize the enzymes to know the action towards substrates of each component of a xylanolytic complex, its regulation and biochemical properties in order to develop more competitive processes. A Trichoderma inhamatum strain, isolated from soil in São Paulo state (Brazil), produces high levels of xylanase in the absence of cellulases [20], an important condition for some industrial applications, as stated above. The influence of some parameters affecting xylanase production by this fungal strain has already been investigated [21]. The present study aimed to purify and characterize the main xylanases produced by this fungus under previously optimized culture conditions.
Materials and methods
2.1. Fungal strain: maintenance and culture conditions T. inhamatum was deposited in the Environmental Studies Center Collection, CEA/UNESP, Brazil. The fungal strain was maintained on Vogel solid medium slants [22] with 1.5% (w/v) wheat bran, at 4°C and cultured periodically. The cultures were inoculated in the same medium with 1.5% (w/v) glucose and incubated for conidia production during 7 days at 28°C. Conidia were harvested, suspended in sterile distilled water and the concentration of the suspension was adjusted to 10 7 conidia milliliter -1 . One milliliter of this suspension was inoculated into Vogel liquid medium pH 6.0 with 1% (w/v) oat spelts xylan as sole carbon source for enzyme production. Cultures were maintained in orbital shaker (120 rpm) at 25°C for 60 h [21]. After cultivation, mycelium was removed by vacuum filtration and the crude culture filtrate was used as source of enzymes.
Enzyme assay
Xylanase activity was determined by incubating enzyme samples with 1% (w/v) birchwood xylan (Sigma, USA; xylose residues ≥90%) in 0.05 M sodium acetate buffer pH 5.5 at 50°C. At suitable intervals, the reaction was interrupted with 3,5-dinitrosalicylic acid (DNS) reagent and the released reducing sugars were measured [23], using xylose as standard. One unit of activity (U) was defined as the amount of enzyme capable to release 1 μmol of reducing groups per min. Specific activity was expressed by the relation between enzyme activity and protein content.
Determination of protein and carbohydrate
Protein was determined by the Lowry method [24], with bovine serum albumin as standard. During the chromatographic steps, proteins were detected by reading absorbance at 280 nm. Total carbohydrate was determined by the phenol-sulphuric acid method [25], with glucose as standard.
Purification of xylanases
The crude culture filtrate (200 mL) was dialyzed against 0.05 M Tris-HCl buffer pH 7.0 for 8 h, with buffer changes each 2 h, in order to exclude small molecules and obtain a buffered solution in this pH. This sample was submitted to ion exchange chromatography on a DEAE Sephadex A-50 column (1.1 × 14.5 cm), previously equilibrated with the same dialysis buffer; at 50 mL/h flow rate. Adsorbed proteins were then eluted with a 0.0-0.5 M NaCl linear gradient in the same buffer. Proteins were detected by reading absorbance at 280 nm and xylanase activity was assayed in the collected 3 mL fractions. Fractions with high xylanase activity were pooled and submitted to electrophoresis (SDS-PAGE). The sample corresponding to the not retained fractions was further dialyzed against 0.05 M ammonium acetate buffer pH 6.8 for 8 h, with buffer changes each 2 h, and then lyophilized. The sample was re-dissolved in a small volume of this buffer and applied to size exclusion chromatography on a Sephadex G-75 (1.8 × 60.5 cm) column equilibrated and eluted in the same buffer, at 19 mL/h flow rate. Proteins were detected by reading absorbance at 280 nm and xylanase activity was assayed in the collected 3 mL fractions. Fractions with high xylanase activity were pooled and submitted to electrophoresis (SDS-PAGE). All purification steps were carried out at 4°C.
Determination of molecular mass under non-denaturing conditions
Molecular masses of the proteins were estimated by gel filtration from a regression curve by plotting log of the molecular masses of the standards against the ratio between their elution volumes and the void volume (V 0 ), estimated using blue dextran. Standard proteins (Sigma, USA) were ribonuclease (15.4 kDa), chymotrypsin (25.0 kDa), ovalbumin (43.0 kDa) and bovine serum albumin (67.0 kDa).
Temperature and pH optima, stability in different temperature and pH
The best pH for activity of the purified xylanases was determined by assaying enzymatic reactions in McIlvaine buffer adjusted to various pH between 3.0 and 8.0, with 0.5 unit intervals at 50°C. For the optimal temperature, enzymatic reactions were carried out with the purified enzymes in 0.05 M sodium acetate buffer pH 5.5 at temperatures from 20 to 60°C, with 5°C intervals.
Thermal stability was determined by verifying residual activity after incubating samples of the purified enzymes without substrate at 40, 50 and 60°C during different periods. The pH stability was determined by verifying the remaining activity after incubating the purified enzymes for 24 h at room temperature. Diluted (1:2; v/v) enzyme samples were incubated with McIlvaine buffer in the pH range from 2.5 to 8.0, with 0.5 unit intervals.
Effect of substances on enzyme activity
The effect of substances was verified by assaying xylanase activity with a variety of compounds dissolved in 0.05 M sodium acetate buffer pH 5.5. The following substances were evaluated at 2 and 10 mM: lead acetate, ammonium chloride, barium chloride, calcium chloride, cobalt chloride, copper chloride, mercury chloride, iron sulfate, magnesium sulfate, manganese sulfate, zinc sulfate, tetrasodium ethylenediaminetetraacetate (EDTA), 1,4-dithiothreitol (DTT), sodium dodecyl sulfate (SDS) and phenylmethylsulfonyl fluoride (PMSF). All assays were carried out in triplicate.
Substrate specificity
The specificity of the xylanases was verified by assaying the activity against different substrates. Xylanase activity was measured on birchwood and oat spelts xylans. Endoglucanase (CMCase) and exoglucanase (Avicelase) activities were assayed in a reaction mixture with carboxymethylcellulose (CMC) and microcrystalline cellulose (Avicel), respectively. Substrates at 1% (w/v) were prepared in 0.05 M sodium acetate buffer pH 5.5 and appropriately diluted enzyme solution. Reducing sugars were quantified with the DNS acid reagent and the absorbance was measured at 540 nm. One unit of activity was defined as the amount of enzyme required to release 1 μmol of product equivalent per min in the assay conditions at 50°C.
Kinetic parameters
The enzymes were incubated with xylan from birchwood and from oat spelts (Sigma, USA; arabinose residues ≤ 10%, glucose residues ≤ 15%, xylose residues ≥ 70%) at concentrations varying from 4.0 to 30.0 mg·mL -1 . The Michaelis-Menten constant (K m ) and maximal velocity (V max ) were estimated from Lineweaver-Burk reciprocal plots [27]. Three experiments were carried out for each substrate concentration in triplicate and the straight line plotted was calculated by linear regression (Microsoft Office Excel, version 12.0) using mean values obtained from each experimental point.
Determination of hydrolysis products
The products of the enzymatic hydrolysis of oat spelts xylan were analyzed by thin-layer chromatography (TLC) on silica-gel G-60 plates (10 × 15 cm), using xylose and xylobiose solutions (1 mg/mL) as standards [28]. The mobile phase was ethyl acetate/acetic acid/formic Table 1 Purification of the xylanases from T. inhamatum.
Purification of T. inhamatum xylanases
Two xylanases were purified from the crude filtrate after growth of T. inhamatum in liquid cultures with xylan, under optimized culture conditions. Xyl II was purified to electrophoretic homogeneity by a single step of ion exchange chromatography, while Xyl I purification required a subsequent molecular exclusion chromatography with Sephadex G-75. The first step (Fig. 1) revealed two protein peaks with xylanolytic activity: Xyl I, corresponding to the not retained fraction, presented 62.7% of the activity, and Xyl II, the retained fraction was eluted with a NaCl gradient and presented 3.7% of the activity.
The sample corresponding to the first xylanase peak, considered the main xylanase produced by T. inhamatum, was subsequently subjected to molecular-exclusion chromatography (Fig. 2), giving rise to two protein peaks, one of them with xylanase activity, representing 12.0% of the initial activity. Xyl I and Xyl II from T. inhamatum were obtained with final specific activities of 3464.2 and 1216.4 U·mg prot -1 and 5.3 and 1.9-fold purification, respectively ( Table 1).
The occurrence of multiple enzyme forms is a common phenomenon and can be considered a specialized function of microorganisms to achieve a more effective hydrolysis of heterogeneous substrates in the nature [2]. Many xylanase forms are produced by T. reesei Rut C-30, for example. The two major xylanases produced by that fungus are Xyn1 and Xyn2, with the latter representing more than 50% of the total xylanolytic activity and both of them are responsible for more than 90% of the specific xylanase activity produced when the fungi was grown on cellulose or xylan [29]. Besides, the presence of several minor xylanases has also been demonstrated [30].
Physico-chemical properties of T. inhamatum xylanases
SDS-PAGE revealed a single band in each sample after the purification steps. The molecular masses estimated by this method were 19 kDa for Xyl I and 21 kDa for Xyl II (Fig. 3). The molecular masses estimated under non-denaturing conditions with Sephadex G-75 were 14.0 and 14.6 kDa for Xyl I and Xyl II, respectively. These values of molecular mass are similar to those observed for many Trichoderma xylanases [14]. The purified xylanases were highly glycosylated, presenting 79% and 62% of carbohydrates for Xyl I and Xyl II, respectively, which justifies some distortion observed in SDS-PAGE.
The activity profiles of Xyl I and Xyl II (Fig. 4a) showed that both enzymes present activity in slightly acid region with optimal activity in pH 5.0-5.5. Both enzymes showed more than 50% of the maximum activity in the pH range from 4.5 to 6.5, and the activity decreased sharply from this range. Optimal activity was observed at 50°C for Xyl I and at 45°C-55°C for Xyl II (Fig. 4b), the former presented more than 50% of the activity between 40°C and 55°C, and the latter between 35°C-60°C. Comparatively, the optima pH and temperature observed for the T. inhamatum xylanases are in accordance with those from many other xylanases from mesophilic Trichoderma strains that are commonly observed in the pH range from 3.5 to 6.0 and in temperatures from 45 to 60°C [14].
Both enzymes were stable at 40°C (Fig. 5), i.e. Xyl I retained 71% and Xyl II retained 77.5% of the activity after 4.5 h of incubation. Xyl I exhibited half-life of 4 min at 50°C and of 40 s at 60°C, while the half-life of Xyl II was 18 min at 50°C and 46 s at 60°C. The purified Trichoderma sp. K9301 xylanase was very stable at 50°C but also rapidly lost activity when incubated at 60°C [15]. The purified xylanases exhibited distinct stabilities in pH from 3.0 to 8.0 (Fig. 6). After 24 h incubation, residual activity of 90% or more was detected in pH from 4.5 to 6.5 for Xyl I, and from 4.0 to 8.0 for Xyl II.
The effect of organic compounds, metallic ions and a chelating agent on the activity of the purified xylanases from T. inhamatum is presented in Table 2. The ion Hg 2+ was a strong inhibitor of the xylanases even at 2 mM, while Cu 2+ also inhibited both enzymes, but this effect was more pronounced only at 10 mM. The inhibition by Hg 2+ seems to be a general property of xylanases, indicating the presence of cysteine thiol groups near or in the active site of the enzyme [31]. The denaturation caused by the detergent SDS resulted in loss of the activity and, in low concentration (2 mM), the effect was more pronounced on Xyl I. The 2+ and Fe 2+ and moderately inhibited by Zn 2+ only at 10 mM·Pb 2+ inhibited the enzymes with more pronounced effect on the Xyl II at 10 mM. EDTA at both concentration and PMSF at 10 mM slightly decreased the activity of both enzymes. The effect of EDTA on both enzymes suggests that they may require divalent ion for catalysis. DTT increased the activity of both enzymes, which can be explained by the prevention of the oxidation of sulfhydryl groups in the presence of this agent or by the reduction of disulfide bridges, restoring their native structure in some specific region or even of the catalytic site.
Substrate specificity and kinetic parameters
The purified enzymes hydrolyzed exclusively xylans with no activity on Avicel or CMC. The activities of Xyl I and Xyl II on oat spelts xylan, a branched arabinoxylan were, respectively, 8% and 16% higher than those on birchwood xylan, which is as less branched xylan with 94% xylose residues [32], indicating the preference of the enzymes for branched and heterogeneous xylan. Both enzymes exhibited Michaelis-Menten kinetics and the corresponding apparent constant values were calculated ( Table 3). The K m values indicated that both enzymes had higher affinity for birchwood than for oat spelts xylan.
With birchwood xylan as substrate, Xyl I presented the lower K m and therefore higher affinity, while with oat spelts xylan, Xyl II had more affinity. For both substrates, the higher value of k cat was that of Xyl II; for both enzymes, higher values of k cat and V max were observed with oat spelts xylan. The ratio k cat /K m for birchwood xylan was 19.9 and 21.7 s -1 ·mM -1 for Xyl I and Xyl II respectively, demonstrating similar efficiencies to hydrolyze this substrate. However, with oat spelts xylan, this ratio was about 2.5-fold greater for Xyl II than for Xyl I, indicating that the former enzyme is much more efficient in degrading this xylan. Two xylanases from Aspergillus giganteus [33] have similar catalytic efficiencies on these substrates, while the Penicillum capsulatum xylanase is 2.7-fold more efficient in hydrolyze oat spelts than birchwood xylan [34].
The mode of action was investigated by identifying the main products of oat spelts xylan hydrolysis by the purified xylanases. Since both enzymes gave products with the same R f values over various incubation intervals, only the TLC profile obtained with Xyl I is shown in Fig. 7. Both enzymes released xylobiose and larger xylooligosaccharides and, thus, they may be classified as endoxylanases. Interestingly, not even after 17 h, release of xylose was verified, in contrast to the xylose release observed after long-term incubation [16]. According to the Table 2 Effect of different substances on Xyl I and Xyl II from T. inhamatum. relative mobility to xylose [28], the two spots with lower mobility than xylobiose corresponded to xylotriose and xylotetrose.
Conclusions
This manuscript presents the first report about the purification and properties of two xylanases from T. inhamatum by a simple and inexpensive procedure. These enzymes were stable over a wide range of pH and the optimal conditions for their activities were around 50°C and pH 5.0 very similar to each other and also to the characteristics observed for the crude enzyme [21]. These two xylanases appear to be differentially modified products from the same gene because they have similar hydrolytic and physico-chemical properties, and differential glycosylation may explain the differences in molecular masses, the capacity to bind to DEAE-anion exchanger and thermal stabilities. The glycosylation explains some cases, but do not completely elucidate the functional and genetic basis for the multiplicity of these enzymes. Furthermore, a comparison of amino acid compositions indicates that three xylanases from Trichoderma harzianum E58 are products of distinct genes [35]. The results indicate possible employment of such enzymes in some industrial processes, which require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries. | v3-fos-license |
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} | pes2o/s2orc | The application of titanium dioxide (TiO2) nanoparticles in the photo-thermal therapy of melanoma cancer model
Objective(s): Photo-thermal therapy (PTT) is a therapeutic method in which photon energy is converted into heat to induce hyperthermia in malignant tumor cells. In this method, energy conversion is performed by nanoparticles (NPs) to enhance induced heat efficacy. The low-cytotoxicity and high optical absorbance of NPs used in this technique are very important. In the present study, titanium dioxide (TiO2) NPs were used as agents for PTT. For increasing water dispersibility and biocompatibility, polyethylene glycol (PEG)-TiO2 NPs (PEGylated TiO2 NPs) were synthesized and the effect of these NPs on reducing melanoma tumor size after PTT was experimentally assessed. Materials and Methods: To improve the dispersibility of TiO2 NPs in water, PEG was used for wrapping the surface of TiO2 NPs. The formation of a thin layer of PEG around the TiO2 NPs was confirmed through thermo-gravimetric analysis and transmission electron microscopy techniques. Forty female cancerous mice were divided into four equal groups and received treatment with NPs and a laser diode (λ = 808 nm, P = 2 W & I = 2 W/cm2) for seven min once in the period of the treatment. Results: Compared to the mice receiving only the laser therapy, the average tumor size in the mice receiving TiO2-PEG NPs with laser excitation treatment sharply decreased. Conclusion: The results of animal studies showed that PEGylated TiO2 NPs were exceptionally potent in destroying solid tumors in the PTT technique.
Introduction
Cancer still remains one of the leading causes of death with increasing incidence all over the world (1). The most pervasive method of cancer treatment is chemotherapy, which normally faces the problems of drug resistance and insufficient efficacy of drug delivery into cancer cells (1,2). Another common method in cancer treatment is surgery. Methods of cancer treatment strongly rely on tumor size, lymph node involvement, and how much the cancer has spread. Surgery in combination with chemotherapy is the primary treatment for cancers (3,4). The advent of nanoparticles (NPs) in biomedical and bioengineering fields made a revolution in the methods of cancer therapy. Nano-scale sizes of NPs improve their ability to be attached and transported to cells (2,5). Nanosized particles have been used in photodynamic therapy (PDT) and sonodynamic therapy of clinical cancer studies. PDT utilizes light absorbing photosensitizers to generate highly reactive oxygen species (ROS) that can cause cell rupture. Stimulated particles could fluctuate the electrons and have them transfer their charge from a state to another one, which produces active oxygen species (6). PDT has been used to treat malignant tumors and abnormal vasculatures (7). Production of toxic singlet oxygen and high photosensitivity of treated patients in this method could limit the PDT technique (4).
Photo-thermal therapy (PTT) by means of NPs promises a new technique to efficiently treat cancer cells without any major limitation or side effects. In particular, NPs play an efficient role in converting the photon energy of laser light into heat due to their specific physicochemical properties and inducing hyperthermia in malignant tissues (2,5). Thus far, a variety of nanostructures such as gold NPs (8), silver NPs (9), and carbon nanotubes (10) have been successfully developed in inducing hyperthermia in tumor tissues. A good candidate with specific characteristics for the PTT of tumors is titanium dioxide (TiO 2 ) NPs.
Recently, TiO 2 has attracted a growing deal of interest (11,12). It is used in pharmaceutical and cosmetic industries, and is generally considered to be biologically inert (13). It is bio-friendly and has exceptional properties, such as high refractive index, and photocatalytic and magnetic properties (14)(15)(16) Such characteristics of TiO 2 stem from the spontaneous formation of an oxide layer on the titanium surface (17). TiO 2 can destroy bacteria, viruses, fungi, and cancer cells (18) and can act as an effective catalyzer for treating malignant tumors (4,19). PDT, drug delivery, cell imaging, biosensors for biological assay, and genetic engineering are some forms of biomedical application of TiO 2 NPs (5). TiO 2 NPs could be a good choice for biomedical applications as agents in converting photon energy into heat in the PTT method, which is due to their super hydrophilicity (20), low-toxicity, good thermal conductivity, good optical absorption, and chemical and thermal stability in vivo (5). TiO 2 nanostructures have been used in drug delivery systems for different anticancer drugs, such as daunorubicin, temozolomide, doxorubicin, and cisplatin (21)(22)(23). To increase the biocompatibility of TiO 2 NPs, polyethylene glycol (PEG) could be attached to their surfaces. PEGylated NPs could escape the Reticulo-Endothelial System (RES) (18,19). In the present study, TiO 2 NPs were evaluated as impressive agents for PTT in vivo. To improve the dispersibility of TiO 2 NPs, a layer of PEG coated the NPs. The efficacy of TiO 2 -PEG NPs in the treatment of melanoma cancer model in the PTT technique was also assessed.
Preparation and characterization of TiO 2 -PEG NPs
The TiO 2 NPs used in this study (particle size: 10-25 nm, purity: > 99%, phase: anatase) were purchased from US Research Nanomaterials, Inc., the United States. To enhance the dispersibility of the TiO 2 NPs in deionized water, they were coated with a layer of PEG (10). Twenty five mg TiO 2 NPs was suspended in 25 mL deionized water. Then, 250 mg PEG 1000 (Sigma-Aldrich, St. Louis, MO, the USA) was dissolved in TiO 2 NPs suspension. The suspension was ultrasonicated for 15 min and then stirred at room temperature overnight to allow the hydrophilic polymer to wrap around the TiO 2 NPs (3). After being stirred, the suspension was centrifuged at 4000 rpm for 15 min to separate the unreacted TiO 2 NPs, and afterward, the supernatant was collected (3,10).
The microscopic image of the TiO 2 -PEG NPs was taken by a transmission electron microscope (TEM) (Philips Electron Optics, the Netherlands). The light absorption spectrum of the TiO 2 NPs was measured by a UV/Vis double beam spectrophotometer (PG Instruments Ltd., T80+ UV-Vis spectrophotometer, Lutterworth, the UK). Thermogravimetric analysis (TGA) was carried out by (METTLER TOLEDO, TGA 2 , Switzerland) under the dynamic atmosphere of an inert gas (N 2 ) at 30 ml/min.
Tumor induction
All experimental standards of this study were endorsed by the Animal Care and Use Committee of Shiraz University of Medical Sciences, Shiraz, Iran, and the experiments were done in accordance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals. All procedures were verified to minimize discomfort to the animals and to use as few animals as possible for statistical analysis. Fortunately, this experiment was approved by the Ethical Committee at Shiraz University of Medical Sciences.
A metastatic murine melanoma cell line, B16/F10 (NCBI C540 was purchased from the National Cell Bank of Pasteur Institute of Iran, Tehran, Iran) was cultured in an RPMI 1640 medium, under 5% CO 2 at the temperature of 37 °C. It was then prepared by 10 % fetal bovine serum, 100 IU/mL of penicillin and 100 µg/ mL streptomycin. Forty female C57BL/6J inbred mice, weighing 25-35 g, and aged 7-9 weeks were selected for the tumor induction. The murine melanoma cells at a number of 0.5 * 10 6 were suspended in 200 µl culture medium and injected subcutaneously into the loose skin over the neck (10). The mice were housed in standard cages under standard conditions with 14:10 hr light/dark cycle (lights on at 6:00 a.m.), at an ambient temperature of 25 ± 2 °C and 30% relative humidity. They were randomly divided into four equal independent groups (N = 10) and had access to normal chow and water ad libitum. Melanoma is a superficial tumor, so its changes could be observed easily during the treatment.
Photo-thermal therapy of tumors
Two weeks after the injection of the murine melanoma cell line, the melanoma tumors had sufficiently grown (approximately 1 cm 3 ) to start the treatment. The animals were anesthetized by injecting Ketamine and Xylazine intramuscularly (IM). The tumor regions were shaved and measured by a caliper and an ultrasound machine (Ultrasonix SonixOP; Burnaby, BC, Canada). The tumor size was estimated through the following equation: Tumor volume = (L/2)*W 2 (mm 3 ) (3, 10) In this equation, L and W indicate the length and width of the tumor, respectively. The treatment started according to the following grouping: Group Ι (TiO 2 +laser): 200 µl/cm 3 (tumor volume) TiO 2 -PEG NPs (1 mg/ml) were injected directly into the tumor and then excited by a laser diode.
Group ΙΙ (Laser therapy): The laser therapy was done without any pre-treatment with the NPs.
Group IV (Control): This group did not receive any treatment.
Groups Ι and ΙΙ were irradiated by a continuous wave (CW) near-infrared (NIR) laser diode (DAJCO, Shiraz, Iran) with these specifications: wavelength = 808 nm; power = 2 W; spot size = 1 cm 2 ; and intensity = 2 W/cm 2 for seven min once in the period of the treatment (10,24). However, the control cases were not irradiated. The tumor sizes were measured three days after the laser excitation. After the period of the treatment, the animals were euthanized and their masses were excised for histopathologic examination (five cases of TiO 2 +laser group were euthanized after three months of follow-up).
Histopathological examination
The specimens were treated, formalin fixed paraffin embedded (FFPE) blocks were provided, and the slides were stained with Hematoxylin and Eosin (H&E) method. The specimens were sampled for microscopy evaluation.
Statistical analysis
The numerical results of this study were presented as mean ± standard deviation (SD). The normality of the results was analyzed by the one-sample Kolmogorov-Smirnov test. Significant differences between the values were statistically tested by Student's t-test in each group. Multiple comparisons at multiple time points were tested by ANOVA with Repeated Measures. The statistical analyses were performed using SPSS® statistical software for Windows®, version 20.0 (SPSS Inc., Chicago, IL, USA). A P-value of < 0.05 was regarded as significant. Figure 1 shows the TEM image of PEG-coated TiO 2 NPs. Accordingly, a continuous layer of PEG with the average thickness of a few nanometers was formed on the surface of the TiO 2 NPs.
Preparation and characterization of the TiO 2 -PEG NPs
The UV-Vis light absorption spectrum of the TiO 2 -PEG NPs is provided in Figure 2. As shown in this figure, absorption occurred in two regions. It reached a peak at the UV range, and then it gradually reduced at visiblenear-IR range. Despite being at the wavelength of 808 nm, photoabsorption of these NPs is relatively low compared to those with a UV wavelength, but due to the deep penetration of NIR wavelength into the body (25), a CW NIR laser diode (808 nm) was used for photo irradiation (10). Additionally, the UV range is dangerous to the body and may cause gene mutation and DNA damage (26).
The TGA measurements provided further evidence regarding the interaction between the PEG and the surfaces of the TiO 2 NPs. Figure 3 shows the thermogram of the physical mixture of the TiO 2 NPs and PEG, and also the thermo-gram of TiO 2 -PEG. In the physical mixture of the TiO 2 NPs and PEG, weight loss occurred at two stages, 224.16 °C and 299.26 °C. TiO 2 -PEG showed a completely different pattern in comparison with the physical mixture of the TiO 2 NPs and PEG. In the TiO 2 -PEG pattern, weight loss occurred at one stage at 312.93 °C.
Photo-thermal therapy of tumors
The tumor sizes were recorded three days after laser excitation. The tumor sizes of different groups (before and three days after the treatment with PTT) were analyzed and the results showed significant differences between groups Ι, ΙΙΙ, and IV (P-value<0.05).
The decrease in tumor volume in group Ι is obvious as indicated in Figure 4. As can be seen in Figure 4, the average tumor volume in the Control and TiO 2 NPs groups increased. However, it significantly decreased in the TiO 2 +laser group. The tumor sizes in the Laser therapy group were not significantly different during the treatment, but the tumors ceased to grow further. The slopes of tumor size against time in the Control, Laser therapy, TiO 2 +laser, and TiO 2 NPs groups were about +103.58 mm 3 /days, +19.07 mm 3 /days, -40.60 mm 3 / days, and +86.70 mm 3 /days, respectively ("+" indicates increase and "-" indicates a decrease in tumor size).
The histopathologic evaluation of the tumors indicates a severe necrosis in the TiO 2 +laser group. Necrosis was the most important discriminator among the cases and its percentage was higher in the TiO 2 +laser group compared to the Laser therapy, TiO 2 , and Control groups, which indicates the noticeable effect of the TiO 2 -PEG NPs in inducing hyperthermia in the tumors when excited with a laser diode. Regressive fibrosis, lymphocytic infiltration, vascular invasion, or neurotropism were not seen in the cases. The histopathologic results are shown in Table 1 and Figure 6.
Five cases in the TiO 2 +laser group were followed up for three months after the treatment to analyze the trend of tissue regeneration. The stages of tumor treatment are shown in Figure 7, which indicates that the hair color of the tumor region changed after three months.
For further examination, three months after the start of the treatment these cases were euthanized and their mass was formalin fixed and sent for histopathologic evaluation. As shown in Figure 8, histopathology of the skin, the soft tissue of the back, and the bone marrow of the treated cases showed that there was no evidence of melanoma cancer in TiO 2 +laser cases after three months of follow-up.
Discussion
Titanium dioxide NPs have a wide variety of applications in medicine and life sciences. These NPs can be used as a carrier for drugs especially anticancer drugs and as an agent for photo-dynamic or PTT of solid tumors. TiO 2 NPs have been used as drug delivery systems for different anti-cancer drugs, such as paclitaxel, doxorubicin, daunorubicin, temozolomide, and camptothecin (18,(27)(28)(29)(30). In these studies, antitumor efficiency could be improved by TiO 2 NPs. However, these NPs tend to aggregate in aqueous media and may cause problems in biological systems. In order to prevent the aggregation of these NPs, their surfaces should be modified. One of the most common methods to prevent aggregation of NPs is to cover them with hydrophilic polymers (31). PEG is one of the best polymers to solve this problem. Through attachment of PEG to the surface of the NPs, the biocompatibility of the NPs would be increased. In addition, PEGylated NPs evade the RES (18). Formation of a thin layer of polymer on the surface of TiO 2 NPs through a simple adsorption method is reported in some studies (18,32,33). We used this polymer to improve the water dispersibility of TiO 2 NPs. The TEM and TGA techniques indicated the formation of a thin layer around the NPs. Then, these prepared NPs were used for PTT of solid melanoma.
The photobiological effects of visible and NIR light rely on their wavelengths and could be affected by the structure, vasculature, thickness, and pigmentation of the skin's strata (34,35). It is reported that a polarized beam in the visible-NIR range can cause biological effects in cells through electron oscillation inducement (36). Optical radiation with a longer wavelength penetrates further into the body than a shorter one (25,37). Visible light is widely absorbed by hemoglobin of the vasculature and the melanin located in the skin (37,38). Infrared beam affects the body through transferring thermal energy into tissues. In the infrared spectrum, scattering increases largely in the body component ; therefore, light will penetrate deeply into the body (34,39). NIR beam deeply penetrates into tissues and can be effectively utilized in cancer treatment. Some nanostructures, such as plasmonic NPs (40) and CNTs (10), can absorb NIR light and effectively convert its energy into heat. The stimulation of NPs causes vibrational stress of electrons and makes them transmit from the ground states to the excited states. The energy caused by electrons' displacement is converted into heat by electron-electron relaxation and electron-photon relaxation (40,41).
Stimulation of TiO 2 NPs with electromagnetic radiation in the range of visible or NIR light causes generation of cytotoxic ROS that induce apoptosis along with increasing the tumor region temperature (42,43). These effects are the principles of PDT and PTT, respectively.
Penetration of the UV-A spectrum in the body is too low. Besides, the UV-A spectrum could have an adverse effect on biological molecules. Therefore, we used NIR wavelength for tumor ablation. NIR wavelength is more transmissive through the body and has low attenuation in biological systems (44,45). Wenjun Ni et al. showed that black TiO 2 NPs are efficient as photosensitizers for PDT to kill bladder cancer cells. They used 808 nm light for irradiation of black TiO 2 NPs (46). In reported studies, the efficacy of TiO 2 NPs after UV and NIR irradiation on the cancer cells was evaluated in the in vitro cell culture medium (32,47). Considering these studies, we performed our research in the in vivo melanoma tumor model. In order to assess the PTT effects of the TiO 2 -PEG NPs, after injection of the TiO 2 -PEG NPs into the tumor, tumor sites were irradiated by an 808 CW laser diode. Results showed that the localized NPs caused significant necrosis due to the deep penetration of NIR beam into the body and good photoabsorption of TiO 2 -PEG at the wavelength of 808 nm. The findings of the animal studies indicated that in the TiO 2 +laser group, not only did the tumor growth cease, but the tumor size also shrank. The
Groups
Tumor size before the treatment Tumor size 3 days after the treatment * * * Figure 4. The tumor volume of different cases before and three days after PTT (* shows statistically significant difference (P<0.05)); the average tumor volume increased in the Control, TiO 2 NPs, and Laser therapy groups, but it decreased in the TiO 2 +laser group histopathological examination showed 70% necrosis in the TiO 2 +laser group, which confirms the good efficiency of the TiO 2 -PEG NPs in the NIR spectrum for the PTT method. Furthermore, following up the TiO 2 +laser cases for three months demonstrates good biocompatibility of these NPs in this technique.
This reported application of the TiO 2 -PEG NPs for in vivo trials could promise a biocompatible agent for this cancer therapy technique. Considering the results, the next step is to assess the efficacy of the TiO 2 NPs in tumor suppression by hyperthermia therapy and to deliver anti-cancer drugs to tumor sites, concurrently.
Conclusion
The present study assessed the application of PEGylated TiO 2 NPs in inducing hyperthermia and necrosis in malignant tumor cells for the PTT technique. The animal trials in this study confirmed the relatively high efficacy of such NPs in destroying solid tumors without any symptom of cancer cells in treated cases. Therefore, the TiO 2 -PEG NPs could be utilized as a potent agent with low toxicity in the PTT technique for ablating solid tumors.
Acknowledgment
This work was financially supported by Shiraz
Conflicts of Interest
The authors declare that they have no conflicts of interest. Authors disclose all relationships or interests that could have direct or potential influence or impart bias on the work. | v3-fos-license |
2018-12-11T03:58:31.458Z | 2014-08-10T00:00:00.000 | 54869114 | {
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} | pes2o/s2orc | Effect of Different Deposition Power of In 2 O 3 Target on the Characteristics of IGZO Thin Films Using the Cosputtering Method
The (In, Ga, Zn)Ox (IGZO) thin films were deposited on glass substrates using cosputtering method in radio frequency magnetron sputtering system. Zn 2 Ga 2 O 5 (Ga 2 O 3 -2 ZnO, GZO) and In 2 O 3 ceramics were used as targets and dual guns were used to deposit the IGZO thin films. Deposition power of GZO target was 80W and deposition power of pure In 2 O 3 target was changed from 70W to 100W, and the deposition time was 30min. The effect of deposition power of In 2 O 3 target on the crystalline, surface, electrical, and optical properties of the IGZO thin films was investigated at room temperature in a pure Ar atmosphere.The cosputtered IGZO thin films showed a very smooth and featureless surface and an amorphous structure regardless of the deposition power of In 2 O 3 target due to the room temperature sputtering process. However, the cosputtered IGZO thin films exhibited transparent electrode properties because they had high transmittance ratio and low resistivity. The value variations in the optical band gap (E g ) values of the IGZO thin film were evaluated from the plots of (αh])2 = c(h] − E g ). We would also show that the deposition power of In 2 O 3 target would have a large effect on mobility and E g value of the IGZO thin films.
Introduction
The hydrogenated amorphous silicon (-Si:H) thin-film transistor (TFT) has been the work material in the activematrix liquid-crystal display (AM-LCD) industry for a long time.The typical plasma enhanced chemical vapor deposition (PECVD) -Si:H TFT has field effect mobility ( eff ) of 0.6∼ 0.8 cm 2 /Vs, subthreshold swing of 0.3∼0.4V/decade, off-state drain current ( Doff ) below 10 −13 A, and on-to-off ratio about 10 7 [1].The -Si:H TFT backplane configuration does not work well in such short time and the pulse/clock signal can be distorted [2].To address this issue, proposed solutions are focused on reducing the gate bus-line RC propagation delay, such as adding gate planarization layer [3], adopting buried bus-line structure [4], or using low resistance Cu interconnection [5].The other emerging area in AM-flat panel display (FPD) is the emissive display such as activematrix organic light-emitting display (AM-OLED), where the organic light-emitting diode (OLED) is directly integrated with the TFT pixel electrode circuit.AM-OLED avoids the need of backlight and its dynamic range of brightness can be controlled at the pixel level, which is ideal for TV applications [6].However, conventional metal oxide semiconductors (TCOs) such as zinc oxide (ZnO) are polycrystalline in nature, even at room temperature.The grain boundaries of such metal oxides could affect device properties, such as uniformity and stability, over large areas.Recently, transparent amorphous oxide semiconductors (TAOSs) thin films, such as Sn-Zn-O (SZO), In-Zn-O (IZO), and In-Ga-Zn-O (IGZO), have received a considerable attention in the largearea FPD industry since they may overcome the difficulties encountered in the amorphous -Si:H and polycrystalline silicon TFTs (poly-Si TFT) technologies [7].For that, over the last several years, there has been great interest in thin-film transistors made of TAOSs.This is mainly due to TAOSs thin-film transistors having unique advantages, such as visible light transparency, largearea uniform deposition at low temperature, and high carrier mobility.The -IGZO thin films were deposited on polyethylene terephthalate at room temperature and exhibited Hall effect mobility exceeding 10 cm 2 V −1 s −1 , which is an order of magnitude larger than for hydrogenated amorphous silicon [8].IGZO thin films are a semiconducting material and they can be used as the TFT backplane of FPDs.IGZO-TFT was developed by Professor Hosono's group at Tokyo Institute of Technology and Japan Science and Technology Agency (JST) in 2003 for crystalline IGZO-TFT [7] and in 2004 for amorphous IGZO-TFT [8].IGZO-TFT has 20-50 times higher mobility than that of amorphous silicon, which has been used for current liquid-crystal displays (LCDs) and electronic papers.Therefore, the IGZO-TFT can improve operation speed, resolution, and size of FDPs, and it is also considered as one of the most promising TFTs to drive OLED displays.In the past, various techniques, such as electron beam evaporation, ion beam assisted deposition, and ion implantation, were used for growth of the IGZO thin films.For example, Nomura et al. got the IGZO thin films by codepositing the Ga:In 2 O 3 and Zn:In 2 O 3 targets to deposit the Ga and Zn codoped In 2 O 3 electrode at room temperature [8].In the past, Ohta et al. used the high-resolution transmission electron microscopy (HR-TEM) to analyze the IGZO thin films and they found that the HR-TEM image of sc-IGZO thin film clearly shows a layered structure [9].Nomura et al. presented that IGZO crystal is composed of alternating stacks of InO 2 − and GaO(ZnO) + layers and the In 2 O 3 concentration has large effect of the characteristics of the IGZO thin films, especially in the electrical properties [10].In this study, we first prepared Zn 2 Ga 2 O 5 and In 2 O 3 targets separately and cosputtering method was used to deposit the IGZO thin films at room temperature on glass substrates.This study examined the structural, surface, optical, and electrical properties of the IGZO thin films as a function of deposition power of In 2 O 3 target.
Experimental
Ga 2 O 3 powder (99.99%) was mixed with ZnO powder (99.99%) to form the Ga 2 O 3 -2 ZnO composition (abbreviated as GZO).After being dried and ground, the GZO powder was calcined at 800 ∘ C for 1 h and then ground again.GZO powder and pure In 2 O 3 powder were mixed with polyvinyl alcohol (PVA) as binder.The mixed powders were uniaxially pressed into pellets of 5 mm thickness and 54 mm diameter using a steel die.After being debindered, the GZO and pure In 2 O 3 pellets were sintered at 1200 ∘ C and 1250 ∘ C, respectively, for 2 h.Glass substrates (Corning 1737) with an area of 2 × 2 cm 2 were cleaned ultrasonically with isopropyl alcohol (IPA) and deionized (DI) water and then dried under a blown nitrogen gas.Then, GZO and pure In 2 O 3 targets were used to deposit the thin films at the same time.Deposition power of GZO was 80 W and deposition power of pure In 2 O 3 was changed from 70 W to 100 W, respectively; room temperature (RT) was used as deposition temperature, and deposition time was 30 min.The base pressure of sputtering chamber was below 5 × 10 −6 Torr and the working pressure was maintained at 3 × 10 −3 Torr in pure Ar (99.99%) ambient.
Thickness and surface morphology of the IGZO thin films were measured using a field emission scanning electron microscopy (FESEM), and their roughness and crystalline structures were measured using atomic force microscopy (AFM) and X-ray diffraction (XRD) patterns with Cu K radiation ( = 1.5418Å).Energy dispersive spectrometer (EDS) and secondary ion mass spectrometry (SIMS) analyses were used to find the variation in the concentration of the IGZO thin films.The optical transmission spectrum was recorded using a Hitachi U-3300 UV-Vis spectrophotometer in the 250-1000 nm wavelength range, while the Hall effect coefficient of the IGZO thin films was measured using a Bio-Rad Hall setup.
Results and Discussion
As the different sintering temperatures are used, the differently crystalline phases will be formed in the IGZO ceramic targets, and the multicrystal phases are only observed in the IGZO ceramic targets.Lo and Hsieh found cubic Ga 2 ZnO 4 spinel and rhombohedral InGaZnO 4 phases are identified in the 1100 ∘ C-sintered sample in addition to the as-prepared oxide powder phases of In 2 O 3 , Ga 2 O 3 , and ZnO [11].However, most of the IGZO thin films will reveal the amorphous phase rather than the polycrystal phases.For example, Jeong and Kim codeposited the Ga:In 2 O 3 and Zn:In 2 O 3 targets to obtain the IGZO thin films, which revealed the amorphous phase [12].Jung et al. deposited the IGZO thin films by using the facing targets sputtering (FTS) method at room temperature; also only the amorphous phase was observed [13].The diffraction intensity of InGaZnO 4 phase was not observed in XRD patters, as Figure 1 shows.The cubic Ga 2 ZnO 4 and spinel rhombohedral InGaZnO 4 phases and the phases of precursor In 2 O 3 , Ga 2 O 3 , and ZnO were also not observed in Figure 1.One weak and broad peak was assigned to the glass substrate and two weak and broad peaks were assigned to the glass substrate.Those results suggest that all the IGZO thin films exhibit the amorphous phase.FESEM was used to examine the surface roughness and morphology of the IGZO thin films.Figure 2 shows the FESEM surface images of the IGZO thin films under different deposition powers of In 2 O 3 target, which indicates that as deposition power was changed, the surface morphologies had no apparent change as well.As the deposition power of In 2 O 3 target was increased from 70 W to 100 W, as Figure 2 shows, morphology of the IGZO thin films exhibited a very smooth surface regardless of deposition power of In 2 O 3 target.Surface morphology of all deposited IGZO thin films showed the nanoparticle structure of IGZO grains.Therefore, all IGZO thin films showed stable and flat amorphous surface features.In order to achieve high performance transparent oxide TFTs or memory devices, the preparation of source and drain electrodes with a smooth surface morphology is very important because surface roughness of the IGZO thin films will influence the leakage current between the semiconducting IGZO active layer and source/drain electrodes.The root-mean-square (RMS) surface roughness was measured to be 0.30 nm by AFM, and measured values were 6.357 nm, 7.856 nm, 6.949 nm, and 7.253 nm as the deposition power of In 2 O 3 target was 70 W, 80 W, 90 W, and 100 W, respectively.This result suggests that the IGZO thin films deposited by the cosputtering method have the low roughness surfaces and can be used to fabricate the high performance transparent oxide TFTs or memory devices.
Figure 3 shows the cross-section observations of the IGZO thin films as a function of deposition powers of In 2 O 3 .Calculating the results in Figure 3, thickness of the IGZO thin films increased with increasing deposition powers of In 2 O 3 target.Thickness of the IGZO thin films was around 178 nm, 202 nm, 235 nm, and 269 nm, as the deposition powers of In 2 O 3 target were 70 W, 80 W, 90 W, and 100 W, respectively.Because the deposition power of GZO target is not changed, the increase in the thickness of the IGZO thin films is caused by the increase of the composition of In 2 O 3 in the IGZO thin films.As the crosssection micrographs shown in Figure 3 are compared, there were different results as the deposition power of GZO target was changed.As the deposition power of GZO target was 70 W, the IGZO thin films grew like a densified group of nanowires, which had the structure of highly oriented nanowires parallel to the substrate normal.
As 80 W was used as the deposition power of GZO target, the IGZO thin films also grew like a densified group of nanowires, but they had the structure of oriented bars with random direction.As the deposition power of GZO target was 90 W and 100 W, the nanowire-aggregated growths were transformed into the irregular plate-shaped growths with no special direction.These results prove that as the deposition power of GZO target in the cosputtering method is changed, the IGZO thin films have different surface morphology.As we know, ZnO-based thin films have the high c-axis orientation in the (00c) direction.As lower deposition power of In 2 O 3 target is used, ZnO will dominate the growth mechanism − and GaO(ZnO) + layers will dominate the growth mechanism.For that, the cross-section of the IGZO thin films shows irregular plate-shaped growths.
In addition, there is no evidence of the segregation of GZO and In 2 O 3 due to the uniform cosputtering of GZO and In 2 O 3 targets using tilted cathode guns.Nomura et al. reported that IGZO crystal is composed of alternating stacks of InO 2 − and GaO(ZnO) + layers and the concentration of In 2 O 3 has a large effect on the crystallization of IGZO thin films [10].For the InO 2 − layer an In 3+ ion is located at an octahedral site coordinated by six oxygens and for the GaO(ZnO) + layer Ga 3+ and Zn 2+ ions are located at trigonalbipyramidal sites and are each coordinated by five oxygens and alternately stacked along the (0001) direction.As the concentration of In 2 O 3 decreases, the crystallization will be inhibited.The results observed from the cross-session images of the IGZO thin films shown in Figure 3 agree with the results of Nomura et al.As Figure 3 shows, as the deposition power of In 2 O 3 target is 70 W, the nanowires were parallel to the substrate normal direction.This result suggests the nanowires are stacked along the (0001) direction.As the deposition power of In 2 O 3 target increases, the structure of nanowires is changed to irregular plate-shaped growths, which suggests the decrease in the crystallization of the IGZO thin films.Due to the large variation in ionization probabilities among different materials, energy dispersive spectrometer (EDS) and secondary ion mass spectrometry (SIMS) are generally considered to be the qualitative techniques.The two methods are used in materials sciences and surface sciences to analyze the composition of solid surfaces and thin films and they can be used to find the variations of atom ratios at the surface (EDS) and at the deposition profile (SIMS) of thin films.Atomic ratio microanalysis in the FESEM is performed by measuring the energy or wavelength and intensity distribution of X-ray signal generated by a focused electron beam on the specimen.With the attachment of EDS, the precise elemental composition of materials can be obtained with high spatial resolution.This suggests that EDS analysis allows one to identify what those particular elements are and their relative proportions, for example, the atomic ratio.Table 1 shows that the atom ratios of Zn and Ga elements decreased and atom ratio of In element increased with increasing deposition power of In 2 O 3 target.These results suggest that as the deposition power of In 2 O 3 target increases, more atoms will be moved from the surface of In 2 O 3 target and then atom ratio of In element increases.
SIMS can analyze the composition by sputtering the surface of specimen with a focused primary ion beam and collecting and analyzing ejected secondary ions.The mass/ charge ratios of these secondary ions are measured with a mass spectrometer to determine the elemental, isotopic, or molecular composition of the surface to a depth of 1 to 2 m.Because SIMS is a high sensitivity surface analysis technique for the determination of surface composition and contaminant analysis and for depth profile in the uppermost surface layers of a sample, it can detect very low concentrations of dopants and impurities.For that, the SIMS analysis was used to find the distribution of Zn, Ga, and In elements in the profile of the IGZO thin films and the results are shown in Figure 4, where the deposition power of In 2 O 3 target was 100 W. The IGZO thin films showed that there were incorporations of Zn, Ga, and In atoms in the IGZO thin films, even when the deposition was carried out at room temperature.The concentrations of In and Ga elements in the profile are almost unchanged as the deposition power was increased from 70 W to 100 W.However, the concentration of Zn element in the profile first decreased and then increased as the analyzed depth increased.However, the relative In concentration in the profile of the IGZO thin films is larger than that of the predicted values obtained from the used targets.The variation in Zn concentration is caused by the fact that as GZO target is deposited at 80 W, the decomposition of GZO will happen and the ZnO will vaporize during the deposition process.For that, the Zn concentration is not uniform in the depth profile.
Figure 5 shows the transmission ratios of the IGZO thin films plotted against wavelengths in the region of 250-1000 nm, with deposition time as the parameter.As the deposition power of In 2 O 3 target was 70 W, 80 W, 90 W, and 100 W, the average transmittance ratio of the IGZO thin films in the range of 400 nm∼700 nm was 87.4%, 86.7%, 87.7%, and 86.6%, and the highest transmittance ratio was 97.4%, 96.9%, 95.6%, and 96.3%, respectively.Those results suggest that as the cosputtering method is used, we can deposit the IGZO thin films with high transmittance ratio.The results in Figure 5 also show that the transmittance ratios in the visible light region are almost unchanged as the deposition power of In 2 O 3 target is changed from 70 W to 100 W.However, as the deposition power of In 2 O 3 target is changed from 70 W to 100 W, the average transmittance ratios of the IGZO thin films in the range of 400 nm∼700 nm have no apparent change.From the results shown in Figure 1, the surfaces of all deposited IGZO thin films reveal a smooth structure and no agglomerated particles are observed, which are the reasons to cause the IGZO thin films having high average transmittance ratios.For the transmission spectra shown in Figure 5, as the different deposition power of In 2 O 3 target was used, the shift of the optical band edge was not really observable and a greater sharpness was noticeable in the curves of the absorption edge.Those results suggest that the optical band gap ( ) values have no apparent change as the cosputtering method is used to prepare the IGZO thin films.Figure 5 also shows that the IGZO thin films deposited on glass substrates had high average transmittance ratios of over 92.7%, 97.6%, 96.3%, and 95.8% in the near-infrared region (700 nm∼1000 nm) as the deposition power of In 2 O 3 target was 70 W, 80 W, 90 W, and 100 W, respectively.In the past, determination of the values was often necessary to develop the electronic band structure of a thin-film material.However, as the extrapolation method is used, the values of thin films can be determined from the absorption edge for direct interband transition.The absorption coefficient was calculated using Lambert's law as follows: International Journal of Photoenergy where and are thin film's transmittance ratio and thickness.The absorption has a maximum at a high energy and decreases with optical energy in a manner similar to the absorption edge of semiconductors.As the extrapolation method is used, the values of thin films can be determined from the absorption edge, which can be calculated using the relation in ( 3): where is the optical absorption coefficient, is the constant for direct transition, ℎ is Planck's constant, and ] is the frequency of the incident photon [14].The linear dependence of (h]) 2 on ℎ] indicates that the IGZO thin films are direct transition type semiconductors.In accordance with (3), as Figure 6 shows, the calculated values of the IGZO thin films increased from 3.71 eV, 3.79 eV, and 3.84 eV to 3.87 eV as the deposition power of In 2 O 3 target was increased from 70 W, 80 W, and 90 W to 100 W, respectively.Because ZnO, Ga 2 O 3 , and In 2 O 3 thin films have different values, the variation in values is believed to be caused by the variation in the composition of the IGZO thin films.When the IGZO thin films are deposited using the cosputtering method, three reasons are believed to influence the carrier mobility of IGZO thin films.First, depositing at room temperature cannot provide enough energy to enhance the motion of plasma molecules, which will improve the crystallization and grain size growth of IGZO thin films, and the defects in the IGZO thin films will be generated during the deposition process.Second, if the agglomerated particles in the IGZO thin films increase, that will cause the decrease in the inhibiting of the barriers electron transportation and the mobility will increase.Third, Hosono showed that, from the electron mobility and concentrations evaluated from the Hall effects for amorphous IGZO thin films with different compositions, the mobility is primarily determined by the fraction of In 2 O 3 content and the highest value of ∼40 cm 2 (V s) −1 is obtained around the samples containing the maximum In 2 O 3 fraction [15].From those reasons, the carrier mobility, carrier concentration, and resistivity of the IGZO thin films are believed to be dependent on deposition power (or concentration) of In 2 O 3 .
However, as Figure 7 indicates the electrical properties of the IGZO thin films, the carrier concentration linearly decreased as the deposition power of In 2 O 3 target was larger than 80 W and the carrier concentration decreased from 3.67 × 10 20 cm −3 to 4.98 × 10 19 cm −3 , respectively.The carrier mobility linearly increased from 5.83 cm 2 /V-s to 63.5 cm 2 /Vs as the deposition power of In 2 O 3 target increased from 70 W to 100 W. Those results suggest that the concentration of In 2 O 3 is the most important factor to influence the mobility of the IGZO thin films and the results agree with the important results investigated by Hosono [15].The resistivity of TCO thin films is proportional to the reciprocal of the product of carrier concentration and mobility : Both the carrier concentration and the carrier mobility contribute to the conductivity.As the deposition power of In 2 O 3 target was changed from 70 W to 100 W, the resistivity of the IGZO thin films was changed from 8.85 × 10 −3 Ω-cm to 7.68 × 10 −4 Ω-cm.The minimum resistivity of the IGZO thin films at a deposition power of In 2 O 3 target of 100 W is mainly caused by the carrier mobility at its maximum.
Conclusions
The characteristics of the IGZO thin films prepared by Ga 2 O 3 -2 ZnO (GZO) cosputtered In 2 O 3 method were well investigated in this study.As deposition time was 30 min, thickness of the IGZO thin films was around 178 nm, 202 nm, 235 nm, and 269 nm, as the deposition powers of In 2 O 3 target were 70 W, 80 W, 90 W, and 100 W, respectively.As the deposition power of In 2 O 3 target was 70 W, 80 W, 90 W, and 100 W, the average transmittance ratio of the IGZO thin films in the range of 400 nm∼700 nm was 87.4%, 86.7%, 87.7%, and 86.6%, and the calculated values increased from 3.72 eV, 3.79 eV, and 3.84 eV to 3.87 eV, respectively.As the deposition power of In 2 O 3 target increased from 70 W to 100 W, the carrier mobility of IGZO thin films linearly increased from 5.83 cm 2 /V-s to 63.5 cm 2 /V-s.The mobility of 63.5 cm 2 /V-s is higher than that of most reported IGZO thin films.We had found that the IGZO thin films deposited by the cosputtering method had the low roughness surfaces and could be used to fabricate the high performance transparent oxide TFTs or memory devices.
Figure 1 :
Figure 1: XRD patterns of IGZO thin films as a function of deposition power of In 2 O 3 target.
Figure 2 :
Figure 2: Surface morphology of IGZO thin films as a function of deposition power of In 2 O 3 target.(a) 70 W, (b) 80 W, (c) 90 W, and (d) 100 W, respectively.
Figure 3 :
Figure 3: Cross-section observations of IGZO thin films as a function of deposition power of In 2 O 3 target.(a) 70 W, (b) 80 W, (c) 90 W, and (d) 100 W, respectively.
Figure 4 :
Figure 4: Second ion mass spectrometry analysis of IGZO thin film; the deposition power of In 2 O 3 target was 100 W.
Figure 5 :
Figure 5: Transmittance spectrum of IGZO thin films as a function of deposition power of In 2 O 3 target.
Figure 7 :
Figure 7: Hall mobility, carrier concentration, and resistivity of IGZO thin films as a function of deposition power of In 2 O 3 target.
Table 1 :
Atom ratios of Zn, Ga as a function of deposition power of In 2 O 3 target. | v3-fos-license |
2020-03-11T15:00:29.378Z | 2020-03-11T00:00:00.000 | 212654390 | {
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} | pes2o/s2orc | Use of the lignocellulose-degrading bacterium Caldicellulosiruptor bescii to assess recalcitrance and conversion of wild-type and transgenic poplar
Background Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. Results Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. Conclusions Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.
wastes, dedicated cultivated feedstocks, and sustainable harvest of forest lands, to name a few sources [10]. In addition, more than half of land-based biomass is carbohydrate in the form of biopolymers, primarily cellulose, a relatively homogenous linear polymer of β-1,4-linked glucose. In angiosperms, the other major carbohydrate components, hemicelluloses, are heterogeneous polymers of primarily xylose along with smaller amounts of arabinose, mannose, rhamnose, galactose, glucose and glucuronic acid [9].
While these substrates are rich in carbohydrate content, the barrier to biomass conversion of the carbohydrate content is the recalcitrance of renewable feedstocks [6], which has been shown to be a strong function of lignin content [16]. In attempts to reduce recalcitrance, transgenic trees and grasses have been generated through a variety of molecular strategies [24], although the efficacy of microbial conversion of these biomasses to fermentation products is highly variable [18,19] and significantly dependent on pretreatment conditions. Such transgenic modifications have ancillary consequences, such as effects on growth. Thus, striking a balance between reducing feedstock recalcitrance, often by lowering lignin content, and achieving excellent growth and fitness under field conditions is a key challenge for developing renewable transgenic biomasses.
In order to determine the suitability of transgenic feedstocks for production of bio-based chemicals, direct deconstruction by microorganisms can provide insight that complements evaluations based on standard simultaneous saccharification and fermentation (SSF) assays utilizing exogenously added enzyme cocktails. Caldicellulosiruptor bescii is an extremely thermophilic bacterium, capable of degrading unpretreated lignocellulosic biomass and deconstructing and metabolizing the cellulose and hemicelluloses [2]. Furthermore, C. bescii has been metabolically engineered to produce non-native fermentation products, such as ethanol [3]. In addition to prospects for using C. bescii as a platform organism for direct processing of unpretreated biomass, there is also the prospect of using this bacterium to screen for the efficacy of transgenic manipulations of lignocellulosic biomass to reduce recalcitrance. Here, previously generated and reported transgenic poplar lines with a broad variation in the lignin content and composition [28] were subjected to fermentation by C. bescii to assess both recalcitrance and convertibility to bio-based ethanol and acetate.
Monolignol targeted transgenic poplar lines
Lignin is the primary component of plant cell walls responsible for biomass recalcitrance [13]. Many studies have attempted to not only reduce its content as a fraction of the biomass, but also to modify its composition and linkage structures by down-regulation of genes in the biosynthetic pathways of monolignols or enzymes involved in lignin polymerization [16]. Monolignol biosynthesis can be viewed in terms of three major steps: (1) production of 4-coumaric acid from phenylalanine; (2) modification of aromatic ring side groups to hydroxy or methoxy moieties at positions 3 and 5; and (3) conversion of the three-carbon branch from an organic acid to an alcohol (Fig. 1). Various genetic strategies to modify the monolignol synthesis pathway have been utilized to generate transgenic poplar lines with reduced recalcitrance (for examples of previous efforts, see Table 1). Most have involved strategies that manipulate a single gene in each transgenic line. Improvements in conversion of the carbohydrate content to sugars from these efforts have likewise been highly variable.
While multiple separate studies have perturbed monolignol synthesis genes in various Populus species, a systems biology-based approach to evaluate the aspects of all of the genes involved in monolignol synthesis provides a more substantial perspective and can be subjected to a more robust analysis. Such a strategy to generate transgenic lines with reduced recalcitrance is likely to be more successful at identifying optimal targets given the intrinsic complexity of monolignol biosynthesis [15]. Along these lines, recent work [28] sought the most promising avenues for modifying P. trichocarpa lignin structure and content with an eye towards favorable wood characteristics and plant fitness. The down-regulation of 21 genes involved in monolignol biosynthesis, individually and by gene-pairs and gene families, were considered as a basis for a mathematical model that predicted wood traits as a function of transgenic changes. A collection of transgenic lines with a broad set of phenotypes was generated and characterized [28]. Selected characterization data, such as carbohydrate and lignin content, from that study are summarized in Table 2, along with previously reported transcript abundance relative to the wild type of the target gene(s). The goal here was to determine how these genetic and transcriptomic alterations in the transgenic poplar lines affected microbial biomass solubilization and bioproduct formation and point to further favorable outcomes for bio-based chemical production.
Biomass preparation
All wild type and transgenic greenhouse grown Populus trichocarpa samples were created and prepared as described elsewhere [28]. The untreated stems of 6-month-old trees were stripped of bark and air dried for approximately 72 h. The dried stem segments were milled utilizing a Wiley Mill and sieved to 40/80 mesh. The 40/80 mesh material was water-washed by adding 1.5 g of material to a 50 mL conical centrifuge tube and filling with deionized water. The centrifuge tube was centrifuged and the supernatant discarded. This was repeated twice more and the pelleted material was dried at 50 °C.
Biomass solubilization
Following the 7-day incubation of C. bescii with the washed biomass, the sealed serum bottles were removed from the shaking incubator and allowed to cool to room temperature. The entire 50 mL contents were transferred
Enzymes for Conversion of Organic Acid to Alcohol
Metabolic pathway grid for monolignol biosynthesis present in angiosperms. Monolignol biosynthesis from phenylalanine with enzymes responsible for conversion of phenylalanine to 4-coumaric acid, modification of the 3′ and 5′ side groups on the aromatic ring, and the conversion of organic acid to alcohol on the three carbon branch at 1′ position on the aromatic ring to a 50 mL conical centrifuge tube and centrifuged, as described above. A portion of the supernatant was sterile-filtered and saved for fermentation product analysis. The remainder of the supernatant was discarded. Each serum bottle was rinsed with deionized water to remove any remaining biomass and this was added to the biomass pellet in the centrifuge tube. The centrifuge tube was filled with water up to 45 mL, shaken to loosen the pellet, and centrifuged again to pellet the biomass. The supernatant was again removed and another wash performed. After the final wash and removal of the supernatant, the pellet was dried at 50 °C and the weight was recorded to calculate biomass solubilization.
Biomass properties
Biomass properties, such as lignin content, carbohydrate content, growth phenotypes and others reported in Table 2, were determined and reported previously [28]. Microbial solubilization and fermentation were generated as part of this study and analyzed in part with the previously reported biomass properties.
Microbial growth on biomass
Caldicellulosiruptor bescii was cultured at 50 mL in sealed serums bottles on 5 g/L DSMZ671 defined media with the washed biomass as the only substrate, as described previously [22]. Cultures were incubated at 65 °C for 7 days (with shaking at 150 RPM) after which fermentation products were analyzed and biomass solubilization was measured, as described above.
Analysis of fermentation products
The sterile-filtered supernatant obtained from the culture was utilized for fermentation product analysis. Results and discussion
C. bescii fermentation of transgenic lines of P. trichocarpa
Based on previous work [28], 17 transgenic samples of P. trichocarpa (Additional file 1: Table S1), along with the wild-type control, were fermented without pretreatment with a metabolically engineered strain of C. bescii in which the adhE gene (bi-functional alcohol dehydrogenase) from Clostridium thermocellum was inserted to enable the generation of ethanol, in addition to its natural fermentation products: acetate, H 2 and CO 2 [29]. Poplar stems (bark removed) were milled and sieved to 40/80 mesh, water washed and dried, and incubated with C. bescii for 7 days at 65 °C. Prior to this study, C. bescii had been examined on two lines of transgenic switchgrass with reduced lignin content, resulting in small improvements in biomass solubilization and fermentation [33]. However, the broader sample set available here-with all samples originating from the same parent line-provided an opportunity to examine C. bescii efficacy as a function of recalcitrance factors, especially the lignin content of the wood. As is shown in Table 2, the transgenic poplar lines that were generated [28] varied significantly in terms of solubilization by C. bescii (15 to 79%) and total fermentation products generated (ethanol plus acetate) by C. bescii (6.8 to 29.6 mM). While two of the transgenic lines (i20-5 and i35-7) with the highest solubilization and conversion by C. bescii have been previously reported [22], this effort aims to extend such analysis to a wider sample set of transgenic poplar lines generated from the same parent line. This includes some lines performing more poorly than the wild-type control (20.1% solubilization and 7.3 mM fermentation products). Overall, fermentation production (mM) by C. bescii directly correlated with biomass solubilization (R 2 = 0.81) (Fig. 2a), and inversely with lignin content (R 2 = 0.79) (Fig. 2b). However, there were some unexpected results. One transgenic poplar sample of interest was the a4-3 line (which targeted the down-regulation of PAL5). With a lignin content of 14.5% versus 21.7% for the wild type wood, an expected improvement in solubilization (56%) compared to wild type (20%) was observed. However, the concentration of fermentation products (9.1 mM) was comparable to that of wild type poplar (7.3 mM), even though previously reported enzymatic saccharification levels were substantially above wild type [28]. The reasons for this are unclear. Yet, upon examining the lignin properties previously reported, the a4-3 line has the highest proportion of spirodienone (β-1) interunit linkages (2.9% vs 2.3% for wild type), while the lines targeting C3H3 that performed substantially better than the wild type had 0.0% for lines i20-5, i69-4, and i69-13 and 0.4% for i20-10 [28]. One possibility is that the a4-3 biomass released a compound that was inhibitory to C. bescii, suggesting that solubilization could be primarily abiotic. Another possibility is that the carbohydrates remained bound to lignin moieties and, while solubilized, were not available in a form that C. bescii could utilize for fermentation.
Another useful comparison for assessment of lignocellulosic substrates is efficacy of the exogenously added enzymatic digestions in comparison to the natively produced degradation enzymes released by C. bescii. The release of glucose and xylose from the wood, via enzymatic saccharification (5 min in water at 180 °C followed by 72 h enzymatic digestion), previously reported [28], correlated with C. bescii conversion to fermentation products (acetate, ethanol) (Fig. 2c, d). It is important to emphasize that prior enzymatic saccharification assays were performed with wood samples that had been pretreated with acetone to remove extractives, while the wood utilized here for the C. bescii treatment was milled without any other form of chemical, thermal, or prior enzymatic pretreatment.
While the overall lignin content negatively correlated to the yield of fermentation products, the type of lignin present can also affect the recalcitrance of the lignocellulosic feedstock. A higher ratio of syringyl to guaiacol subunits (S/G ratio) present in the lignin has previously been suggested to improve the saccharification yield of P. trichocarpa and subsequent ethanol yield from fermentation of the enzymatically saccharified biomass with yeast [32]. For the lines tested here with available data on S/G ratio, the higher ratios correlated weakly with increased fermentation performance (R 2 = 0.41) (Fig. 3). The importance of the S/G ratio has been reported in prior work with various Populus species and ratios of such previous work are included in Table 1. While other work has highlighted the significance of the S/G ratio of lignin [5,23,32], no such a correlation of S/G to recalcitrance was noted with the microbial-based assay utilized in this study.
Growth productivity of lignocellulosic feedstock
A viable lignocellulosic feedstock must not only be more readily digestible, either by a naturally cellulolytic and hemicellulolytic organism, such as C. bescii, or by more traditional enzymatic saccharification treatment, it must also have favorable growth performance and productivity. Lower lignin is generally correlated with growth defects [16]. Herein, we found a similar correlation with those lines demonstrating the highest fermentation performance, i20-5 and i35-7, having stem volumes of 50% and 26% of the wild type, respectively (Table 2). Yet, there are some transgenic lines in which the lower lignin content did not result in a growth defect. Line i20-10 (lignin content 13.3%), developed with the same construct as i20-5, targeting the C3H3 gene but with slightly less down-regulation, had a stem volume of 95% of the wild type, thus demonstrating low lignin composition and excellent fermentation performance without a penalty to biomass productivity.
To account for these parameters, a fermentationgrowth factor was created in which the concentration of fermentation products (ethanol plus acetate, in mM) was multiplied by the stem volume (normalized to wild type) and the overall factor normalized to wild type set at 1.0 (Fig. 4). Transgenic poplar lines generated targeting the C3H3 gene stand out as lines with desirable properties for further improvement of biomass feedstocks. In fact, line i20-10 performs three times as well as the wild-type after accounting for both growth factors and fermentation performance. Thus, this suggests that the C3H3 gene is a highly promising target for low recalcitrance biomass. However, there may only be a narrow transcript window in which the growth phenotype is maintained for a less recalcitrant feedstock. The i20-5 line had a C3H3 transcript level of 13% and i20-10 was similar at 17%, both exhibiting greatly improved solubilization and fermentation with C. bescii. Line i20-2 also targeted the C3H3 gene and the C3H3 transcript level was approximately 50% of wild type. However, despite the transcript reduction to 50%, this line demonstrated lignin content, wood composition, and enzymatic saccharification results in line with wild type [28]. Thus, more control of transcript levels may be required to generate lignocellulosic feedstocks with the desired properties.
Many previous efforts to generate transgenic poplar, such as those listed in Table 1, have been performed by RNA interference using Agrobacterium based genetic techniques, which does not allow fine down-regulation control due to random genome integration of transgene. Thus, more surgical genetic tools are needed to exert precise control of transcript level, localization, and impact on specific cell types. CRISPR-based genome editing may be the solution to more strategic control of monolignol biosynthesis and, hence, the desired reduction in biomass recalcitrance.
Conclusions
There have been attempts to reduce recalcitrance in various potential lignocellulosic feedstocks via other methodologies that do not involve the monolignol biosynthetic pathway. Examples include the overexpression of xylem development regulatory genes [8], down-regulation of pectin synthesis [1], and overexpression of cell wall degrading enzymes, such as xyloglucanases [17], glycosyl hydrolases [31] and xylanases [21]. Natural variants with desirable properties have also been considered [14,23,32]. However, these approaches have not yet achieved the reductions in feedstock recalcitrance obtained through the use of systems biology-based approaches for generating transgenic plants with strategic properties.
Utilization of a direct screen of lignocellulosic feedstocks, such as transgenic wood, by an organism capable of both deconstructing plant biomass and fermenting the carbohydrate content is an effective and informative alternative to assessment by enzymatic saccharification and fermentation. This is feasible utilizing lignocellulosic fermentative microbes, such as C. bescii, that not only digest and metabolize the hexose and pentose saccharide portions of unpretreated plant biomass but can themselves be genetically engineering to generate useful products. Consequently, as described herein, this microbial based assay provides insight into how genetic alterations to the transgenic plant affect biomass solubilization and conversion. While the fermentation capabilities of C. bescii require further improvement for consideration of this organism for commercial use, the utilization of such a screen provides another informative tool for characterizing biomasses proposed in a lignocellulosic feedstock bioprocess.
Additional file 1. Poplar biomass samples utilized in this study. Fig. 4 Fermentation-growth factor of transgenics compared to wild type for C. bescii fermentation. Fermentation-growth factor is the product of estimated stem volume (from [28] and fermentation product titer from C. bescii treatment). The wild-type control was set to 1.0 to normalize data. The four best performing lines (i20-5, i20-10, i69-4, and i69-13) all targeted the C3H3 gene. *Stem volume data not available for a12-10 and i15-3 such that fermentation-growth factor could not be calculated | v3-fos-license |
2018-12-08T15:34:16.788Z | 2018-01-01T00:00:00.000 | 105198861 | {
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} | pes2o/s2orc | Optimization of dimethyl ether production process synthesis using superstructure analysis
Current world energy consumption is likely to increase over time. This is due to the growth of industry and transportation. The most important and most used energy sources are crude oil and natural gas. The consumption of energy is increasing continuously due to the economic expansion of the world fleet. At present, prices of primary energy sources such as oil and natural gas tend to increase. In addition, oil and gas are limited and likely to run out in the future. Currently, research and research on alternative energy is ongoing. To find the best alternative energy to replace in the future. Dimethyl ether is a substance that can be used as a substitute for liquefied petroleum gas (LPG) because of its similar physical properties. Most of them are used as fuel in vehicles. In addition, dimethyl ether is easier to liquefy than liquefied petroleum gas, giving advantages in terms of storage and transport, and a higher cetane value that can be used in the vehicle. Dimethyl ether is a substance that will burn completely. Dimethyl ether production has a wide variety of options. If the best option is difficult to analyse because of the complexity of the solution. Superstructure analysis will help to find alternatives for the production of dimethyl ether. Superstructure will identify the most economical alternative. The mathematical model is applied to the existing production process and new alternatives. In this work, the alternatives to produce dimethyl ether are displayed and the optimum alternative are chosen.
Introduction
Current world energy consumption is likely to increase over time due to the growth of industry and transportation. The most important energy sources are crude oil and natural gas which are globally consumed at approximately 5,700 million tons equivalent of crude oil per year [1]. At present, prices of primary energy sources such as oil and gas are in an increasing trend. Moreover, oil and gas are non-renewable and likely to run out in the near future. Current research on alternative energy to find the best alternative energy is ongoing.
Dimethyl ether (DME) is a substance that can be used as a liquefied petroleum gas (LPG) substitute because of their similar physical properties. DME is easier to liquefy than the LPG, giving advantages in terms of storage and transportation; moreover, it also has higher cetane value and smaller molecule which will be completely burnt benefitting the vehicle usage. It's also a nitrogen and sulphur lacked substrate; therefore, combustion of DME does not produce small particulates matters (PM), nitrogen oxides (NOx) and sulphur oxide (SOx). It can be said that DME is a clean alternative energy that is ideal for vehicle usage. The production of DME includes a wide variety of options ranging from simple reactordistillations, reactor-distillation-pervaporation, single reactive distillation, or multiple reactive distillations. Due to the complexity of the synthesis and design of the process, the optimum processing pathway is hardly accomplished.
The process synthesis and design through superstructure optimization could help identifying the most economical alternative via mathematical method. The conventional production pathways, as well as, newly generated alternatives are combined into the form of superstructure, the generic mathematical model is applied and solved using mixed-integer linear programming (MILP) embed in the GAMs software.
Methodology
The integrated business and engineering framework for synthesis and design of processing networks [2,3] has been applied into this work. The potential alternatives for synthesizing and designing processes are presented as processes intervals; they are arranged in the form of superstructure as displayed in fig. 1. Raw materials are presented in the first column; while products are embodied in the last column. The processing steps sequentially proceed from the left-to the right-hand side of the superstructure, converting and/or separating the raw materials to the products.
Whereas, each process is presented through a generic process interval description displayed in fig. 2. Eqs. 1-7 are incorporated generic equations depicting the operation of a unit or a group of units.
The mathematical model is formulated as mixedinteger linear programming problem with the objective function, the logical constraints, and the variable bounds defined as following by eqs. 8-12. max ( ) The objective function of this study is maximizing annual profit, in term of earnings before interest and taxes (EBITA), subject to logical decisions on choosing the unit operations and raw material flowrate.
Conventional DME production
The conventional production of DME by dehydration of methanol with the capacity 110 Mta has been reported by Luyben [4]. The process consists of an adiabatic reactor, two distillation columns, and feed preheater as displayed in fig. 3. Feed, 153 Mta of methanol, is preheated to 275 °C and then fed into the adiabatic reactor with solid catalyst. Methanol is undergoing the dehydration reaction; 82% of methanol is converted to DME with water as side product as displayed in eq. 13. 3 3 The reactor product is used for preheating the feed, then cooled down before sending to distillation column. The first column separates DME from methanol and water; DME, as top product, comes out at 99.999% purity. The methanol and water mixture at the bottom of the first column is then sent to second column. The unreacted methanol is separated in this column and recycled back to mix with the feed.
The capital cost of the conventional process is approximately 3.4 MM$, with the net energy cost 2.6 MM$/y.
Processing alternatives
Pervaporation with alpha-alumina membrane has been reported employing for the separation between water and methanol by Chapman et al. [5]. Water-methanol are separated completely at 50 °C with 0.57 kg/m 2 ·h flux. This unit can be used as a substitution for the second distillation column.
Reactive distillation (RD) has also been reported for the production of DME, both from pure methanol [6] and methanol in glycerol [7]. The RD can be applied as a combined reactor-separation to enhance the methanol conversion; as well as, employed as product enhancer after the conventional reactor; moreover, it can also be used to convert the recycled methanol to reduce the main reactor load [8]. Products of RD could be methanol, water, DME, or mixtures of different compositions of these compounds depending on the whereabout of the column. In this study, the RD is employed as the single unit process, and as enhanced-substitution of both distillation columns.
The important mixing, reacting, and separating parameters of the conventional and alternatives processes are reported in tables 1a-c, and the DME production superstructure is displayed in fig. 4. Fig. 3. Conventional DME production [4]
Results
Single reactive distillation operation has been chosen as the optimum production pathway for the dehydrogenation of DME, displayed as greyed box in fig. 4. Comparing to the conventional process, the optimum pathway requires only one preheater and one column; moreover, due to the parallel reacting-separating operation of the RD, the reaction is shifted toward the product side, resulting in the increasing of the raw material conversion. An initial estimation is found that the capital investment of the optimum process is 2.3 MM$, 32% lowering from the conventional process; additionally, the total operating cost (including net energy cost) is approximately 2.2 MM$/y, 17% lower from the conventional operation.
The optimum process is then simulated in an open source process simulator DWSIM v.5.1. The result of the detailed simulation, as presented in fig. 5, indicates that the 54 stages-RD column can produce up to 110 Mtonne/y of above 99.99% purity of DME with the column operating pressure 11 atm., column reflux ratio 6.2 and the column diameter 2.1 m. Water, at above 99.99% purity, is also produced as a side product. The conversion of methanol reaches 100% with 34 reactive stages, and the methanol raw material is fed to the column at the 9 th stage.
The detailed simulated result confirms the feasibility of the superstructure optimization methodology. The obtained design reduces capital and operating costs; moreover, the amount of waste generated is also reduced due to the enhanced conversion. On the other hand, there fig. 6; more energy could be conserved by recycling the heat in the water from the column bottom.
Conclusion
In this work, the integrated business and engineering framework for synthesis and design of processing networks has been adopted for the synthesis and design of the DME production.
The conventional DME production process, as well as, processing alternatives are collected and organized in form of the superstructure. The mathematical model has been formulated as the mixed-integer linear programming problem following the generic structure-interval model.
The MILP problem has been solved with the objective to maximize the annual profit (in term of EBITA). The obtained result selects single RD unit as the optimum design for the production of DME. The design has also been validated with the detailed simulation. The result achieves 32% reduction of the capital investment and 17% reduction of the operating cost.
Despite achieving the optimum design with various improvements, the process can be further intensified for an even better result. The intensification step, specially, regarding energy intensification should also be included into the framework in the future. | v3-fos-license |
2019-09-26T09:00:23.447Z | 2019-07-05T00:00:00.000 | 204097136 | {
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} | pes2o/s2orc | In vivo biocompatibility and immunogenicity of metal–phenolic gelation
Coordination-driven supramolecular in vivo assembly of metal–phenolic hydrogels.
Introduction
Supramolecular biomaterials and hydrogels have gained great interest in biomedical engineering for applications such as drug delivery, tissue engineering, regenerative medicine and immunology. 1,2 Supramolecular hydrogels are assembled by interlinking small molecules in a noncovalent fashion, for example through metal coordination. 3 One such example is the coordination between metals and phenolic compounds, which has previously been used to engineer conformal coatings and thin lms, 4-6 as well as to stabilize hydrogels prepared using presynthesized macromolecular building blocks. [7][8][9] Metalphenolic materials are of increasing interest across the chemical sciences for the preparation of functional materials in diverse application areas such as adhesives, self-cleaning surfaces, separation, catalysis, crystallization, sensing, drug delivery and imaging. [10][11][12] Increasing our understanding of the biological interactions of metal-phenolic materials is paramount for continued research in this emerging area of materials science, as it is a prerequisite for understanding their toxicology, environmental impact and potential in biomedicine.
Recently, metal-phenolic supramolecular gelation was demonstrated through the direct gelation between naturally occurring, unmodied polyphenols and group IV metal ions (Ti IV or Zr IV ). 13 This coordination-driven supramolecular assembly can be initiated by simple mixing at ambient conditions in various solvents over large concentration ranges and metal-ligand stoichiometries. 13 The result is robust and adaptive gels, which can be used for controlling concurrent assembly processes (e.g., crystallization of metal-organic frameworks or pharmaceuticals), or for in situ co-gelation of diverse dopants. 13,14 While the in vitro cytotoxicity of this new class of materials based on metal-phenolic supramolecular gelation has been studied and observed to be negligible, 13 in vivo studies are yet to be reported.
In vivo gelation, where hydrogels form spontaneously in situ under physiological conditions (e.g., at the site of injection), is of broad interest for biomedical applications due to its minimal invasiveness and high translational potential. 15,16 Examples include thermosensitive poloxamer-based gels used for antibiotic delivery to the inner ear, 17 hydrophobically modied poly(ethylene glycol) gels for ophthalmic applications, 18 and cell sheet transplantation using thermosensitive gelatin-based hydrogels. 19 Many biomedical applications also use gel systems such as calcium-alginate gels which are based on ionic crosslinking of the biomolecule alginate with Ca 2+ . Calciumalginate gels typically form rapidly upon mixing. 20,21 While strategies to modulate the gelation kinetics exist (e.g., using calcium-releasing liposomes to trigger gelation 22 or phosphate salts to slow gelation time 23 ), pre-formed alginate gels such as implantable beads remain the most commonly used in both pre-clinical and clinical research. [24][25][26][27][28] In contrast, gelation times for recently introduced 13 metal-phenolic gels-prepared through coordination of the biomolecule tannic acid (Fig. S1 in ESI †) with Ti IV -can be readily tuned from less than 1 minute to more than 1 day in a robust gelation process that is insensitive to a large range of conditions. We thus hypothesize that these Ti IV -tannic acid gel systems ("TiTannic gels") are suitable candidates for in vivo gelation and future biomedical applications.
Here, we demonstrate that metal-phenolic supramolecular gelation occurs in vivo and investigate the host response to the material over 14 weeks (Fig. 1). Liquid precursors (tannic acid solution and Ti IV solution) were prepared and lter-sterilized. The composition was tailored to achieve a gelation time of around 15 minutes, which was deemed suitable for allowing careful injection. Prior to animal studies, the gel system was characterized using electron microscopy and Raman spectroscopy, and permeability and porosity were assessed using a glucose permeability assay and particle tracking analysis, respectively. For the animal studies, the two sterile precursors were mixed immediately prior to subcutaneous injection in the anks of immunocompetent mice. At specied time points during the 14 week period, external and internal photographs of the injection sites were taken and histological sections were prepared. Additionally, tissue samples were collected for titanium biodistribution studies using mass spectroscopy. Titanium remained largely at basal levels for most studied tissues and time points, indicating low to negligible titanium accumulation. Finally, drug loading and elution studies were performed in vitro using the corticosteroid dexamethasone, and drug elution from the TiTannic gels was observed over a period of >10 days, which can be compared to <1 day for the Pluronic F127 hydrogels prepared as controls.
Taken together, these results demonstrate that metalphenolic supramolecular gelation occurs in vivo, and provide essential in vivo characterization for this emerging class of materials. The observed in vivo responses are largely comparable with those previously reported for conventional calciumalginate gels. [24][25][26][27][28] Additional benets of the TiTannic system, including readily tuneable gelation time, robustness and adaptability, and the easy, low-cost, off-the-shelf, and scalable preparation process, make this new class of material of significant interest for diverse biomedical applications.
Results and discussion
Electron microscopy, Raman spectroscopy and rheology of the hydrogels Metal-phenolic supramolecular gelation occurs spontaneously upon mixing of polyphenols (e.g. tannic acid, TA) with group IV metal ions (e.g. Ti IV ). This class of material was recently introduced and has been characterized with rheological methods, optical microscopy, UV-vis spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction among other techniques. 13,14 To further explore the nanostructure of the gel, we performed a freeze-fracture procedure using liquid nitrogen, followed by freeze-substitution in acetone and lyophilization before scanning electron microscopy (SEM) imaging (Fig. 2). The results show a structure with porosity on the micrometer and nanometer length scales ( Fig. 2a and b). It should be noted, however, that whilst the freeze-substitution and lyophilization process may affect the pore structure compared to the pristine hydrated material, the technique can still provide valuable insight into pore structure and levels of hierarchy. 29 Raman spectroscopy has been extensively used to investigate materials prepared using coordination-driven assembly and coordination compounds. 30,31 We applied a Raman spectroscopy probe with continuous spectral acquisition (with 1 second integration time) to obtain Raman spectra in situ during gelation ( Fig. S2 †), the results are shown in Fig. 2c and d. When comparing Raman spectra of the individual components (TA solution and Ti IV solution) with that of the formed gel (TA + Ti IV ), two enhanced peaks, at 1361 cm À1 and at 1490 cm À1 , are observed (Fig. 2c). These peaks increased in intensity during the rst 30 seconds aer mixing, and then remained largely stable during the whole experiment (1 hour). The observed changes correspond to previously reported Raman shis for metal-phenolic systems, 32 which were assigned to skeletal modes of the substituted benzene rings and stretching of carboxylate groups of the phenolic compounds interacting with the metal ligands. Metal-phenolic networks can also be redox-active as has been recently reported by an electrochemical study, 33 and future studies combining Raman spectroscopy with electrochemical analysis may be of interest to elucidate these molecular interactions further. The TiTannic hydrogels were measured using rheology (Fig. S3 †), displaying a storage modulus (G 0 ¼ 12.6 kPa) signicantly larger than the loss modulus (G 00 ¼ 0.595 kPa). An amplitude sweep was conducted to determine the linear viscoelastic region and crossover points of the hydrogels, whereas the frequency sweep showed frequency independent behavior of the hydrogels. The time sweep showed gelation occurred within 15 minutes, with the hydrogels ageing slowly over $15 hours. Gelation, as determined using vial inversion (Fig. 1a), occurred within 15 minutes and is consistent with the gelation kinetics measured by rheology (Fig. S3 †). At earlier time points (e.g. 30 seconds aer mixing) the mixture is still liquid and will not remain stationary if the vial is inverted. The Raman probe provides spectral information of a local volume ($1 mm from the probe tip) and these data therefore suggest that local coordination occurs rapidly (within 1 minute), which is in agreement with previously reported metal-phenolic nanoscale lms and coatings. 4 Shorter range (nanoscale) interactions in the material may thus be completed within seconds, while longerrange interactions needed to form the stable gel were observed to take around 15 minutes for the composition used here. It is noted that for other compositions, gelation time can vary drastically, from <1 minute to >1 day. 13 Hydrogel porosity and permeability To further investigate the porosity of the hydrated material, particle tracking analysis was used (Fig. 3). In this method, microparticles are dispersed in the gel (through co-gelation) and their movement is tracked over time using optical microscopy. This information can then be used to explore the porosity and functional tortuosity of a material. 34, 35 We compared the movement of embedded 1 mm polystyrene particles in Pluronic F127 hydrogels and in TiTannic gels at 37 C. Pluronic F127 gelation is a well-established gelation system (in this study acting as comparative control samples) that is thermoresponsive and where the gelation occurs through a micellarpacking mechanism. 36,37 In the Pluronic F127 gel, a particle movement of around 50 mm (median displacement distance) per minute was observed (Fig. 3c). That particles can easily move around is expected as the micellar-packing structure can accommodate substantial exibility and movement inside the gel. 36,37 In contrast, for TiTannic gels the median displacement distance per minute was 10-fold lower (<5 mm; Fig. 3c). The largely stationary behavior of particles embedded in the TiTannic hydrogels indicates that the effective hydrated pore size is smaller than the particle diameter (i.e., 1 mm). This difference in effective pore size between the two different gel systems may be explained by the differences in chemistry and assembly mechanism: whereas Pluronic F127 gels are composed of macromolecules (poloxamers, >10 kDa) that form micelles, 38 TiTannic gels are instead assembled through molecular coordination between metal ions and low molecular weight polyphenol (TA, 1.7 kDa). For future studies it may be of interest to expand on these results using nanoparticles and super-resolution microscopy, as has been recently done for other types of gel materials. 34,35 Aer having established that microparticles remain largely stationary in TiTannic gels, we investigated the diffusion rate of a small molecule: glucose (0.18 kDa). Glucose-permeable hydrogels are of interest clinically, as these types of gels are being explored to encapsulate insulin-producing cells for the treatment of diabetes. 23,24,28 In the present investigation, we developed a glucose permeability assay ( Fig. 4) to compare glucose permeability of the TiTannic gel system to Pluronic F127 gels, an in vivo gelation system which has previously been explored for the treatment of diabetes. 39 In the glucose permeability assay, the gel was cast in a porous well insert (membrane with 0.4 mm pores) and PBS was added both underneath and above the gel following gelation (Fig. 4a). Concentrated glucose was then added above the gel, while a glucose probe was situated underneath the gel (Fig. S4 †). In the empty well control (i.e. free diffusion), the glucose concentration was measured as equilibrated within the rst three hours, and for the TiTannic control sample (no glucose added) the signal remained low, as expected (Fig. 4b). For both Pluronic F127 and TiTannic gels, retarded diffusion relative to the empty well (free diffusion) was observed. While there is a large overlap in the observed glucose concentration increase during the rst few hours (Fig. 4b), Pluronic F127 gels appeared to stabilize at longer time points (>10 hours) at a higher concentration than the TiTannic gels ( Fig. S5 †). Pluronic F127 gels are known to dissolve over time which may contribute to this difference. 36 Polyphenols are known to interact with carbohydrates, 40 and this may contribute to the observed lower glucose level detected. Nevertheless, the results demonstrate that Pluronic F127 gels and TiTannic gels are both permeable to glucose. TiTannic gel (b). Red lines correspond to the movement of a single particle during 1 minute. White arrows indicate red trace lines in (b); these particles remained largely stationary (<5 mm distance travelled). (c) Displacement distance measured using particle tracking analysis. Dots are individual data points and the lines indicate the median.
TiTannic gels are stable and well tolerated aer subcutaneous injection in mice
For initial assessment of TiTannic gel tolerability, an individual mouse was injected subcutaneously on the right ank with 50 mL of gel precursors. Precursors were always mixed immediately before injection and had a pH of around 7 (see Section 1 in ESI †). The initial assessment using an individual mouse was followed by a second mouse injected subcutaneously with 100 mL of gel precursors. The gels formed a solid disc-shaped mass within $15 minutes, which was palpable underneath the skin. As no systemic or local adverse effects were observed for these rst mice, a subsequent group of mice was subcutaneously injected with 100 mL of TiTannic gel precursor and culled at selected time points up to week 14 post-injection. The injection site was investigated and resected for histological assessment. Distant tissue samples were collected (including brain, heart, kidney, liver, lung and spleen) to measure systemic levels of titanium. Gross macroscopic assessment at termination showed negligible changes in external as well as internal appearance of the injection site ( Fig. 5a and b). The small variation in gel mass ($0.2-0.3 g) between animals is likely due to variation introduced during injection and processing for histology (Fig. 5c). No substantial swelling or shrinkage of the gels was observed, and gel sizes remained largely constant over the duration of the experiment (Fig. 5d). These results show that TiTannic gels are stable in vivo, which is in agreement with the in vitro disassembly studies ( Fig. S6 and S7 †).
TiTannic gels elicit a mild but persistent foreign body reaction
Aer excision, gels were processed for histology and stained with haematoxylin & eosin (H&E) to assess immune cell accumulation around the gel and Picrosirius red to visualize collagen deposition and the formation of a brotic capsule. The entire gel underneath the skin and embedded in subcutaneous tissue is shown in Fig. 6 and S12. † Negligible cell inltration or collagen deposition was observed aer 3 days but increased by week 1 showing a clear disorganized mononuclear inltrate scattered with individual collagen bers around the gel. This inammatory reaction was then observed to follow the common course of a foreign body reaction: mononuclear cells organized into layers of epithelioid histiocytes gave the foreign body capsule a homogenous appearance. Foreign body giant cells also started to appear at the border of the gel. From week 2 onwards, areas of increasing overlap between the gels and surrounding tissue were observed (Fig. 6). A thin but homogenously organized brotic capsule was visible at later time points. As expected, most cells surrounding the gel stained positive for CD45, a pan-leukocyte marker (Fig. 6c).
A quantitative time course of the development of the foreign body reaction and representative histology images are shown in Fig. 7 and S11, † and quantication is presented in Fig. 8. The thicknesses of inammatory inltrate (Fig. 8a) and brotic layer (Fig. 8b) followed a similar curve with an early increase and a gradual decline over time. As expected, brosis developed with a brief delay as a result of immune cell inltration. On the other hand, the thickness of the overlap layer between gel and cellular inltrate increased over time (Fig. 8c), as did the number of foreign body cells ( Fig. 8d and S13 †).
The above indicates that TiTannic gels are largely stable in vivo, with limited degradation observed during the experiment, which is in agreement with the in vitro results ( Fig. S6 and S7 †). However, interestingly, increasing interactions with surrounding tissue and cells over time was observed. For example, it appears that small pieces of gel broke off the bulk material and were being taken up by surrounding cells, which gel. An empty well (no gel added) was used as a control, representing free diffusion of glucose through the well insert. The dotted black line corresponds to 2.0 mM glucose concentration which is the equilibrated glucose concentration on both sides of the well insert membrane (i.e. full permeability). TiTannic gels without any glucose added were used as controls to confirm that the gel itself did not induce any substantial signal with the glucose probe. Dots represent average values of duplicates and the shaded areas represent the standard deviation.
may eventually lead to disassembly and degradation of the gel over extended periods of time. The disassembly of TiTannic gels can occur through pH-induced changes in the molecular interactions of the materials, and/or through the presence of competing ligands (Fig. S6 †). For metal-phenolic materials in general, the disassembly behavior can be tuned by tailoring the composition (e.g. using blends of phenolic ligands). 6,10,33 An interesting research direction is, therefore, the engineering of metal-phenolic gels with disassembly proles tailored to specic applications, such as rapid biodegradation. Metalphenolic hydrogels can also be prepared by replacing titanium with zirconium as the coordinating metal. 13 Exploring these zirconium-based hydrogels would be of interest for future studies as zirconium-containing materials have shown improved performance (even compared to well-performing titanium-containing materials) in some biomedical applications. 41
Titanium accumulation is low in distal tissues
Coordination-driven assembly strategies are based around noncovalent interactions, which are dynamic with a stability that depends on the local environment and the time scale. 2,3 For example, TiTannic hydrogels can disassemble rapidly (within minutes to hours) if exposed to extreme pH or to competing ligands ( Fig. S6 †), but remain stable for months when immersed in sterile cell culture media and PBS (Fig. S7 †). While no large changes in gel size or mass were observed during our in vivo studies (Fig. 5), small amounts of material leakage from the TiTannic gels could still occur. Tannic acid is a naturally abundant polyphenol (found in many plants, fruits and vegetables) and is being investigated for its intrinsic therapeutic effects and its ability to enhance the delivery of drugs. 42,43 The other component, titanium, is a metal commonly used in the design of implants (e.g., for dental implants and joint replacement). [44][45][46] Detection of increased tissue levels of titanium is of interest clinically to aid in the assessment of implant status and prognosis, where increased levels of titanium may indicate increased implant wear. 46 In the current study, we used mass spectroscopy to evaluate titanium levels at distal tissues to the injection site (Fig. 9). For brain, heart, kidney and lung tissues no large differences were observed in titanium concentration for tissue samples from mice which had been subcutaneously injected with TiTannic gels compared to tissue samples from mice which had not been exposed to TiTannic gels. The titanium concentration in these tissues was below 50 ng Ti per g of tissue (Fig. 9). This is in agreement with previously reported results for unexposed rodents. 47 A background level is expected as titanium is abundant in our environment, especially in the form of TiO 2 (e.g. in pigments, sunscreens, toothpaste, paints) with median daily adult intake in the UK being approximately 2.5 mg. 48,49 For patients with titanium-containing implants, so tissue Ti concentrations of around 1 Â 10 6 ng Ti per g tissue have been reported directly adjacent to the implant, and concentrations around 6500 ng Ti per g for tissues collected 3 cm from the implant. 45 In the present study, increased Ti concentrations were observed for liver and spleen tissues at the longer time points (Fig. 9), with concentrations of around 50-100 ng Ti per g of tissue. The overall low titanium concentrations observed is in Titanium concentration in blood and urine samples collected from the animals were also determined using mass spectroscopy (Fig. S8 †). At earlier time points (3 days and 1 week) a 5-10-fold increase in Ti concentration was observed for the blood samples obtained from animals subcutaneously injected with TiTannic gel compared to blood obtained from unexposed animals: from around 1 mg Ti per L of blood to 5-10 mg per L. Basal concentrations on the order of 1 mg Ti per L of blood is routinely reported for unexposed rodents and patients./ 50 Ti concentration in urine samples was low (close to limit of detection) for all samples studied (Fig. S8 †), as expected as the metal is not readily excreted into the urine. 46 Taken together with the observed Ti accumulation in the liver (Fig. 9), this suggests that a possible excretion mechanism may instead Fig. 9 Biodistribution studies of titanium reveal low levels in most distal tissues, with some accumulation observed in liver and spleen. Quantification of titanium levels was performed using mass spectroscopy following tissue digestion. Control samples (ctrl) are from animals that were not exposed to TiTannic gels. Tissues from animals exposed to TiTannic gels were collected 3 days (d3) and 1, 10 and 14 weeks (w1, w10, w14, respectively) following subcutaneous injection of TiTannic gel. Data points indicate tissues from an individual animal (N ¼ 2-5), and horizontal lines indicate median value. be through the feces. 51 Feces were not collected and analyzed in the current study, but may be of interest for future studies.
TiTannic gels exhibit more sustained drug release compared to Pluronic F127 gels Hydrogels that can be formed in situ under physiological conditions (i.e., in vivo gelation) are of interest for diverse biomedical applications, including drug delivery, tissue engineering and regenerative medicine. 15,16 As the results presented herein demonstrate that TiTannic gels are suitable for in vivo gelation, we hypothesized that they may be of interest for drug delivery applications. To test this, we compared the in vitro drug loading and release properties of TiTannic gels to Pluronic F127 hydrogels. Pluronic F127 gels are currently being explored as an in situ gelation system to treat otitis media (ear infection). 17 In the current study, we loaded Pluronic F127 gels and TiTannic gels with the corticosteroid dexamethasone, and measured drug release using liquid chromatography (Fig. 10). For Pluronic F127 gels, >75% of the drug was released within 3 hours. In contrast, for TiTannic gels >75% of drug was released over 6 days (a >40-fold difference compared to Pluronic F127 gels). Drug release was still at a detectable level (around 1.5-2%) from the TiTannic gel even aer 28 days, while for Pluronic F127 no drug release was observed aer 2 days. This difference in release rate may be due to the ability of phenols to interact strongly with diverse biomolecules and pharmaceuticals, which may retard the release and enable sustained drug delivery. 42,43,52,53
Conclusions
In this study, we demonstrate that metal-phenolic supramolecular gelation can occur successfully in vivo, and provide essential in vivo characterization for this emerging class of hydrogels. Following characterization with optical and electron microscopy, Raman spectroscopy, and rheological methods-as well as a study of hydrogel porosity and permeability-we assess biocompatibility and immunogenicity following subcutaneous injection in immunocompetent mice. We show that TiTannic gels are stable and well-tolerated, and elicit a mild but persistent foreign body reaction. Through mass spectroscopic analysis of tissue samples, we show that titanium accumulation in distal tissues remains low over the 14 week study period. Finally, we show that TiTannic gels exhibit more sustained release (from <1 day to >10 days) compared to a commonly used in vivo gelation system (i.e., Pluronic F127 hydrogels) when loaded with the clinically used corticosteroid dexamethasone. Taken together, these results provide a solid foundation for further exploration of TiTannic gels in biomedical areas such as drug delivery and regenerative medicine.
Materials
Tannic acid (TA; Sigma-Aldrich product #403040), titanium(IV) bis(ammonium lactato)dihydroxide solution (Ti-BALDH; Sigma-Aldrich product #388165), sterile dimethyl sulfoxide (DMSO; Sigma-Aldrich product #D2650), sodium hydroxide (NaOH Sigma-Aldrich product #S8045), Pluronic F-127 (Sigma-Aldrich product #P2443), polystyrene microparticles (10% particle dispersion in aqueous solution, 1.1 mm diameter particles; Sigma-Aldrich product #LB11), dexamethasone sodium phosphate (Sigma-Aldrich product #BP108), and 12well cell culture plates (Sigma-Aldrich product #CLS3513) were purchased from Sigma-Aldrich (USA). The received DMSO was purchased pre-sterilized while other components could be lter-sterilized (0.2 mm syringe lter) aer dilution but prior to mixing and usage, if needed. Sterility of DMSO and solutions following sterile-ltration was maintained by working in a biosafety cabinet using standard aseptic procedures. 54 Millicell cell culture well inserts (0.4 mm pore size, 12 mm diameter; Merck Millipore product #PICM01250) were purchased from Merck Millipore (Ireland). Dulbecco's phosphatebuffered saline (PBS), Dulbecco's modied eagle medium (DMEM), and fetal bovine serum (FBS), were purchased from Thermo Fisher Scientic (USA). Absolute ethanol (>99.8%) was purchased from Honeywell (Germany). Sunower oil was obtained from a local grocery store (Tesco, UK). Deionized and ltered (0.2 mm pore size) water was used for all experiments (unless otherwise stated) and obtained from a Triple Red water system (Avidity Science, UK). Micropipettes calibrated and certied at least annually were used for all experiments. The MIRIBEL standard 55 for reporting bio-nano science research and the ARRIVE guidelines 56,57 for reporting research with animals were consulted during the preparation of this manuscript.
Preparation of TiTannic gels
Full details of the diverse hydrogels and organogels that can be prepared using the TiTannic gel system, including under which conditions and compositions (incl. molar ratios and gelation concentrations) different gelation times can be achieved, has been previously described. 13 Briey, a 50 mg mL À1 TA solution was prepared in deionized water and the pH was raised to $7 using NaOH (1 M, aq.). Separately, DMSO was added to Ti-BALDH until a nal concentration of 20 vol%, this Ti-BALDH/ DMSO mixture is the Ti IV solution. TA solution and Ti IV solution was mixed through vortexing at a TA : Ti IV molar ratio of around 1 : 2.5 for a gelation time around $15 minutes. See Section S1 in the ESI † for additional details including a step-bystep protocol.
Preparation of Pluronic F127 gels
Pluronic F127 was added to cold PBS (4 C) to reach a concentration of 18 wt%. This solution was stored in the fridge (4 C) to keep the solution in a liquid-like stage: at 37 C the Pluronic F127 solution turns into a gel. 58,59 Particle tracking analysis Pluronic F127 and TiTannic gels were prepared as described above to a nal volume of 480 mL, which included 10 mL of 10% polystyrene microparticle dispersion which was dispersed in the gel precursor prior to gelation for homogenous distribution throughout the nal gel. Aer gelation for 1 hour at 37 C, microscopy videos of the particles inside the gels were acquired in bright-eld mode using an Olympus BX51 microscope at 10 frames per second for 60 seconds. Particle tracking of individual particles was performed on the acquired videos using the "Particle Tracker" plugin 60,61 of Fiji, 62 with the output being distance (mm) travelled per individually tracked particle during 60 seconds. The results were plotted using GraphPad Prism 8 (GraphPad Soware, USA).
Rheology
Rheological measurements of the hydrogels were recorded using an Anton Paar MCR302 rheometer tted with a 25 mm stainless steel parallel plate (PP25) and Peltier temperature controlled hood (P-PTD200/80/I). All measurements were recorded at 298 K, with water added to the Peltier hood to prevent sample evaporation. Time-sweeps were performed with frequency ¼ 10 rad s À1 , strain ¼ 0.1% and sampling rate ¼ 5 seconds. Amplitude sweeps were performed with frequency ¼ 10 rad s À1 , log ramp of strain (0.01-100%) and sampling rate ¼ 33 points per decade. Frequency sweeps were performed with strain ¼ 0.1%, log ramp of frequency (0.1-100 rad s À1 ) and sampling rate ¼ 33 points per decade. The results were plotted using OriginPro 2017 and are presented in Fig. S3. †
TiTannic gel dissolution study
An oil bath using sunower oil was prepared by pouring the oil into a petri dish until a height from the bottom of the dish to the liquid surface of around 1 cm was obtained. TiTannic gel was prepared as described above. Immediately upon mixing and vortexing TiTannic gel components, 10 mL droplets were manually pipetted directly into the oil bath, resulting in largely spherical aqueous droplets inside the oil bath. These liquid droplets were allowed to gel for 1 hour to form TiTannic gel beads. The gel beads were then transferred into a 50 mL tube of ethanol using disposable transfer pipettes with an opening larger than the bead size. The 50 mL tubes were kept on a tube roller shaker (to ensure continuous mixing) for 1 hour, aer which the ethanol was removed and replaced with fresh ethanol. This was repeated three times. During this process the gel beads were washed from oil and sterilized. This washing process was then repeated using sterile PBS (instead of ethanol). Aer sterilization in ethanol, tubes were only opened inside a biosafety cabinet and handled using aseptic technique and sterilized disposable transfer pipettes. Aer the nal washing step, the beads were added into a sterile petri dish containing PBS which was placed on top of a micrometer ruler and photographs were taken from above the petri dish. The photographs were processed using Fiji, 62 where the ruler was used to dene distance in each photograph which could then be used to measure the size of each bead ($2 mm in diameter). Following this time point zero measurement, 10 gel beads were added into a 50 mL tube containing 50 mL of PBS, and 10 gel beads were added into a 50 mL tube containing 50 mL of cell culture media (DMEM) supplemented with 10% v/v fetal bovine serum. At predetermined time intervals, the gel beads were aseptically transferred into a sterile petri dish and the size of each gel bead was again measured following the procedure outlined above. Aer each measurement, the gel beads were re-dispersed into 50 mL of fresh PBS or fresh DMEM to continue the incubation. During incubation the tubes were kept on a tube roller shaker. Aer the last time point, the results were plotted using Graph-Pad Prism 8 (GraphPad Soware, USA) and the results are presented in Fig. S7. † In addition to the study described above, additional gel beads were also added to vials containing PBS, 1 M NaOH, 1 M HCl, or a 0.4 g mL À1 aqueous solution of pyrocatechol (Fig. S1b †). Using pyrocatechol to initiate competing ligand-mediated gel disassembly has previously been reported. 14 Photos of the vials were taken rst with all vials containing only PBS (time point ¼ 0 hours) and then at pre-determined time intervals (5 minutes, 1 hour and 24 hours) aer replacement of PBS in three of the four vials with NaOH, HCl or pyrocatechol aqueous solutions. Results are presented in Fig. S6. †
Raman spectroscopy
Raman spectra were obtained using a custom-built Raman spectroscopy system (Fig. S2 †) consisting of a multimodal Raman spectroscopy probe with a central excitation ber surrounded by seven collection bers (EmVision) connected to a 785 nm diode laser with maximum output of 500 mW (B&W TEK Inc.) for Raman excitation and collected using a ber-coupled QEPro spectrograph with a 1200 grooves mm À1 grating and a 50 mm slit (OceanOptics Inc., Dunedin, FL). The Raman probe has a spot size of around 500 mm and, for comparison, 785 nm light penetration through tissues (depending on the tissue) is on the order of 1 mm. 63,64 Raman spectra were acquired continuously with a 1 second integration time and 100 mW output power using custom, in-house developed scripts (MATLAB 2017a, Mathworks, USA). Spectral processing was also performed in MATLAB and consisted of wavelength calibration using a NeAr lamp, system spectral response correction for CCD dark noise and ber-optic probe background, uorescence background subtraction (Whittaker lter, l ¼ 100 000), normalization to the area under the curve, and smoothing using a rst-order Savitzky-Golay lter with a frame length of 7.
Drug loading and release
Three samples of 100 mL Pluronic F127 solution were prepared (each in a 1.7 mL microcentrifuge tube) following the standard protocol described above and kept on ice, with the difference that 60 mg dexamethasone was mixed into each liquid Pluronic F127 sample (nal concentration was 60 mg dexamethasone per 100 mL gel). The samples were then gelled for 1 hour by moving these tubes into a heating block kept at 37 C. 500 mL of PBS (pre-warmed to 37 C) was then added into each tube to start the elution study.
TiTannic gel was prepared following the standard protocol described above with dexamethasone added to achieve a nal concentration of 60 mg dexamethasone per 100 mL TiTannic gel. 100 mL of TiTannic gel mixture was added into each of three 1.7 mL microcentrifuge tubes, this was done immediately upon mixing the TA solution (containing dexamethasone) with the Ti IV solution, before gelation had occurred. The mixtures were then allowed to gel for 1 hour. 500 mL of PBS was then added into each tube to start the elution study. The elution study was also repeated for TiTannic gels but instead of 60 mg dexamethasone per 100 mL gel, 6 mg dexamethasone per 100 mL gel was used to increase the dexamethasone signal per time point to facilitate HPLC detection (as the observed release rate from TiTannic gels were much slower than for the Pluronic F127 gels). In this repeat study, double the amount of Ti IV solution was used as the higher concentration of dexamethasone present affected the gelation time of the TiTannic gel, as assessed with vial inversion testing.
During the elution study, all tubes were incubated in a heating block kept at 37 C. A sample of fresh PBS (unexposed to the gels) was kept and dened as time point ¼ 0. At each predetermined time point, 500 mL of the PBS was aspirated from each sample and replaced with 500 mL fresh PBS (pre-warmed to 37 C). Samples were stored at À20 C until samples from all time points had been collected. Quantication of the dexamethasone concentration of each sample was performed using HPLC, as described below.
High-performance liquid chromatography (HPLC)
HPLC samples were measured using an Agilent 1260 Innity Quaternary LC equipped with a Phenomenex Gemini NX C18 column (150 Â 4.6 mm, 5 mm pore size and 100Å particle size). HPLC grade formic acid, water and acetonitrile were used for all analyses and were obtained from Merck. Samples were separated using an isocratic elution of 40% v/v acetonitrile in water with 0.1% v/v formic acid over 10 minutes at a ow rate of 1 mL min À1 , injection volume of 20 mL, detection wavelength of 240 nm, and a column temperature of 40 C. The concentration of dexamethasone was determined from a linear calibration curve of the peak areas (Fig. S9 †). Samples were analyzed using Agilent OpenLab CDS soware to measure peak areas (Fig. S10 †). The results were plotted using GraphPad Prism 8 (GraphPad Soware, USA).
Scanning electron microscopy (SEM)
TiTannic gels were prepared (following the standard protocol described above) in 1 mL cut-off syringe tops for easy gelmoulding and removal. Samples were cut from the bulk gels using a biopsy punch and immersed in liquid nitrogen for 1 minute until completely frozen before freeze fracturing using a scalpel. A piece approximately 2 Â 2 Â 2 mm in size was taken for further processing. The sample was freeze substituted using a Leica EM AFS2 (Leica Microsystems, Germany) by immersion in pure acetone precooled to À90 C and the sample was ramped to 0 C at 5 C per hour. The sample was removed from the acetone and lyophilized, mounted on a SEM stub, and sputtercoated with a 10 nm layer of gold. The sample was imaged using an Auriga Zeiss Crossbeam (Carl Zeiss AG, Germany) in SEM mode at 54 sample tilt (tilt corrected) using the in-lens secondary electron detector and 1.6 kV accelerating voltage. The images were processed using Fiji. 62 Permeability assay A porous well insert (membrane with 0.4 mm pores) was secured in a well of a 12-well cell culture plate lled with stirred 3.5 mL PBS solution. The well insert, containing either no gel (i.e., empty well control), or gel (either 0.2 mL of TiTannic gel or 0.2 mL of Pluronic F127 gel), was positioned so that the permeable bottom was fully immersed in the PBS solution. An in-house fabricated glucose sensor was used to monitor glucose concentration outside of the well insert (Fig. S4 †). Aer a few minutes of stabilization (as monitored by continuous read-out from the glucose sensor), 0.3 mL of either PBS (negative control) or 20 mM glucose solution (in PBS) was added on top of the well insert, i.e. on the opposite side of the gel in relation to the glucose sensor. The response of the glucose biosensor was continuously recorded over time with measurements acquired approximately once per second. Evaporation was reduced using a paralm cover during the assay.
The glucose sensors used in this work are based on and adapted from electrochemical glucose biosensors described elsewhere. [65][66][67] All monitoring was controlled using in-house potentiostats and a PowerLab 8/35 data acquisition device (ADinstruments, UK), controlled by LabChart Pro (ADInstruments). Data analysis was also performed using LabChart Pro. Current measurements were converted into concentration values using pre-and post-experiment glucose calibrations (by measuring known concentrations of glucose). The results were plotted using GraphPad Prism 8 (GraphPad Soware, USA).
Mice
All animal procedures were performed with UK Home Office approval (UK HO PPL P6F4D9876, holder Dr Susanne Sattler) and conformed to the UK Animals (Scientic Procedures) Act, 1986, incorporating Directive 2010/63/EU of the European Parliament. Healthy, immunocompetent, 8-10 week old, male or female mice of a previously described 68 hybrid strain (C57Bl/ 6J Â FVB/N Â NOD/Shilt) were used. Procedures for the husbandry and housing of animals follow the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and UK Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientic Purposes. Mice were housed in Allentown XJ individually ventilated cages, with ECO2 bedding (Datesand Group, UK) and a 12 : 12 light : dark cycle. RM1 and RM3 chow (Special Diet Service) were provided ad libitum. Environmental enrichment was provided including cardboard tubes, wooden chewing blocks, and tissue. The maximum housing density was 5 mice per cage if <25 g and 3 mice per cage if $25 g. No animals were involved in previous procedures.
In vivo TiTannic hydrogel injection 100 mL of a sterile preparation of TiTannic hydrogel was injected under aseptic conditions. Mice were anaesthetized using 2% isourane and hair was removed from a <1 cm 2 area on both anks and treated with Betadine surgical scrub (Fisher Scien-tic, UK) before injection. TiTannic hydrogels were injected subcutaneously using a sterile 25-gauge hypodermic needle. Mice were maintained under general anesthesia for a total of 10 minutes to allow the gel to form homogenously. Recovery was monitored closely until the mice moved freely and were observed to start feeding again. TiTannic hydrogel with the surrounding skin and subcutaneous adipose tissue layer as well as tissue for Ti detection were isolated aer schedule 1 culling of the mice at dened time points.
Histology
TiTannic hydrogels with surrounding tissues were weighed and measured and xed in 10% w/v neutral buffered formalin (NBF) overnight, dehydrated in an increasing gradient of ethanol and embedded in paraffin. Five mm sections were cut, de-waxed and rehydrated in an ethanol gradient. Sections were stained with haematoxylin and eosin (H&E) and Picrosirius Red/Fast Green. All reagents were purchased from Sigma-Aldrich. Sections were also stained for the pan-leukocyte marker CD45 using antimouse CD45 antibody clone: 30-F11 (BioLegend, UK) and detection using HRP-labelled goat anti-rat IgG (Vector Laboratories, USA). The DAB substrate kit (Vector Laboratories, UK) was used according to manufacturer's instructions. Aer nal dehydration, slides were mounted with DPX mountant (Sigma-Aldrich) and analyzed using a Nikon Eclipse TE2000 inverted microscope or a LMD7000 microscope (Leica microsystems, UK). Measurements were obtained using Fiji 62 and included the total area of the gel, the thickness of the layer of inltrate and brosis surrounding the gel, and the thickness of the material/ cell overlap layer. The number of foreign body giant cells were counted manually as shown in Fig. S14. †
Titanium measurements and biodistribution
Titanium was measured in blood, urine and tissue samples with a Thermo Element 2 magnetic sector eld HR-ICP-MS (Thermo Fisher Scientic, Germany). Calibration standards were prepared by dilution from a custom stock solution (QMX Laboratories Limited, UK) with a titanium concentration traceable to NIST SRM 3162a Lot 130925. Separate matrixmatched calibrations were prepared for each sample type to minimize matrix effects.
150 mL samples of blood or urine were mixed with 150 mL of water and 4.5 mL of assay diluent: 0.5% (v/v) tetramethylammonium hydroxide (electronics grade, Alpha Aesar, US), 0.005% (v/v) Triton X-100 (Romil, UK) and 2.5 mg L À1 gallium (Alpha Aesar, US). Blood and urine calibrators comprised 150 mL of each standard with 4.5 mL of assay diluent and 150 mL of either debrinated horse blood (TSC Biosciences, Buckingham, UK) or deionized water (urine calibrators and blank).
Tissue samples up to 150 mg in weight were accurately weighed (Sartorius CP124S analytical balance) and made up to 300 mg with deionized water. Tissue calibrators comprised 150 mL of each calibration standard and either 150 mL debrinated horse blood (TSC Biosciences, UK) or deionized water (blank). 1 mL of 25% (w/w) aqueous tetramethylammonium hydroxide (electronics grade, Alpha Aesar, US) was added to the tissue samples and calibrators and incubated for 48 hours at room temperature. 1% (v/v) nitric acid containing 5 mg L À1 gallium (Alpha Aesar, US) was added to partially neutralize the sample prior to analysis.
The diluted samples and calibration standards were sequentially sampled using an ESI-SC FAST autosampler (Elemental Scientic, US) and introduced to the HR-ICP-MS with a PTFE Nebulizer (Elemental Scientic, US) and cyclonic spray chamber (Thermo Scientic). Ti 47 and Ga 71 were measured in medium resolution mode. The counts per second (cps) data for Ti 47 cps were normalized to Ga 71 cps and calibration curves plotted in Microso Excel.
During the period of study, the laboratory was enrolled in the Quebec Multielement External Quality Assessment Scheme for blood titanium measurement and submitted results that were close to target and well within the acceptable range.
Conflicts of interest
The authors declare no competing interests. | v3-fos-license |
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} | pes2o/s2orc | Accident Damage Analysis Module (ADAM): Novel European Commission tool for consequence assessment—Scientific evaluation of performance
This paper summarises the scientific evaluation of the performance of a novel modelling tool, the Accident Damage Assessment Module (ADAM), developed by the Joint Research Centre (JRC) of the European Commission (EC) to assess the consequences of an industrial accident resulting from an unintended release of a dangerous substance. The ADAM tool is specifically intended to assist Competent Authorities in the European Union (EU) and European Economic Area (EEA), and their supporting research institutions, responsible for the implementation of the Seveso Directive in their countries, as well as governmental and research organisations of EU Accession and Candidate Countries, and European Neighbourhood Policy countries involved in chemical accident prevention and preparedness. In particular, the tool provides decision-making support to various functions associated with industrial risk management, enforcement and oversight, including risk analysis, land-use and emergency planning, inspection and monitoring, and the preparation and review of safety reports. Consequence assessment models are characterised by high level of complexity and of uncertainty. It is therefore of paramount importance to assess their limits. The scientific evaluation was conducted across the entire consequence assessment cycle, including the source terms and the physical effects calculations associated with the concentration toxics after airborne dispersion, the thermal radiation of chemical fires, and the explosion of vapour flammable clouds. The evaluation described in this paper was conducted on a series of relevant scenarios, by benchmarking the outcome of ADAM with the results obtained by similar software tools and with the experimental data obtained on a series of reference field campaigns, as taken from the literature. © 2019 The Authors. Published by Elsevier B.V. on behalf of Institution of Chemical Engineers. This is artic an open access
Introduction
In the European Union (EU), establishments that process, handle or store such chemicals in sufficient volume to have accident potential are covered under the Seveso Directive (2012/18/EU). The European Commission (EC) Joint Research Centre has developed the Accident Damage Analysis Module (ADAM) for consequence assessment, to assist EU Member States, Accession and Candidate Countries in implementation of the Seveso Directive (Wood et al., 2008) as well as a support to Neighbourhood Policy Countries involved in chemical accident prevention and preparedness related activities. ADAM became an official tool of the European Commission via the Commission Decision of 28/09/2017. It has been designed to support safety authorities in applying consequence and risk assessment for chemical accident prevention as well as to make decisions on land-use planning, emergency planning, and to assess site risk management during inspection.
Jointly funded by the EC Joint Research Centre (JRC) and the EC Directorate General on EU Humanitarian Aid and Civil Protection (DG ECHO), the ADAM tool is intended to provide competent authorities easy access to the complex technical aspects required to assess potential severity of fires, explosions and toxic releases on major chemical hazard sites, to improve risk management and protection of workers, communities and the environment from such incidents, consistent with the requirements of the Seveso Directive. The tool is also intended to help accelerate capacity of Enlargement and Neighbourhood countries and other third countries to align their national programmes with the Seveso Directive. The ADAM tool became ready for launching early this year and is being gradually deployed to countries who have expressed interest. More specifically, ADAM makes use of models that simulate all possible developments of an unintended release of a hazardous substance from the loss of containment to the final hazardous events, and estimate the physical effects produced by these events. These models cover the different aspects of consequence assessment, by including the unintended release, dispersion, fire and explosion of hazardous substances. The understanding of the performance of the predictive models is essential for the decision making process and a proper evaluation of these models is necessary to demonstrate their credibility and their limits of applicability. In general, the terms verification and validation (V&V) are currently used for this purpose, where the former aims at demonstrating that the tool accurately describes the model as designed whilst the second refers to the accurate correspondence of model prediction and observations. However, as also addressed by Chang and Hanna (2004) the random nature of the accident consequences leads to a certain irreducible inherent uncertainty, which implies "models can only be confirmed or evaluated by the demonstration of good agreement between several set of observations and predictions". In this paper, we refer to this activity as the "evaluation" (or sometimes the "scientific evaluation" to make a distinction from other types of evaluations that might be applied to a software tool, for example, by users).
Such an evaluation is not very straightforward. The main difficulty is associated with the need to establish whether the measurement data of the experimental trials used to evaluate model performance are accurate enough. In some circumstances, it would even be incorrect to assume that a perfectly accurate model would reproduce measured data. For instance, some fluctuations in the environmental conditions occurring during the field tests might influence significantly the test results and cannot be accounted for in the simulations. For this reason, instead of comparing each single prediction with the corresponding observation, a possible way forward to approach the problem is to group the observations and predictions according to a certain criterion, and then to compare the averaged results. In this respect, a model can be only confirmed or evaluated by showing the good agreement between some set of observation data and predictions.
The present paper summarises the evaluation conducted on the overall consequence assessment cycle of ADAM, from the critical event, which consists of the unintended release of the dangerous substance (ADAM Module 1) to the physical effects associated with such a release, by including the airborne dispersions of toxics, fires, and vapour cloud explosions (ADAM Module 2). This evaluation was conducted on a series of relevant scenarios, by benchmarking the outcome of ADAM with the results obtained by similar software tools and with the experimental data obtained on a series of reference field campaigns, as taken from the literature.
More specific information on the technical aspects of ADAM and on validation of its models are reported elsewhere (Fabbri et al., 2017;Fabbri et al., 2018).
Evaluation methodology
Different evaluation methodologies have been suggested for atmospheric dispersion modelling, which provide a quantitative and objective means of comparing observed and predicted physical parameters. Much less exists in other areas of consequence modelling such as methodologies predicting the effects of fires and explosions as well as phenomena derived from the source term (Coldrick, 2017). Amongst the existing evaluation methodologies, the method described in detail by Chang and Hanna (2004) was used for the evaluation of ADAM. The method is based on a range of comprehensive statistical performance measures, including the fractional bias, the normalised mean square error, the geometric mean, the geometric variance, the correlation coefficient, and the fraction of prediction within a factor of two of observations (see Table 1). Although this methodology was developed to evaluate the performance of dispersion modelling, as explained by its authors, the statistical performance measures that they use are generic enough to be applied to other contexts as long as predictions and observations are paired (Chang and Hanna, 2004). These performance measures have been therefore applied to models implemented in ADAM, i.e., for physical effects (fires and explosions), and source term related phenomena (rainout and pool evaporation).
In order to visualise the overall performance behaviour, the geometric variance VG is often graphed as a function of the geometrical mean bias MG (alternatively NMSE vs FB). As a comparison, a parabolic line that represents the minimum possible value of the geometric variance (VG) for a certain value of the geometrical mean bias (MG) is reported. A model that "perfectly" matches with observations would be placed at the vertex of the parabola (i.e., at the 1,1 point in the case of VG vs MG and at 0,0 in the case of NMSE vs FB).
When necessary to establish the confidence intervals on the different performance measures, in order to assess the significance of model differences, a Bootstrap resampling technique was employed (Efron, 1987). This procedure essentially involves random sampling from the original data set (i.e., observations and prediction pairs) with replacement from the original sample. The purpose is to generate a larger number of new sample sets of the same size as the original data set. This approach is normally suggested since the above parameters are not easily transformed by standard procedures to a normal distribution. In the present case, the Bootstrap method was applied by resampling 10,000 estimates to determine the 95% confidence intervals for each performance level.
Source term (ADAM Module 1)
The first module of ADAM refers to the implementation of all models necessary to characterise the accident source term, consisting of the estimation of the amount of the hazardous substance involved in the release process, including the release dynamic, and all the release parameters including those associated with the postexpansion process. This estimate requires the knowledge of the type of substance involved in the accident, the storage properties (i.e., quantity, temperature, and pressure), the type and mode of rupture, and the release time.
Most of the source terms models are quite well established and are somehow uniform amongst the different tools for consequence assessment. In these cases, the ADAM model implementation was simply verified by benchmarking the results obtained on a series of representative accident scenarios with those obtained using similar software tools (i.e., PHAST of DNV and EFFECTS of TNO). For the more complex evaluation of post-expansion phenomena typical of pressurised-liquefied substances, such as droplets' formation and rainout, a quantitative evaluation was conducted by comparing the ADAM outcome with the experimental observations available in the literature, and using the statistical performance measures as described in the previous section. It should be highlighted that all simulations conducted using other software tools for benchmarking purposes were always performed by the JRC independent of the software developers.
Every loss of containment is characterised by the type and properties of the contained substance, the containment geometry, and the rupture type. Therefore, benchmarking of the different models within ADAM requires an approach that addresses all the different ways that an accident can vary. In particular, by combining the initial state of the substance (i.e., compressed gas, non-boiling liquid, and liquefied-pressurised gas) and the three different types of damage mechanisms (i.e., failure from vessel hole, from pipe connected The Fractional Bias (FB) is a measure of mean bias and indicates systematic errors, which allows assessing whether the model underestimates or overestimates the measured values. FB is based on a linear scale and the systematic bias refers to the arithmetic difference between Cp and Co. Geometrical mean Bias (MG) MG = exp(ln (Co) −ln (Cp)) The Geometrical mean Bias (MG) is also a measure of mean bias and indicates systematic errors, but differently from FB that is based on a linear scale is based on a logarithmic scale. Its use is normally preferred in dispersion related applications because of the wide range of magnitudes involved.
Normalised Mean Square Error (NMSE) The Normalised Mean Square Error (NMSE) is a measure of the overall scatter around the true value and accounts for unpredictable fluctuations. It reflects both systematic and unsystematic (random) errors.
The Geometrical Variance (VG) is, analogous to the NMSE, a measure of the overall scatter around the true value. It is based on a logarithmic scale and its use is normally preferred in dispersion related applications because of the wide range of magnitudes involved.
The correlation coefficient (R) reflects the linear relationship between two variables. It is insensitive to either an additive or a multiplicative factor. A perfect correlation coefficient is only a necessary, but not sufficient condition for accuracy. Fraction of Predictions within a factor-of-two (FAC2) FAC2 = fraction of data that satisfies 0.5 ≤ Cp Co ≤ 2 The fraction of predictions within a factor of two of observations (FAC2) is the most robust measure, because it is not overly influenced by high and low outliers.
Cp values predicted by the models.
Co observations from the experimental trials. overbar stands for the average over the dataset.
C standard deviation over the dataset. to a vessel, and catastrophic rupture of a vessel), there are nine different release categories to evaluate. The present paper shows the results of benchmarking on selected cases, whilst the benchmarking conducted on all the different release categories is reported elsewhere (Fabbri et al., 2018).
Source term
For compressed gases, Fig. 1 reports the flow rate dynamic in the case of a release of ethylene from a 25 mm hole in a 0.5 m 3 'bullet' type tank (i.e. cylindrical with hemiheads), where the compressed gas is stored at an absolute pressure of 250 psi at a temperature of −25 • F. The comparison shows a good agreement between ADAM and PHAST 7.22. The curve calculated by EFFECTS 5.5 is less conservative and matches with the curve calculated by ADAM using the ideal gas assumption. The same release type was also calculated by varying the hole diameter as shown in Fig. 2.
The release of non-boiling liquid can be simulated by assuming that the tank is either 'sealed' or atmospheric. In the first case, a depressurization phenomenon will take place within the vessel during outflow, which will last until the internal pressure added to the hydrostatic weight equals the external pressure. This leads to an oscillating behaviour of the flow rate resulting in a gurgling outflow. Both PHAST 7.22 and ADAM model the depressurization of a non-boiling liquid using both assumptions. On the contrary, EFFECTS uses the atmospheric assumption. In order to make the comparison, ADAM was therefore run twice, by using both assumptions. The result for a release of benzene stored at a temperature of 300 K in a horizontal tank (10 m height, 5 m diameter, 70% filling) from a 100 mm hole located at 1 m from the tank bottom is given in Fig. 3. In both cases, ADAM reproduces quite well the results of the other tools, even if in the 'sealed' tank case, the outflow provided by PHAST, which was obtained by setting the 'vacuum relief valve' with the 'not operating' option, is somehow more erratic.
The case of releases of pressurised-liquefied gases are more complex if compared to the previous ones. The sudden depressurisation after exit produces the jet flash and leads to a two-phase release. This process is characterised by droplets' formation and subsequent rainout of a part thereof, which might alter significantly the cloud dispersion phenomenon. The estimate of the initial flow rate calculated with ADAM was compared with the simulation conducted with PHAST on a couple of reference scenarios, which are described by Kukkonen (1990). The first consisted of a release from the fullbore rupture of a pipe connected to a vessel at a distance of L =2 m from the pipe inlet (pipe roughness =0.05 mm; pipe diameter D =40 mm; released substances: chlorine, propane, ammonia, sulphur dioxide, hydrogen fluoride). The second reference scenario is the release from the full-bore rupture of a pipe connected to a vessel at variable distances from the pipe inlet (pipe roughness =0.05 mm; pipe diameters D =40 mm and D =20 mm; storage temperature 15 degrees Celsius; storage pressure at saturation; released substance: chlorine). The results of this benchmarking are given in Fig. 4 and Fig. 5, which show a good agreement between ADAM and PHAST in all involved cases. Differently from the case presented by Kukkonen (1990), who proposed a coefficient of contraction of Cd = 0.5 to account of the pipe inlet losses, the calculation herewith presented was conducted by assuming no losses at the pipe inlet, as it was not possible to input this information in the short-pipe model of PHAST.
The droplet size estimation was evaluated by comparing the tool predictions with the observations of a series of scaled experiments on different substances (i.e., water, gasoline, cyclohexane, n-butane, propane) as conducted at Cardiff University . Amongst the different methods implemented in ADAM for the calculus of the initial droplet size, the Phase III JIP SMD correlation was used . The results are shown in Fig. 6, where the predicted values of the initial SMD (i.e., Sauter Comparison between ADAM and PHAST on the release from pipe connected to a vessel containing chlorine. Flow rate vs. pipe length at rupture for two pipe diameters (i.e. 20 mm and 40 mm). Pipe roughness =0.05 mm, no losses inside pipe. Mean Diameter) are given as a function of the observed values. For a comparison, the predicted values calculated by Witlox et al. (2010), which were obtained by using the same correlation, are also reported. The results are very similar. A more quantitative analysis was conducted by applying the statistical performance measures described in Section 2, and are shown in Table 2. The rainout calculus module was evaluated against the experimental tests conducted by the American Institute of Chemical Engineers, Center for Chemical Process Safety (CCPS), which were executed in two phases and on several substances (i.e., water, CFC-11, methylamine, chlorine, and cyclohexane) (Johnson and Woodward, 1999). The rainout ADAM model was applied by using the modified CCPS correlation for evaluating the initial droplet size and the ADAM procedure to calculate the final jet vapour fraction. This is based on the selection of the proper droplets distribution function and calculating the evaporation during the droplets flight (see Annex I and Fabbri et al., 2017). An example of the comparison of the different rainout calculations against the CCPS chlorine data is shown in Fig. 7. The different curves are obtained by using the different droplets' distributions that can be selected in the ADAM option menu. In particular, the solid line was obtained by using the log normal distribution with the value for the geometric spread equal to 1.4, as suggested by Woodward (2014). The other curves were calculated by using the Rosin-Rammler distribution, with fixed parameters as suggested by Elkotb (1982) or calculated by using the Kay et al. (2010) procedure. For comparison, the rainout prediction with the Lautkaski correlation, that does not require the full calculation, but can be selected in the ADAM option menu, was also reported (Lautkaski, 2008).
In order to assess the performance of the different droplets' distributions used in the rainout calculus module, the statistical measures described in Section 2 were applied on the overall set of alternative distributions implemented in ADAM. The result is given in Table 3, which shows that the log normal distribution performs the best (higher FAC2 lower FB and NMSE, overall), and for this reason is addressed as the recommended distribution in ADAM for the calculus of the rainout.
To depict the different performance behaviours, the graph reporting the geometric variance VG as a function of the geometrical mean bias MG is often reported (Chang and Hanna, 2004). This is shown in Fig. 8 for the current case. The 95% confidence intervals on MG were calculated using the Bootstrap technique that indicates that differences in performance across the different distributions are significant.
Pool spreading and vaporisation
The recommended model for pool spreading and vaporisation models implemented in ADAM is GASP (Gas Accumulation over Spreading) developed at the UKAEA (nowadays ESR Technology) on behalf of the UK Health and Safety Executive (HSE) (Webber, 1990). This model is based on the numerical solution of a series of coupled equations for the integral properties of the pool and is applied to both continuous and catastrophic releases. The spreading model is based on the analytical solution of the shallow water equations generalised to include the effect of friction in laminar and turbulent conditions, which was introduced to overcome the negative inertia limitation typical of similar integral models. The detailed set of coupled equations providing the pool radius are reported by Webber (1990). The expression providing the evaporation/vaporisation rate per unit surface from a plane liquid surface into a turbulent boundary layer is given by Brighton's expression (Brighton, 1987).
For completeness, ADAM implemented also alternative models, which are widely in use for the determination of pool spreading and vaporisation. For pool spreading, a method introduced by several authors and recently described in detail by Raj (2011) was selected. For vaporisation, the alternative model to GASP implemented in ADAM is given by the well-known Mackay and Matsugu formula (MacKay and Matsugu, 1973).
The above models have already been the object of validation by their developers, however, since their implementation is characterised by a certain level of complexity, a separate evaluation was conducted. In particular, some selected test cases emphasising either the spreading or the vaporisation mechanisms were chosen. In addition, separate evaluations were conducted for pool spreading on land and on water, since the models involved are quite different.
For the case of spreading on land, the evaluation was conducted using the tests of Belore and McBean (1986), which consisted of the release of water over a plywood surface. Due to the very low vaporisation rate, vaporisation is negligible and it is possible to decouple the spreading mechanism from vaporisation in the considered time lapse. The input data used for the simulations and the results on the three tests (i.e., 29, 29 and 30) are reported elsewhere (Fabbri et al., 2018). These results show that the GASP model implemented in ADAM performs quite well for both tests 28 and test 29, whilst it tends to overpredict the pool radius for test 30. By contrast, the Raj model implemented in ADAM overpredicts for all the selected tests. This is in agreement with the deductions of Webber who clearly emphasised that the Raj equation rightly "expresses the resistance effect of displaced water, and has absolutely no justification for pools spreading on land" (Webber, 2012). Fig. 9 shows the result obtained for test n. 29.
For pools spreading on water, the reference tests were those provided by Dodge et al. (1983), which involved both instantaneous and continuous releases of a series of volatile and non-volatile hydrocarbons. Due to the considerable number of trials, it was possible to determine the statistical performance measures described in Section 2 for the whole series of tests, and the results are reported in Table 4. Differently for the case of spreading on land, the Raj model implemented in ADAM performs slightly better than GASP, since the performance measures are significantly different within their 95% confidence interval. As a general result, the ADAM version of GASP is recommended for pool spreading on land whilst the Raj variant is recommended in case of pool spreading on water.
For evaluating the pool vaporisation module on land, the trials conducted by Kawamura and Mackay (1987) were selected. These consisted of releases of seven volatile substances in bund, in which the spreading mechanism has no relevance (i.e., with a complete pool formation occurring in fractions of second). Details of the input data use for the simulation as well the procedure used to calculate evaporation rates per unit surface averaged over the whole experiment duration are reported elsewhere (Fabbri et al., 2018). The result of this simulation is depicted in Fig. 10, which provides a comparison between predicted and experimental data. The results obtained by using ADAM GASP and ADAM MacKay & Matsugu models are also compared to the values obtained by Fernandez (2013) using the PVAP-MC model, which is one of the implemented models in PHAST. Overall, using the Brighton value for the pool roughness length (i.e., 0.00023 m) (Brighton, 1987), the performance of ADAM GASP is very good. (This value is the recommended value in ADAM).
For pools on water, the vaporisation module was evaluated against the data of the U.S. Bureau of Mines (Burgess et al., 1972), that referred to several tests involving instantaneous releases of LNG, liquid nitrogen and liquid methane. The number of tests and the measured averaged evaporation rates (calculated during the first 20 s after the release), together with the input parameters used for the simulation are given in Table 5. This table reports also the average vaporisation rates as calculated with ADAM GASP under the two different assumptions i.e., in presence of a film boiling between the pool and the water substrate, or not. ADAM GASP reproduces quite well the experimental results for the LNG and Methane trials without the film boiling assumption (i.e. with a devi- (Burgess et al., 1972) ation of ca. 5%), with the exception of the case of liquid nitrogen for which the film boiling assumption seems much more appropriate.
Physical effects (ADAM module 2)
The second calculus module of ADAM estimates the physical effects resulting from the accidental development following the loss of containment. As such, they depend on the hazardous event involved, and in the Seveso context, they can be summarised as follows: • concentration of a toxic after airborne dispersion; • thermal radiation of chemical fires; • overpressure/impulse of the vapour flammable cloud explosion.
This calculation is normally influenced by the atmospheric conditions (i.e., air temperature, air stability, wind speed) and by other parameters, such as the average time for vapour dispersions or the ignition time or ignition location for flash fires and vapour cloud explosions. Since the models implemented in the calculation are strongly dependent on the modelling assumptions, all cases were evaluated by comparing the model output with the data of reference experimental trials.
Atmospheric dispersion
ADAM uses an in-house modified version of the SLAB model for modelling atmospheric dispersion of toxic and flammable clouds. SLAB was developed by the Lawrence Livermore National Labo-ratory. This model is based on the solution of spatially averaged conservation equations of mass, momentum, energy, and species, and produces spatially averaged cloud properties via the calculus of similarity function coefficients as a function of the downwind distance (Ermak, 1990).
Before introducing new methodological aspects, the original SLAB code was replicated in C++, and restructured in a more efficient way. A first verification was conducted by comparing the software outcome with the original code on a very large number of reference scenarios and by varying all input parameters in a wide range, in order to make sure that the replicated code would be equivalent to the original. After this verification, some major methodological improvements were introduced into ADAM, as described in detail in Fabbri et al. (2017) and summarised in Appendix B. These modifications make the ADAM-SLAB software in some way different from the original SLAB. As such, it was necessary to carry out a new evaluation exercise of SLAB as modified and implemented in the ADAM tool. The evaluation was conducted against a recognised series of large-scale field experiments, that includes the following: • Burro& Coyote (LNG releases into a water basin) (Koopman et al., 1982a, b, (Goldwire et al., 1983a. • Desert Tortoise (ammonia horizontal jets) (Goldwire et al., 1983b). • Goldfish (hydrogen fluoride horizontal jets) (Blewitt et al., 1987).
The experimental concentrations measured through sensors positioned at different locations during the trials formed the observations' data set (Co) to be compared with the model predictions (Cp).
For the trials involving finite duration releases, sensor raw data, providing concentration vs time, were processed to obtain average values within the release time duration. For the comparison, time average simulations were conducted with ADAM at the same sensors' locations. It was preferred to consider the sensor mean concentrations rather than sensor maximum concentrations in order to make use of the overall sensor curve instead of relying on a single point, which might be more sensitive to stochastic deviations (e.g., outliers). Due to the inherent local fluctuations of the concentration caused by wind direction deviations and turbulence, the construction of the pairs set based on the single observation/prediction couples for each sensor position does not make very much sense. For this reason, as sensors were displaced into equidistance arcs, data were grouped to form pairs (observed and simulated data) for each separate arc. In such a way, the average concentration of sensors located on the same arc was coupled to the average of model calculations carried out for each single sensor location on the same arc. The result of this comparison is given in Fig. 11 that shows the scatter plot of observation/prediction pairs together with the ideal behaviour (solid line) and the FAC2 curves (dashed lines).
For the Thorney Island instantaneous releases, the pairs were formed from the centre-line maximum concentrations, and the result of the comparison is given in Fig. 12, where the different trials are distinguished.
In order to quantify all the above results, the statistical performance measures defined in Section 2 were calculated for the different trials' set. Clearly, differently from Fig. 11 and Fig. 12 that provide the log-values of concentrations, the performance measures were calculated by starting from the real values of concentrations, as defined on Table 1. The outcome of this calculus is given on Table 6, which shows the general good performance of ADAM. In general, all performance measures are within the acceptability range, with the exception of the Goldfish trials, for which ADAM under predicted the concentration. For Coyote trials, the fractional bias and the geometrical mean bias were just above the acceptability threshold. The overall performance can also be represented in terms of the geometric variance, VG vs the geometrical mean bias, MG (Fig. 13). Each point representing each field cam- . 13. Model performance indicators, geometrical mean bias MG and geometric variance VG for concentration prediction and observations. The horizontal lines on MG represent 95% confidence intervals; the solid parabola is the "minimum" VG curve. The vertical dashed lines represent the 'factor-of-two' between mean predictions and observations. paign is given with its MG 95% confidence interval as calculated by using the Bootstrap technique. Overall, the results show that the performance of ADAM is very good and in line with the results of similar consequence assessment tools (Witlox et al., 2018).
Flammable effects
This section deals with the evaluation of flammable effect models implemented in ADAM, which are all based on semi-empirical correlations. For fire-related effects, the flame geometry, the radiative properties and the calculation of the radiant flux at receptor location were not analysed separately, but were studied in terms of the overall final effect. Due to modifications introduced by ADAM, both pool and jet fire calculations differ from the original set of reference models and thus, a full evaluation of these calculations within ADAM was necessary. Specifically, these modifications included the property procedure for the calculus of the view factor, and an alternative expression to address the effects of atmospheric absorption (Fabbri et al., 2017). For fireballs, since the implemented model is very well established and simple to verify by hand calculations, its evaluation was not reported. Vapour cloud explosions in ADAM are based on a set of established scaled curves for peakoverpressure and blast duration (or equivalent positive impulse) that refer to different blast severities. The outcome of ADAM was verified against the original scaled curves and evaluated against a series of experimental trials.
Pool and jet fires
For pool fires, three different semi-empirical models are implemented in ADAM (i.e. TNO modified, Shokry and Beyler, and Mudan). The three models differ amongst each other for the use Fig. 14. Model performance indicators, for the pool fire trials. The alternative pool fire models included in ADAM were tested separately. The horizontal lines on MG represent 95% confidence intervals; the solid parabola is the minimum possible value of the geometric variance; the two vertical dashed lines indicate the 'factor-of-two' limit between predictions and observations. of different correlations for the flame length, the flame tilt, the drag diameter, and the surface emissive power. Details on these correlations are given in the related references and in the ADAM technical guidance (Fabbri et al., 2017). The evaluation was carried out against experimental trials on LNG and LPG reported by Johnson (1992) and by Welker and Cavin (1982), respectively. Separate simulations were conducted for the three different models implemented. Details on the input data and assumptions used for the simulations are reported elsewhere (Fabbri et al., 2018). As for the previous cases, the evaluation results are expressed in terms of the associated performance measures and the diagram of the geometric variance (VG) plotted against the geometrical mean bias (MB) that are reported in Table 7 and Fig. 14, respectively. On the basis of these results, the Modified TNO method performs best amongst the models implemented, and as such, it has been established as the recommended method in ADAM. To further illustrate the general trend, scatter plots of the observation/predictions pairs are also shown in Fig. 15, where ADAM simulations on the Modified TNO and Mudan methods are also compared with the simulations of Johnson (1992). For jet fires, ADAM implements the model proposed by Chamberlain (1987), with Johnson's variant for horizontal jets (Johnson et al., 1994). In this model, the flame shape is represented as a tilted frustum of cone, radiating as a solid body with a uniform surface emissive power. Since the Chamberlain model was developed for gaseous substances, in order to extend its validity to two-phase releases (e.g., LPG), Cook's correction was applied (Cook et al., 1990). The detailed expression of the parameters that fully characterise the jet flame i.e., the flame lift-off distance from the Johnson (1992). Scatter plot of observation/prediction pairs. orifice, the flame length, the frustum length, the width of the of the frustum bases, the deflection angle of the flame, and the proprietary procedure developed for calculating the view factor are reported in the ADAM Technical Guidance (Fabbri et al., 2017). The evaluation was carried out against experimental trials involving natural gas releases (Chamberlain, 1987;Johnson et al., 1994). Specifically, for vertical jets, one reasonable source of test data is reported by Chamberlain (1987), which contains data from large-scale trials carried out at onshore oil installation in Cumbria (Trial n.4). For horizontal jets, the field trials carried out at the British Gas test site reported in the paper by Johnson et al. (1994) were used. These involved four different tests involving natural gas horizontal jets (B, C, D, and E), originated from different orifice diameters and under different operative and environmental conditions. Again, information on the input data and assumptions used for the simulations are reported in detail elsewhere (Fabbri et al., 2018). Fig. 16 and Fig. 17 show the comparison of thermal radiation data predicted by ADAM vs the observed values for vertical and horizontal jet fire trials, respectively. As a reference, the original Chamberlain (1987). Scatter plot of observation/prediction pairs for the different tests (i.e. A, B, and C). Johnson et al. (1994). Scatter plot of observation/prediction pairs for the different Tests (i.e. B, C, D, and E). Chamberlain (1987) and Johnson et al. (1994) were also included in the graphs. The overall evaluation is reported in Table 8, in which the whole set of statistical performance measures is provided. Similarly to the case of pool fires, ADAM shows good performance.
Vapour cloud explosions
The evaluation of the vapour cloud explosion module of ADAM was conducted on the two alternative models implemented in ADAM i.e., the Multi Energy (ME) and the Baker-Strehlow-Tang (BST) methods (Fabbri et al., 2017). For the ME method, the ten scaled curves of the peak overpressure and of the positive phase duration, that correspond to the 10 blast strength levels, were extracted from the original graph produced by the model developers (Mercx and van den Berg, 2005). Each scaled curve was digitised by producing 1000 points and uploaded on the ADAM code. The same was done for the 9-scaled-curves of the peak overpressure and positive impulse of the BTS method, which were extracted from the original work of the model developers (Tang and Baker, 2000). Since the available 9-scaled curves are associated to flame speeds that do not cover all Mach values of Pierorazio's table (Pierorazio et al., 2005), intermediate curves were created by using the spline interpolation method. All scaled curves uploaded in ADAM were carefully verified against the original graphs by superimposition.
The evaluation was completed against the BFETs and EMERGE experiments and on the Shell Deer Park accident case by replicating the detailed comparison of the ME and BST simulations with the reference data conducted by Fitzgerald (2001). Details on the comparisons of results from individual tests and on the input data used for the simulation are reported in Fabbri et al. (2018).
The outcome of the simulations on BFETS tests performed with ADAM by using the BST and ME methods are shown in Fig. 18 and Fig. 19, respectively. These simulations are also compared to those carried out by Fitzgerald (2001) on the same reference tests. In general, ADAM calculations confirm Fitzgerald's results, that is, the BST tends to underestimate the BFETS data, whilst overpressure estimated by using the ME method was found to be more accurate. A clear improvement of the BST performance was indicated when the method of Xu et al. (2009) was applied. This result provides the ground correction factor implemented in PHAST. For this reason, this correction was also implemented in ADAM as an option that can be selected in the ADAM menu.
Concerning EMERGE experiments, similarly to the conclusions of Fitzgerald's paper, all small and medium scale tests were considered not realistic for industrial scenarios. Fig. 20 shows the overpressure simulation as a function of the distance from the edge of the congestion zone for EMERGE Tests L1, 2 (methane, high congestion. Confinement 3D). As for the previous case, the ME method provides the best estimate.
Finally, Fig. 21 shows the simulation and comparison with the observed overpressure data resulting from a large VCE of an accident case occurred at the Shell Company plant in Deer Park, Texas, for which far field overpressures were estimated, based on different observed damage levels. The simulation shows that both ME and BSI methods performed similarly for this case. A point to note is that the estimate of overpressures in vapour cloud explosions by using BST and ME methods are generally oversimplified especially in the near field, due to the presence of obstacles and the challenge of replicating site geometries in a test environment. The difficulty arises from the presence of varying types of obstructions (e.g., walls, pipes, vessels and other equipment) that the vapour release might encounter. For each obstruction, its particular dimensions, as well the dimensions of the open space around it (based on the distance(s) from other obstructions in the vicinity or the source of the release) can influence the development of the vapour cloud, including its density and dispersion qualities but are not accounted for by the integral models used for the simulation. This is one of the main reasons of typical discrepancies between the simulations conducted using ME and BST methods and the experimental tests.
Conclusions
The application of statistical performance measures for evaluating and comparing consequence analysis methodologies provided valuable support to evaluation of the development of the ADAM tool. In particular, this approach was important for validating recommendations in the ADAM tool for calculating physical effects and source term related phenomena.
The results of the validation efforts gave substantial support to recommended default options in the ADAM tool to users for calculation of physical effects and source term related phenomena. These recommendations are intended in particularly to aid users that may not feel expert enough to select their own calculation method. The validation exercises showed that ADAM's recommendations for calculating rain-out, pool vaporisation, atmospheric dispersion, flammable effects, pool and jet fires, and vapour cloud explosions perform well against other methodologies in replicating consequences using actual accident data.
It should be noted that these results do not suggest that other methodologies developed to calculate these effects should never be used. Indeed, all the methodologies have strengths and weaknesses for particular scenarios. For this reason, ADAM also includes the choice of other well-known and respected methodologies as options for expert users who are experienced in recognising situations where these methodologies are best applied. assess the substance response within the vessel during depressurisation (van den Bosch and Duijm, 2005). These equations are described in detail in the ADAM Technical Guidance report (Fabbri et al., 2017). The post-expansion parameters, typical of releases resulting from compressed gases or superheated substances (i.e., liquefied-pressurised substances) are obtained by assuming a 1D homogeneous jet (i.e., with no phase-slip) in a thermal equilibrium and resolving two alternatives set of conservation equations i.e. mass/momentum/energy or mass/momentum/entropy (Britter, 1994(Britter, , 1995. Flash velocity is estimated by combining mass and momentum conservation equations, whilst the vapour quality after flash is obtained via the energy conservation equation in the first case, or via the isoentropic condition in the alternative case (Fabbri et al., 2017). Despite of the attempts to establish which of these two approaches is best (Britter, 1994), a definite conclusion has yet not been established. In ADAM, both approaches are used depending on the specific selected correlation for the estimate of droplets size as explained below. For compressed gases, ADAM always makes use of the isentropic assumption, since it seems to provide more reliable results (Witlox and Bowel, 2002).
After post-expansion, the liquid mass fraction of the jet will break into droplets by mechanical and/or flashing brake-up. The first is normally produced on subcooled liquids relative to ambient conditions, that are also under enough pressure as they share forces resulting from the velocity difference between liquid and air, the second is typically produced on superheated liquids during a depressurisation process. The understanding of droplet formation and entrainment in the vapour cloud is particularly important to assess their influence on the cloud dispersion behaviour. In addition, droplet size is directly associated with liquid mass fraction falling onto the ground after release (rainout), which is cause of reduction of airborne concentration and extension of duration of the dispersion phenomenon. The modelling of this process is rather complex, since the atomisation produces many different final droplet sizes that follow a certain distribution. In ADAM we use both the Log-Normal (Woodward, 2014) and the Rosin-Rammler equations to model the distribution of droplet sizes in a cloud. These distributions are fully described by one or two adjustable parameters together with a characteristic droplet size. In the context of consequence assessment, this characteristic size is defined by the Sauter Mean Diameter (SMD or d p32) : where D is the diameter of droplets generated by the depressurisation. The choice on SMD with respect to other means is because droplet size distributions with the same SMD have the same volume to surface ratio, which is very important in the rainout process.
The different heuristic correlations, used by ADAM to determine the value of SMD for a determined release scenario, make use of the post expansion data, calculated by the source term module. They can be selected in the option menu and are briefly described in Table A1.
Once the SMD value is determined for the release under investigation, ADAM produces the estimate of the rainout, i.e., the total mass fraction that falls on the ground after release. This is conducted by considering the overall droplet distribution selected in the option menu (i.e. Log-Normal or Rosin-Rammler) with initial mean size equal to SMD and calculating the mass fraction of single droplets of the distribution reaching the ground, by combining the droplet motion equation (Holterman, 2003) with the equation describing rate of decrease of droplet diameter due to evaporation in air (Williamson and Threadgill, 1974). The resulting mass Witlox et al., 2007Witlox et al., , 2010 fraction of the jet that remains airborne is given by the following expression: where p(D) is the density function of droplets' distribution, and x sd rain is the mass fraction of the droplet of diameter D that remains airborne. The overall procedure for this calculation is described in detail elsewhere (Fabbri et al., 2017). For completeness, ADAM incorporates also other approaches based on empirical correlations of de Vaull and King (1992) and Lautkaski (2008), which can be selected in the option menu in alternative to the full calculation.
Appendix B. Dispersion modelling. Modified version of SLAB
In ADAM, the SLAB algorithm has been rewritten with more efficient code, that increases significantly the number of points to produce consequence maps with better spatial resolution. All environmental data, which are coded and fixed in the original software, are taken directly from the ADAM database that correspond to the specific environmental conditions of the scenario under study. In addition to these modifications, ADAM introduces some major modelling improvements, and specifically: Fig. B1. Catastrophic releases in presence of rainout. The total mass released Q is given by the sum of a vapour, Q V = x f Q and a liquid, Q L = (1-x f )Q, phase. After rainout a pool is formed, Q pool = X L rain Q L , whilst part of the liquid droplets remain airborne Q aerosols = (1-X L rain ) Q L .
• alternative calculus of the average concentration for instantaneous releases; • calculus for time-varying releases; • inclusion of the contribution from pool evaporation in case of rainout; Alternative calculus of the average concentration for instantaneous releases The calculus of the time-average concentration is performed by using the following equation: where C is the instantaneous concentration, recalculated to account the meander effect, t av is the averaging time and t pk the time of peak concentration. This calculus is rather trivial for continuous or finite duration releases, but the full integration has to be done in the case of puff dispersion, typical of instantaneous releases. The original SLAB code simplified this calculus by performing a variable transformation from time to downwind distance via the velocity of the puff centre-of-mass, and resolving the above integral. However, and as demonstrated by Fabbri et al. (2017), this approximation has the inherent error that the peak concentration time at a certain location does not necessarily occur when the centre-of-mass of the puff is centred in such a location. Besides, the centre-of-mass of the puff will never be located for negative values of x, since it propagates downwind. Consequently, the estimated average concentration will be always zero backwind that is obviously not possible since a small part of the toxics will always diffuse backwind, for catastrophic releases. Overall, this approximation will tend to produce a correct average concentration only in the far field, a lower concentration in the near region, and complete wrong results both in proximity of the emitting source and backwind (Fabbri et al., 2017). For this reason, the SLAB approximation is not applied in ADAM, and the full integral is calculated using a special algorithm based on both Romberg and Gaussian integration methods to optimise the calculus time.
Calculus for time-varying releases
For scenarios involving finite duration releases (i.e. horizontal/vertical jets, and pool evaporation), SLAB is based on the assumption that the flow rate (or evaporation rate) is constant within the release time range, and zero outside. Since ADAM calculates the actual source term, which includes its time dependence, it is necessary to couple the time-varying release with the dispersion model. In order to achieve this, the time-varying release is divided into a number of discrete time segments, and the dispersion associated therewith is calculated accordingly. As suggested in the Purple Book (2005), the segments used to approximate the flow rate are characterised by constant values, which correspond to the outflow average in the selected time segment. The segment duration is defined in such a way that all segments have equal areas (i.e. the released mass is equal in each segment time) and the sum of all segment areas is the same as the area under the flow rate curve (i.e., the total released mass). ADAM allows splitting the timevarying release source term into a maximum number of twenty segments, with a default value of five, and it produces the calculus of the concentration by using the following procedure: 1 The release is replaced by N segments with outflow rates q i (i refers to the i-th segment) and time duration t i . = Q tot /(N q i ) , where Q tot is the total mass released. 2 N different dispersion calculations are separately conducted for hypothetical releases with outflow rates q i and durations t Di = min( t i , Q tot /q i ) , where t i is the total duration of the release. 3 Since the concentration produced by the time varying release, has to be in between the concentrations produced by the hypothetical releases with higher and lower outflow rates, the overall concentration is estimated by a weighted average of those associated to the single releases, with the released masses Q i as the reference weights.
A more exhaustive explanation of the above procedure and limitations is reported in the ADAM technical guidance (Fabbri et al., 2017).
Contribution from pool evaporation in case of rainout
Since ADAM provides an estimate of the rainout, the dispersion is calculated by using a recombination process. In particular, the dispersion of the vapour jet (or the vapour part in a catastrophic release) is combined with the vapour resulting from the evaporation of the rainout pool. These two phenomena are considered as independent, and ADAM separately estimates the effects associated with each of them. The overall concentration is conservatively estimated by adding up each contribution. In order to apply the recombination method, the dispersion calculation is applied twice. The first is applied to the primary phenomenon, i.e., the one associated with the direct vapour source, whilst the second is associated with the secondary phenomenon, i.e., the evaporation for the pool resulting from the rainout. When the release involves a pure liquid, the primary dispersion phenomenon is driven by the vapours originating from the pool evaporation whilst the secondary and minor phenomenon results from the jet vapours produced by the mechanical brake-up of liquid droplets. The input parameters to apply are selected according to the simplified scheme depicted in Fig. B1, which typically refers to a two-phase catastrophic release. Since after rainout, the droplets that remain airborne will be added to the vapour phase in the dispersion-related phenomenon, the masses used as SLAB input variables for the primary and secondary dispersions are: where Q is the total mass released, and x rain is given by: where X L rain the liquid fraction after rainout (i.e. Q pool /Q L in the figure), and x f the vapour quality after flash.
For the part of dispersion associated with the direct vapour, since in SLAB the substance aerosols are considered as embedded/transported by the cloud, the liquid fraction to be fed to SLAB in the recombination process is provided by ADAM as follows: Clearly for the part of the dispersion associated with pool evaporation mechanism (i.e. generally the secondary dispersion), this parameter will be fixed to zero as the evaporated phase has no liquid portions. For continuous releases, the above relations are also valid, with the condition of substituting the mass with the flow rate. | v3-fos-license |
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} | pes2o/s2orc | Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates trans-retinol to 3,4-dehydroretinol
Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all-trans-retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5–2.3 (on kcat/Km) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.
Cytochrome P450 (P450) 2 enzymes are rather ubiquitous in nature. The mammalian P450 enzymes are important in drug and carcinogen metabolism (2,3) and also have critical roles in steroid metabolism (4). In addition, P450s have important roles in the metabolism of vitamins A, D, E, and K (5).
Of the 57 human P450 genes, 50 code for proteins that are localized mainly in the endoplasmic reticulum, and seven code for P450s that are nucleus-encoded and transported to mitochondria (5). These seven P450s utilize adrenodoxin (Adx), a ferredoxin, as a source of electrons, instead of the flavoprotein NADPH-P450 reductase used by the P450s in the endoplasmic reticulum ( Fig. 1). In addition, a complement of some of the microsomal P450s is also found in mitochondria, and these P450s also utilize electrons from Adx (6,7). Of the seven mammalian mitochondrial P450s, X-ray crystal structures of three have been reported (11A1, 11B2, and 24A1) (8 -11). Only P450s 11A1 and 11B1 have been extensively characterized in terms of their interactions with Adx and their catalytic mechanisms (12)(13)(14)(15)(16)(17)(18)(19)(20)(21).
In this work, we established the localization of human P450 27C1 in skin. Both 3-and 4-hydroxyretinol were characterized as minor products of P450 27C1. Kinetic isotope effect (KIE) studies with 3-and 4-deuterated retinol showed contributions of C-H bond breaking as a rate-limiting step in catalysis of desaturation and the hydroxylations. Kinetic analysis of other steps in the reaction cycle indicated that P450 27C1 reduction by Adx also contributes in terms of rate limitation.
Identification of P450 27C1 in skin by mass spectrometry and immunochemical methods
MS profiling was done with human liver and skin samples, based on previous work showing P450 27C1 mRNA in the liver (23) and the demonstration of retinol desaturation activity in human skin epidermis (41). Because P450 27C1 is considered to be a mitochondrial protein (due to sequence similarity to P450s 27A1 and 27B1 and the demonstrated reduction by Adx (24)), we utilized tissue homogenates instead of microsomal fractions. Human P450 27C1 is 43% identical to P450 27A1 (a liver enzyme (5)) and 38% identical to P450 27B1 (a kidney enzyme (5)), based on Uniprot.org alignment (http://www.uniprot.org/ align). 3 Purified recombinant P450 27C1 (see supplemental Fig. S1) was used to guide the selection of migration distances selected for analysis (the M r is less because 66 residues were deleted from the N terminus in the construct that we used (GenBank TM accession number AC027142; March 28, 2000) (and two new ones were added at the N terminus and His 6 at the C terminus) (23)). The P450 27C1 sequence listed in Uniprot Q4G0S4 (and also as NG007986, NM_001001165, and AC027142 elsewhere) begins with MRSVLRQ, is only 372 residues long, and is missing the N-terminal 106 residues of our expressed P450 27C1 (23), based on the original sequence of Nelson (http://drnelson.uthsc.edu/cytochromeP450.html) 3 (95), which denotes the shortened sequences in that website. Initial LC-MS/MS of tryptic peptides from a human skin sample showed the presence of P450 27C1, with 51% sequence coverage in this 372-residue region (supplemental Table S1) (Fig. 3). We hypothesized that the 372-amino acid entry in the databases is an error due to misassignment of the methionine start site and did extensive proteomic analysis with a second skin sample (i.e. different individual) to define the protein found in human skin.
Polyclonal antibodies were raised against P450 27C1 and purified by adsorption with immobilized P450 27C1. Immunoblotting consistently showed the presence of two bands in all five human skin samples examined (Fig. 4A) but no detectable bands in any of the human liver or kidney samples, only background staining (Fig. 4, B and C) (the limit of detection was 0.1 pmol of P450 27C1). (Note that the tissue samples were not from the same five individuals.) Because strong denaturing conditions were required to solubilize human skin samples, conventional methods for antibody pull-down experiments were not possible for validating the specificity of the antibody. Accordingly, we digested the proteins in the gel regions corresponding to antibody binding (Fig. 4)
P450 27C1 oxidation of retinol
protein) Ϫ1 and from 5 to 11 pmol P450 27C1 (mg of protein) Ϫ1 for the lower band. Further proteomic analysis of the two gel regions was done. Peptides corresponding to both bands were identified, validating the specificity of our antibody (Fig. 5). The coverage was 37% in the upper band and ϳ40% in the lower one (depending on exactly where the N terminus is). The identification of the peptide MQTSAMALLAR (in the higher M r band) ( Fig. 5 is 524 -537, and the same peptide is shown again in supplemental Fig. S3. Identified fragment ions are annotated and color-coordinated (blue, y-ions; purple, b-ions). Heavy lines, peptide fragments identified; dashed lines, missed fragment ions. Included is the precursor ϩ3 ion (inset, isotopic distribution) identified and selected for fragmentation. The spectrum was generated using SeeMS for MS 2 data integrated into IDPicker software and Xcalibur Qual Browser for MS 1 data. See also supplemental Table S1. Fig. 4) was used, and the two immunoreactive bands were located by comparison with a part of the same gel in which the proteins were transferred to a sheet of nitrocellulose and located by immunostaining (Fig. 4). A, predicted full-length amino acid sequence of P450 27C1, with highlighted and underlined peptides identified in the higher-M r band of Fig. 4; see supplemental Figs. S2 and S3 for the N-terminal and most C-terminal peptides identified. B, predicted full-length amino acid sequence of P450 27C1, with highlighted and underlined peptides identified in the lower-M r band of Fig. 4. See Fig. 3 and supplemental Table S1 for more data on peptide analysis with a sample from a different individual.
is. We did identify the peptide SLAAMPGPR (amino acids 70 -78 in Fig. 5B) in an analysis. The C terminus is nearly intact, with the peptide THGLLTPGGPIHVR identified (in both bands; Fig. 5 and supplemental Fig. S3). Therefore the cleavage site is postulated to be near residue 70 (Fig. 5C), in that the M r of the lower band is very similar to that of recombinant P450 27C1 (Fig. 4A), which is 58 residues shorter than the full-length sequence shown (23). No P450 27C1 peptides were detected in a (single) human liver sample when a similar analysis was done under similar conditions (results not shown).
Identification of reaction products
Oxidation of all-trans-retinol to 3-and 4-OH products-We previously reported that human P450 27C1 oxidized retinol to a product that co-eluted with 4-OH retinol (Fig. 2), in addition to the desaturation product 3,4-dehydroretinol (25). However, we did not have any independent evidence for its identity. We synthesized both 3-and 4-OH retinol (supplemental Figs. S4 and S5) and found that both of these alcohols are minor products, based on co-chromatography by HPLC and UV and mass spectral comparisons. Because of the trace amounts of these products formed, we did not determine steady-state kinetic parameters for their production. At a substrate concentration of 7 M, the molar ratio of products formed was 100:3:2 for 3,4-dehydroretinol, 4-OH retinol, and 3-retinol, respectively (Fig. 6). Incubation of 3-or 4-OH retinol with P450 27C1 (in the presence or absence of NADPH-Adx reductase (ADR), Adx, and NADPH) did not show any formation of 3,4-dehydroretinol, indicating that neither of the hydroxy compounds is an intermediate in the formation of dehydroretinol.
Other reactions-In previous studies, we found that retinoic acid and retinaldehyde (oxidized forms of all-trans-retinol) were substrates for P450 27C1, with lower catalytic activities compared with retinol (23,24). We also tested two known biologically important retinyl esters as substrates with P450 27C1. Retinol acetate and retinol palmitate (20 M) were incubated in the presence of P450 27C1, ADR, Adx, and NADPH. After mild KOH hydrolysis of reaction mixtures, 3,4-dehydroretinol was detected in the reaction with retinol acetate but not with retinol palmitate (data not shown).
Individual reaction steps and catalytic mechanism
Substrate binding-We had previously shown that human P450 27C1 forms a tight complex with all-trans-retinol, shifting the heme Soret peak from the low-to high-spin iron form ("Type I" difference spectrum), with an estimated K d of ϳ5.6 nM (25). The rate of binding was measured using two different approaches. Equimolar concentrations of P450 27C1 and the substrate all-trans-retinol were mixed in a stopped-flow spectrophotometer (Fig. 7A), and the data (Fig. 7B) were fit to a second-order equation, in that the binding reaction is first-order in both P450 and substrate (42). The estimated k on value was 1.9 ϫ 10 5 M Ϫ1 s Ϫ1 (Fig. 7C). In an alternative approach, first-order rates were estimated with varying concentrations of substrate and plotted versus the substrate concentration (43) to obtain a k on value of 1.6 ϫ 10 5 M Ϫ1 s Ϫ1 and k off of 0.022 s Ϫ1 (Fig. 7D). This k off value is subject to considerable error because of the need to use Ն1 M P450 in these stopped-flow measurements. A trapping assay was used in which a 1:1 molar mixture of 2 M P450 27C1 and trans-retinol was mixed with 20 M ketoconazole, an inhibitor that was measured to have a K d of 7 Ϯ 4 M and gives a spectrally distinct complex with P450s ("Type II") (44). The observed rate was 0.017 s Ϫ1 , or 1 min Ϫ1 (compared with a rate of 1,200 Ϯ 240 min Ϫ1 for ketoconazole binding to P450 27C1 in the absence of dehydroretinol; results not shown).
Reduction by Adx-Adx forms a complex with ADR and also has been known to be involved in electron delivery to mitochondrial P450s (45). Tight complexes with Adx have also been demonstrated with mitochondrial P450s 11A1 (12) and 11B1 (13), involving changes of low-to high-spin iron. However, we did not observe any spectral perturbation of P450 27C1 by the addition of Adx (in the absence or presence of equimolar amounts of all-trans-retinol), although the P450 27C1 activity is clearly dependent upon Adx for electrons (data not shown). Preliminary assays were done by mixing photoreduced Adx with ferric P450 27C1 under an anaerobic CO atmosphere (supplemental Fig. S7). Considerable evidence for Adx acting as a mobile electron carrier has been published with another mitochondrial P450, P450 11A1 (13). In our experiments, the estimated rate of reduction (at 23°C) was 1.3 Ϯ 0.3 min Ϫ1 in the absence of the substrate retinol and 3.6 Ϯ 0.3 min Ϫ1 in the presence of 5 M retinol. We concluded that the presence of the substrate enhanced the rate of reduction, and further measurements were all done in the presence of substrate.
In a system with a 1:10:1 molar ratio of ADR/Adx/P450 27C1, the rate of reduction (in the presence of retinol) was 21 Ϯ 1 min Ϫ1 at 37°C (Fig. 8). (Rates of reduction of ADR by NADPH and Adx by ADR have been measured previously (32 and 5.4 s Ϫ1 , respectively, at 15°C (21)). (In the steady-state kinetic assays, the enzymes were much less concentrated than in these anaerobic experiments, with 7.5-fold less ADR, 3-fold less Adx, and 75-fold less P450.) Product release-A trapping assay was used in which a 1:1 molar mixture of 2 M P450 27C1 and dehydroretinol was mixed with 20 M ketoconazole (see above). A first-order rate of 54 Ϯ 5 min Ϫ1 was estimated for the release of the product dehydroretinol at 23°C.
Another important approach to determining whether a step following product formation is rate-limiting is to establish whether or not there is a kinetic burst (43). When we extrapolated to time 0, no burst was seen (Fig. 9). We conclude that neither product release nor any step following product formation is rate-limiting.
KIEs
Deuterated retinols were synthesized (with Ͼ95% atomic excess; see supplemental material, including supplemental Fig. S6) and used to test the hypothesis that C-H bond breaking is a rate-limiting step in the desaturation or hydroxylation of retinol. We do not have information about the stereospecificity of hydrogen abstraction from retinol (which is pro-chiral at C-3 and C-4), so the dideuterated retinol derivatives 3,3-d 2 and 4,4-d 2 retinol were prepared, as well as tetradeuterated 3,3,4,4-d 4 retinol. (The synthesized 4,4-d 2 and 3,3,4,4-d 4 retinols also had deuterium at the side chain carbinol position (C-15), but this is irrelevant in the assays in that no oxidation occurs there.) The KIE experiment that is most informative about a ratelimiting step is a noncompetitive intermolecular design, in which k cat and K m are measured with varying concentrations of protiated and deuterated substrates and used to calculate D V and D (V/K), defined as H k cat / D k cat and ( H k cat / H K m )/( D k cat / D K m ), respectively, with H and D indicating parameters measured with protium and deuterium at a site (1,46,47). 4 Relatively small but reproducible changes were observed in the rates of desaturation when using deuterium-labeled substrates; low KIEs were observed for desaturation with 3,3-d 2 , 4,4-d 2 , and 3,3,4,4-d 4 retinol (Fig. 10). Most significantly, a KIE of ϳ2 ( D (V/K)) was observed when deuteriums were present at C-3 of the substrate (3,3-d 2 and 3,3,4,4-d 4 ), whereas the D (V/K) for 4,4-d 2 retinol was calculated to be only ϳ1.5.
We considered the possibility that deuterium scrambling might occur after abstraction of a hydrogen atom from C-3 via a radical intermediate (or, alternatively, from a carbocationic intermediate that might be formed from further electron transfer (37)) ( Fig. 2). Accordingly, we analyzed the desaturation and 4-hydroxylation products of 4,4-d 2 retinol by LC-HRMS. The masses detected at 100,000 resolution were consistent with those of the expected products, providing evidence against migration of deuterium from the C-4 to the adjacent C-3 site (Fig. 12).
Discussion
We have established that P450 27C1 is located in the skin, is involved in the desaturation of retinol, and also forms minor amounts of 3-OH and 4-OH retinol. Within the catalytic cycle, the reduction of ferric iron and C-H bond-breaking steps both contribute to limiting the reaction rates, but substrate binding and product release do not.
Skin is a major organ, in terms of collective body mass, and metabolism there is of interest with many drugs (applied cutaneously) and with a number of endogenous chemicals. Regarding P450s, there is some evidence that P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1 2S1, 2U1, 2W1, 3A4, 3A5, 4B1, 4F2, 4F3, 4F22, 11A1, 17A1, 24A1, 26A1, 26B1, 26C1, 27A1, and 51A1 are expressed in human skin samples or in derived cells (in culture) (48 -54). However, almost all of the data are at the mRNA level. Only in a few cases has protein expression been reported, and then only at the immunohistochemical or enzyme activity level (52,54). Previous studies by Törmä and Vahlquist (41) demonstrated the presence of retinol 3,4-desaturation activity in human skin epidermis, as well as in human keratinocytes, melanoma cells, and HeLa cells (55). In the present study, we identified P450 27C1 in human skin samples using proteomic and immunochemical methods (Figs. 3-5) but did not find P450 27C1 in the human liver and kidney samples that we analyzed (Fig. 4, B and C). Earlier studies with an oligonucleotide probe selective for P450 27C1 (compared with P450s 27A1 and 27B1) indicated the presence of P450 27C1 mRNA in several human tissues, including the liver, kidney, and pancreas (23). Skin mRNA had not been included in that study. One possibility for the apparent difference in tissue localization is that the oligonucleotide probe used previously (23) was not specific, although that seems unlikely in the context of the selectivity demonstrated versus P450 27A1 and 27B1 sequences. An alternative possibility is that mRNA is formed in several tissues but not translated into protein, which would not be surprising in light of the overall concordance between mRNA and protein expression (56). The possibility also exists that the mRNA could be alternatively spliced or transcribed in other tissues. In reviewing the Human Protein Atlas (http:// www.proteinatlas.org), 3 the highest level of mRNA expression of CYP27C1 is in skin (57). Some other sites in which higher than baseline levels were seen include the uterine cervix, urinary bladder, and vagina (57). Trace but apparently real levels of CYP27C1 mRNA were found in many other tissues, including those that we reported previously (23). Our conclusion is that we were observing these low levels of expression in our earlier data (23), which was misleading in that skin was not included. Although expression in retinal epithelium is critical in the biology of dehydroretinol in fish (24), the literature does not indicate the presence of dehydroretinoids (vitamin A 2 ) in human eyes (57). 3,4-Dehydroretinoic acid is also formed by human P450 27C1 (25), but the biological activities of retinoic acid and 3,4-dehydroretinoic acid are similar in human keratinocytes (58). In human skin, dehydroretinol accounts for ϳ25% of the retinoid pool (59,60), but the function remains unknown. Levels of dehydroretinol are elevated in psoriasis (60) and squamous cell carcinoma and keratocanthoma (61), and the desaturation activity is induced by exposure to UVA/B light (62). In our analyses, the levels of the two forms of P450 27C1 only varied ϳ2-fold among the five human samples analyzed (Fig. 4A). This level of variation is similar to that commonly seen for P450s involved in normal physiological processes (63) and differs from the wide variation in levels of the P450s that are involved in the metabolism of xenobiotic chemicals (5, 63).
We utilized the original human CYP27C1 sequence (Gen-Bank TM AC027142; March 28, 2000) (Fig. 5) in consideration of what is actually expressed in human skin. The Uniprot Q4G0S4 372-residue sequence is clearly wrong. No P450 this short has ever been identified to our knowledge, even in bacteria, and it would not be expected to be catalytically active. The 482-amino acid version that we expressed (25) and used in catalytic assays (Figs. 6 -9) is similar in M r to the smaller protein found in the skin (Fig. 4A). The larger protein (Fig. 4) contained the peptides that correspond to residues 1-537 (Fig. 5A), in that the peptides for residues 1-11 and 524 -537 were characterized. The smaller protein contains residues 70 -78 (Fig. 5B), so this peptide must be near the N terminus, but we cannot determine exactly where P450 27C1 was cleaved. We presume that the smaller protein is catalytically active, in that its size is similar to that of the recombinant protein (Fig. 4A). Which of these proteins is present in mitochondria is currently unknown. The program MitoFATES (64) (mitf.cbrc.jp/MitoFates/cgi-bin/top.cgi) 3 predicts a TOM20 recognition motif at residues 9 -14 and an MPP cleavage site at residue 66, the latter of which is what we had serendipitously designed in our original Escherichia coli expression construct, leaving an N terminus similar to that observed for murine P450 27B1 (23). Further work is needed, however, to characterize the transport of a specific sequence into mito-
P450 27C1 oxidation of retinol
chondria. Our current results indicate the methionine at residue 1 as the start site for protein translation (Fig. 5A).
Desaturation is a relatively infrequent event in P450 oxidations, at least compared with hydroxylation, and we were interested in defining rate-limiting steps. Very little work has been published on rate-limiting steps in reactions by the seven mitochondrial P450s (11A1, 11B1, 11B2, 24A1, 27A1, 27B1, and 27C1), including KIE studies (5, 65). The literature indicates that the KIEs observed for some microsomal P450 desaturation reactions are relatively high (33,66), depending on the experimental design. We measured D V and D (V/K), the latter of which is considered the most useful KIE experiment to establish to what extent C-H bond breaking is rate-limiting in a reaction (1,46,47). The KIE values are relatively low, in terms of the values seen in some (but certainly not all) P450 oxidations (47), but nevertheless the KIE values are significantly greater than unity, indicating some contribution to rate limitation.
Several other steps in the catalytic cycle were also measured. The rate of substrate binding was measured to be 1.6 -1.9 ϫ 10 5 M Ϫ1 s Ϫ1 , using two approaches (Fig. 7). The reaction appeared to be monophasic but is slow in terms of what is expected for a diffusion-limited reaction (67). Further, the apparent rate constant is at least 1 order of magnitude less than those measured for substrate/ligand binding to some human microsomal P450s (68 -70) and bacterial P450 101A1 (P450 cam ) (71). The slower rate measured for P450 27C1 may reflect the existence of a slow step, following initial binding, that leads to the spin state change, as in the case of P450 3A4 (69,72). Regardless, substrate binding and product release are considerably faster than the overall oxidation, and the absence of burst kinetics already indicates that a step following product formation cannot be ratelimiting (Fig. 9). The rate of reduction of ferric P450 27C1 is somewhat faster than overall catalysis (Fig. 8), and we propose that it can contribute in part as a rate-limiting step. Preliminary analysis of a simplified KinTek Explorer mechanism (supplemental Fig. S8) indicated that the simulated rate of the overall reaction was increased when the rate constant for the reduction step was raised above the rate observed in our work, in support of the view that the reduction of ferric P450 27C1 is a partially rate-limiting step. The reduction rate was stimulated in the presence of the substrate retinol (ϳ3-fold) (supplemental Fig. S7); sometimes P450 reduction rates are stimulated by substrates and sometimes not (73). It is of interest to note that Tuckey et al. (19) concluded that the reduction of mitochondrial P450 11A1 by Adx was rate-limiting in human placenta, as well as in reconstituted systems (74). To our knowledge, of the seven mitochondrial P450s, only with P450 11A1 has a ferrous-O 2 complex been characterized (and at low temperature) (17). We attempted to characterize such a P450 27C1 complex by mixing photoreduced P450 27C1 with O 2 but were not successful. It would be desirable to measure the rate of Adx reduction of this complex, but no such mitochondrial P450 reaction has been characterized in detail yet.
The KIE values for desaturation (Fig. 10) and 4-hydroxylation are relatively low. We have not estimated the intrinsic KIE, D k, in either case but suspect that it may be considerably higher. Thus, the values of 1.5-2.3 we measured for D (V/K) (Fig. 10) may be attenuated due to the tight binding of retinol (previously estimated K d of 5.6 nM (24) and k off of 1 min Ϫ1 (this work)). This represents a case of high forward commitment to catalysis (C f , using the conventions of Northrop (1) and as elaborated by Northrop (1) and elsewhere (47)), (when there is no commitment to reverse catalysis). Thus, although it might not be intuitive, raising C f (i.e. decreasing the substrate k off ) lowers the observed D (V/K) value for a reaction such as a P450-catalyzed oxidation, which is essentially irreversible. Therefore, the D (V/K) values measured here are probably lower than the D k and may not reflect a higher KIE. The KIE and other deuterium experiments provide some insight into steps associated with the oxidation of retinol (Figs. 10, 11, and 13). We propose that abstraction of a C-4 hydrogen atom is the first step in both 4-hydroxylation and desaturation, in that both processes had low KIEs (Fig. 10). 3-Hydroxylation is a separate reaction and begins with abstraction of a C-3 hydrogen atom. This is an unfavorable reaction, possibly because, unlike the C-4 abstraction, it does not yield an allylic radical. The measured KIE values were much higher, and hydrogen atom abstraction is clearly rate-limiting for the minor 3-hydroxylation reaction (Fig. 11F). We were unable to measure a K d for binding of the product dehydroretinol to P450 27C1, but the measured k off rate was 50 min Ϫ1 , and no burst kinetics were seen (Fig. 10). HRMS results indicate that the 3and 4-position radicals do not equilibrate (Fig. 13). The "metabolic switching" to yield more 3-OH retinol takes place with the substrate, not the C-4 allylic radical. In other words, slowing C-4 radical formation yields more retinol for the C-3 abstraction. C-3 abstraction is a minor pathway and, accordingly, not substantial enough to yield a noticeable shift to 4-hydroxylation or desaturation. Apparently, C-3 hydrogen abstraction is slower than Adx reduction. In the C-4 pathways, Adx reduction is slow enough to contribute to reaction rate limitation.
P450 27C1 oxidation of retinol
desaturases are important in oxidation of fatty acids and some other alkanes, and a few residues have been shown to alter the balance of desaturation and hydroxylation (27,77,78). Some mammalian P450s are also capable of (-)desaturation of fatty acids (34). The bifurcation of desaturation and hydroxylation has also been considered at a theoretical level (27,37,38). Kumar et al. (37) concluded that desaturation involved carbocationic intermediates, generated by further electron transfer from an initial carbon radical. Desaturation is proposed to occur because of stereoelectronic inhibition of the oxygen rebound process (37). The conclusion that desaturation occurs only through a carbocationic intermediate (37) may or may not be correct, and our results do not address this issue directly. It is of interest to note, however, that no shifts of the 3-or 4-deuterium atoms were observed (Fig. 12). This lack of allylic scrambling does not necessarily negate the hypothesis about carbocations. Ji et al. (38) did not consider carbocations but concluded that the difference in O-H versus C-O bonds formed is critical in the outcome, and Cooper et al. (27) also favored a radical mechanism instead of carbocationic. This is concluded to be important, and -conjugation, which occurs in the desaturation of retinol, lowers the excitation energy. In these theoretical papers (37,38), the difference between hydroxylation and desaturation can ultimately be rationalized in the context of the structure of the enzymesubstrate complex.
One alternative mechanism of desaturation that can be considered is shown in Fig. 14, in which the initial reaction is an epoxidation of the cyclohexenyl ring, followed by two basecatalyzed abstractions of the C-3 and C-4 hydrogens. This mechanism could be consistent with all of our data on isotope effects, and there is no single experiment that we have done that would rule this out. We are inclined against this mechanism in that the epoxide would be expected to be relatively stable, as in the case of cyclohexene oxide (79) and vitamin K epoxide (80). Additionally, we did not find any additional LC-MS peaks with m/z 285 (Fig. 6). If this were the mechanism, then the hydroxylation reactions would be separate events, not as shown in Fig. 13, and discussion of competition between hydroxylation and desaturation would be a moot point.
The effect of limited changes in amino acids on the partitioning between desaturation and hydroxylation has been appreciated in non-heme iron monooxygenases for some time (77,78). A similar balance has been observed in two bacterial P450s (102A1 and 199A4) by Whitehouse et al. (39) and Bell et al. (40), respectively, in which a 5-amino acid change in P450 102A1 or a single-amino acid change in P450 199A4 (F185V) could change the ratio of hydroxylation to desaturation. Although no three-dimensional structure of a P450 27C1-retinoid complex is available yet, we believe that it might be instructive as to the proclivity toward desaturation and suggest specific site-directed mutagenesis experiments.
In summary, we have characterized several aspects of human P450 27C1, a retinol desaturase found in skin. The enzyme also catalyzes 3-and 4-hydroxylation as minor events, and the balance of these reactions is probably due to the allylic nature of the reaction and to the juxtaposition of the substrate in the active site. Small but significant deuterium KIEs were calculated, and metabolic switching of minor products was observed, leading us to conclude that abstraction at C-4 may occur first. Rate limitation at other key steps in P450 oxidation of retinol was considered, including substrate binding and product release, as well as reduction. We conclude that at least two reaction steps, C-H bond breaking and Adx reduction of the ferric enzyme, contribute to limiting rates of the desaturation of retinol by P450 27C1.
Tissue samples
All work was done with the approval of the Vanderbilt institutional review board, which considers these particular studies exempt if sample donors are not identified. The human liver and kidney samples were obtained through the Nashville Regional Organ Procurement Agency. Two human skin samples, from the abdominal area of an adult male (sample W, code A16-6) and female (sample P, code A16-4), were obtained from Prof. Joseph Corbo (Washington University School of Medicine, St. Louis, MO) (these were used in Fig. 4). A third sample, from the breast area of an adult male, was also obtained from the same source and used in the work presented in Fig. 3. Three other skin samples (all from adult female breast) were obtained from the tissue procurement source at Vanderbilt University Medical Center and are coded 37, 51, and 57 (these were used in Fig. 4, along with samples W and P from Washington University). The identities of the individuals were not disclosed.
Tissue samples were homogenized using a two-step procedure. The first step was done with an IKA disperser homogenizer (on ice) (4 ϫ 10 s). The resulting mixture was then processed with a motorized glass-Teflon Potter-Elvehjem tissue homogenizer (on ice) (4 ϫ 15-s pulses, with ϳ4 up-and-down strokes during each). Approximately 400 mg of human liver and kidney was homogenized in 10 ml of 50 mM potassium phosphate (pH 7.4) buffer. Much more disruptive methods were required for human skin samples: ϳ400 mg of human skin was homogenized in 6 ml of lysis buffer (6 M urea, 2 M thiourea, 4% (w/v) sodium CHAPS (w/v), 0.1% SDS (w/v)). Unhomogenized skin tissue was filtered out through glass wool, and the homogenates were centrifuged at 2,000 ϫ g for 10 min, and the resulting supernatant was centrifuged at 16,000 ϫ g for 20 min (at 4°C). The supernatant was collected and precipitated with 5 volumes of cold acetone and chilled overnight at Ϫ20°C. The precipitated proteins were pelleted at 16,000 ϫ g for 20 min (at 4°C), washed once with chilled 50% acetone (aqueous) with vortex mixing, and centrifuged again at 16,000 ϫ g for 10 min. The resulting pellets were resuspended in 50 mM Tris buffer (pH 7.4) containing 2% SDS (w/v) and heated at 95°C for 5 min. The concentrations of protein in all tissue samples were estimated using a BCA assay (Bio-Rad) or Pierce 660-nm protein assay (with detergent compatibility reagent) (Thermo Fisher Scientific).
Antibodies
Rabbit antibodies were raised by Cocalico Biologicals (Stevens, PA). Three rabbits were immunized with a total of 6 mg of purified P450 27C1 (see above; further purified by by DEAE and hydroxylapatite chromatography to remove some impurities). The rabbits were injected with purified protein in PBS, followed by three boosts over a period of 49 days. A test bleed was collected, and antibody strength was tested using an immunoblot with purified P450 27C1 as an antigen. Unless further boosting was required for more antibody production, the rabbits were then boosted a final time before exsanguination. The sera were compared, and the best serum was used based on specificity and titer.
Rabbit anti-27C1 antibody was purified by affinity chromatography with purified recombinant protein (84). Purified 27C1 was coupled to CNBr-activated Sepharose (GE Healthcare) according to the manufacturer's instructions. The coupled beads were incubated with diluted antiserum from rabbit number 497 (1:10 dilution in PBS, v/v) at room temperature overnight with end-over-end rotation. The suspension was washed extensively with PBS until the A 280 was Ͻ0.01. Bound antibody was eluted with 0.20 M glycine-HCl buffer (pH 2.5) in 1-ml fractions and neutralized with 1.0 M Tris-HCl buffer (pH 8.0). Fractions containing antibody (monitored by A 280 measurements) were combined, and protein concentration was measured using a BCA assay (Pierce/Thermo Fisher Scientific).
Immunoblotting (Western blotting) of tissue homogenates was carried out using the purified 497 antibody and reagents purchased from LI-COR (Lincoln, NE). Proteins from skin (20 g), liver (50 g), and kidney (50 g) were separated using SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (85) and stained with SyPro Ruby Blot Stain (Thermo Fisher Scientific) for total protein visualization using a PharosFX Plus molecular imager (Bio-Rad). Higher protein loads of the skin homogenates led to poor resolution from the keratins. The membranes were blocked with LI-COR Odyssey blocking buffer. Immunoblots were optimized with a 1:200 dilution (v/v) of the immunopurified rabbit antiserum (ϳ1 g of protein/ml) and a 1:10 4 dilution (v/v) of goat antirabbit 800CW near-infrared (IR) dye for the secondary antibody incubation (LI-COR protocol). Fluorescence signals were detected and quantified using a LI-COR Odyssey infrared imaging system (Thermo Fisher Scientific).
Proteomic analysis of skin homogenates
Proteins in the skin homogenate samples (prepared above) were separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide, w/v), and bands were visualized with Coomassie SimplyBlue SafeStain (Thermo Fisher Scientific). Recombinant P450 27C1 was used to guide the selection of migration distances selected for analysis (the M r is less because it is 58 residues shorter (23)) as well as immunoblots done on portions of the same gel (Fig. 4). The molecular weight region corresponding to recombinant P450 27C1 and the higher region, which was suspected to contain a higher-M r P450 27C1 protein, was excised and processed for in-gel digestion. In-gel trypsin digestion was performed as described (86). Extracted peptides were analyzed on a nanoLC Ultra system (Eksigent Technologies, Dublin, CA) interfaced with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). Approximately 2 g of peptides were reconstituted in 0.1% HCO 2 H and pressure-loaded (1.5 l min Ϫ1 ) onto a 360-m outer diameter ϫ 100-m inner diameter microcapillary analytical column packed with Jupiter octadecylsilane (C18) resin (3 m, 300 Å; Phenomenex) and equipped with an integrated electrospray emitter tip. Peptides were then separated with a linear gradient composed of 0.1% HCO 2 H in H 2 O (solvent A) and 0.1% HCO 2 H in CH 3 CN (solvent B) as follows: 2% B held from 0 to 10 min and increased from 2 to 45% B from 10 to 55 min at 500 nl P450 27C1 oxidation of retinol min Ϫ1 . The spray voltage was set to 2.0 kV, and the heated capillary temperature was set to 200°C. HCD MS/MS spectra were recorded in the data-dependent mode using a Top 2 method with an inclusion list containing m/z values corresponding to P450 27C1 tryptic peptides (Fig. 5) determined in silico with Skyline software (87). MS1 spectra were measured with a resolution of 70,000, an AGC target of 1e6, and a mass range from m/z 300 to 1,500. HCD MS/MS spectra were acquired with a resolution of 7,500, an AGC target of 1e5, and normalized collision energy of 35. Peptide m/z values that triggered MS/MS scans were dynamically excluded from further MS/MS scans for 20 s with a repeat count of 1. In some cases, a Q Exactive mass spectrometer (Thermo Fisher Scientific) was used for parallel reaction monitoring analysis. Tryptic peptides from the recombinant P450 27C1 protein were used to determine retention time windows to monitor for the skin homogenate. Retention time windows were set at Ϯ1 min for each peptide in the C-terminal region. Peptides from the previously undetermined N-terminal region were monitored for the entirety of the LC gradient. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from m/z 300 to 1,500. HCD MS/MS spectra were acquired with a resolution of 17,500, an AGC target of 5e5, and NCE of 27. For C-terminal peptides, a maximum injection time of 200 ms was used with a loop count of 10. An isolation width of m/z 1.5 with an offset of m/z 0.1 was used. For N-terminal peptides, a maximum injection time of 120 ms was used with a loop count of 18. Skyline software (87) was used to monitor both precursors and products from the parallel reaction monitoring analysis. Raw data files were analyzed using MyriMatch (88) against a decoy protein database consisting of forward and reversed human Uniprot/Swissprot database (version 20160620). The precursor ion mass tolerance was 10 ppm, and the fragmentation tolerance was 20 ppm for the database search. Methionine oxidation (15.9949 Da, dynamic) and cysteine modifications by iodoacetamide (carbamidomethyl, 57.0214 Da, static) were searched as modifications. The maximum Q values of peptide spectrum matches were adjusted to achieve either a peptide or a protein false discovery rate Ͻ5% using IDPicker software (version 3.1.642.0) (89).
Spectroscopy
UV-visible absorbance spectra were recorded using OLIS/ Cary 14 and OLIS/Aminco DW-2 spectrophotometers (On-Line Instrument Systems, Bogart, GA). Stopped-flow measurements were made with an OLIS RSM-1000 instrument operating in the rapid-scanning mode (1,000 spectra s Ϫ1 , with signal averaging for runs Ͼ4 s). In general, the slit widths were 1.24 mm (nominal 8-nm bandwidth), the gratings were 600 lines mm Ϫ1 (spectral width 300 nm), and the wavelength center was 450 nm. The temperature was controlled with a jacketed water bath and an external Jubalo F-25 circulating water bath (temperatures are noted for individual experiments). Collected spectra were analyzed with single-variable decomposition (SVD) software (OLIS Global Works) to estimate rates, using appropriate models in the manufacturer's software. The number of replicate traces and the S.D. are indicated for each experiment.
Anerobic kinetic experiments were conducted by loading the stopped-flow instrument with samples from tonometers that had been degassed and placed under a CO atmosphere as described in detail elsewhere (46,90,91). The syringes of the stopped-flow apparatus had previously been scrubbed with an anaerobic solution of a mixture of protocatechuate dioxygenase and 3,4-dihydroxybenzoic acid (92,93).
Incubations with P450 27C1
Incubations were done at 37°C in a shaking water bath. Typically, incubations for steady-state reactions were based on work done with other mitochondrial P450s and included 0.02 M P450 27C1, 5 M Adx, and 0.2 M ADR in 50 mM potassium phosphate buffer (pH 7.4) (0.5-ml final volume) (25), along with varying concentrations of all-trans-retinol and an NADPHgenerating system (94). All procedures with retinoids were done in amber glass vials under dim light because of the photosensitivity. Retinol was dissolved in ethanol, and the final concentration of ethanol in the reactions was Յ1% (v/v). We found that the presence of 30 M L-␣-dilauroyl-sn-glycero-3-phosphocholine did not enhance the initial reaction rate but yielded a longer period of linearity of product formation. Typical reactions were done for 2 min and quenched by the addition of 2 volumes of tert-butyl methyl ether containing 20 M butylated hydroxytoluene (to prevent chemical oxidation). Samples were resuspended in 50% ethanol (aqueous, v/v) for chromatographic separation (100 l).
Chromatography
HPLC with UV-visible absorbance detection was routinely used for measurement rates of oxidation of retinol in steadystate kinetic assays. Products were generally separated using a Waters Acquity UPLC system and an Acquity UPLC-BEH octadesylsilane (C18) column (50 mm ϫ 2.1 mm, 1.7 m) with a solvent system consisting of 4.9% CH 3 CN, 0.1% HCO 2 H, and 95% H 2 O (solvent A) and 95% CH 3 CN, 0.1% HCO 2 H, and 4.9% H 2 O (solvent B) (all v/v). Samples were injected with a 20-l loop, and a linear gradient was used for analysis of 3,4-dehydroretinol: 60 -66% B (v/v) over 10 min at a flow rate of 0.3 ml min Ϫ1 . For detection of all P450-dependent oxidation products the following was used: 40 -45% B over 5 min followed by 60 -66% (all v/v) over 10 min at a flow rate of 0.3 ml min Ϫ1 . Similar UPLC systems were used for mass spectrometry measurements for enzyme incubations.
LC-MS experiments for detection of retinol oxidation products
LC-MS analyses of retinol oxidation products were done with samples introduced following UPLC as described above. For detection of oxidized products, a Thermo LTQ mass spectrometer was used; for high resolution mass measurement, a Thermo LTQ-Orbitrap mass spectrometer was required at 100,000 resolution. In both cases, the mass spectrometer was operated in the atmosphere pressure chemical ionization (APCI) positive ion mode and was tuned with authentic alltrans-retinol. The tune settings are as follows: sheath gas flow rate, 40; auxiliary gas flow rate, 10; sweep gas flow rate, 0; capillary temperature, 300°C; APCI vaporizer temperature, 350°C; source voltage set to 6 kV; source current, 5 A; capillary voltage, 10 V; tube lens, 55 V.
Author contributions-K. M. J. synthesized the retinoids and did all of the enzymatic assays. T. T. N. P. purified P450 27C1 and ADR, purified and characterized the antibody, and performed the immunoquantitation experiments. K. M. J. and M. E. A. did the proteomic analysis of P450 27C1 expression in tissues, with K. M. J. F. P. G. conceived the project, purified Adx, and did the anaerobic stoppedflow measurements with K. M. J. F. P. G. and K. M. J. wrote most of the manuscript. | v3-fos-license |
2017-09-15T21:00:45.179Z | 2008-01-01T00:00:00.000 | 27486611 | {
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} | pes2o/s2orc | HPLC microfractionation of flavones and antioxidant ( radical scavenging ) activity of Saccharum officinarum L
The antioxidant activity of sugarcane (Saccharum officinarum L.) juice towards DPPH reagent was determined (EC50) and the main compounds with radical scavenging activity in juice and leaves extracts were identified by HPLC-UV/PAD analysis combined with HPLC microfractionation monitored by TLC using β-carotene and DPPH as the detection reagents. In sugarcane leaves, luteolin-8-C-(rhamnosylglucoside) (1) was the most important compound with radical scavenging activity; in sugarcane juice, the flavones diosmetin-8-C-glucoside (2), vitexin (3) schaftoside (9), isoschaftoside (10) and 4',5'-dimethyl-luteolin-8-C-glucoside (11) were the most relevant compounds. The content of juice flavonoids (0.241 ± 0.001 mg total flavonoids/mL juice), comparable to other food sources of flavonoids, suggest the potential of sugarcane as a dietary source of natural antioxidants. However, the low antioxidant ability of sugarcane juice (EC50 = 100.2 ± 2.6 g L-1) also points to the need for further studies about the dietary intake of sugarcane flavonoids and its effects on human health.
Introduction
Flavonoids, which are part of the daily human diet, are found in several fruits, vegetables and beverages of plant origin.The study of flavonoid-rich foods has attracted the interest of researchers due to their promising physiological activities, among them as natural antioxidants against free radicals.Their pharmacological properties also account for the growing interest in studying the content of flavonoids in numerous juices, fruits and plant foods. 1 However, many natural products from Latin American countries, including standardized extracts with a large number of components, still require adequate scientific or clinical research.A recent review illustrates these challenges and presents a compilation of reports concerning the development of a nutritional supplement or functional food from standardized whole-crude mango (Mangifera indica L., Anacardiaceae) extracts. 2ugarcane (a popular name that includes several species of the genus Saccharum L., Poaceae 3 ) is the most important source of sugar and of raw material for the production of ethanol fuel in Brazil, and is also cultivated commercially in other tropical countries.Former studies on Saccharum spp.4][5] The structural elucidation of several flavones (aglycones, C-and O-glycosides) by LC-UV-MS from Saccharum officinarum L. commercially cultivated in Brazil and also of transgenic plants has been reported by our research group. 6,7An HPLC-UV method for the quantification of their flavonoids was fully described, 8 and this quantitative method was applied to analyze commercially cultivated samples of S. officinarum L. (leaves and bagasse), transgenic plants (leaves) and also sugarcane juice purchased from street vendors (known as "garapa" in Brazil).The flavonoid content in sugarcane juice samples was comparable to other flavonoid-containing beverages such as orange juice and black tea.Moreover, the flavonoid content of leaves and bagasse was considerably high in comparison with other flavonoid sources reported in the literature, such as apples. 8These previous findings about sugarcane juice flavonoids have therefore motivated further studies, which are reported herein.
A mixture of high molecular weight alcohols from sugarcane wax has shown antioxidant properties on in vivo models (rats). 9Other studies also using in vitro rat models and antioxidant activity assays of phenolic compounds present in sugarcane juice have indicated the potential of sugarcane for beneficial health effects. 10The free radical scavenging activity of manufactured sugar products and the identification of polyphenolic compounds by LC-MS has also been reported. 11n view of the potential role of sugarcane as a dietary source of flavonoids as well as its possible use as a functional food, this study involved an investigation into the antioxidant activity of Saccharum officinarum L. juice ("garapa") and the identification of active compounds from leaves and juice, using HPLC microfractionation 12 combined with TLC monitoring of the compounds with antioxidant activity ( -carotene test) and free radical scavenging activity (DPPH assays). 13HPLC microfractionation consists of collecting the peak from an analytical-scale HPLC separation for more detailed examinations of the compounds on the microgram scale.HPLC microfractionation is a chemical screening strategy that has the disadvantage of requiring more extensive handling of the sample, but which has the advantage of offering the chromatographic conditions developed for hyphenated analyses such as LC-UV-MS and online information to validate structural assignments, combined with the simplicity of TLC bioassays. 12,13
Plant material
Leaves of Saccharum officinarum L. (Poaceae) were obtained from a commercial plantation in Araraquara, SP, Brazil.The plant material was dried to constant weight at about 40 °C prior to extraction.Samples of sugarcane juice, obtained by crushing cleaned and peeled sugarcane stems, were purchased from an organic plantation at Embrapa Agropecuária Sudeste, São Carlos-SP, Brazil.The juice samples were poured into plastic bottles and frozen at -10 o C for storage.Immediately prior to sample preparation, the juice samples were thawed and homogenized.
Sample preparation and quantification of sugarcane juice flavonoids by HPLC-UV/PAD
Sample preparation and quantitative HPLC analyses of total flavonoids in the sugarcane juice followed a procedure previously fully described and optimized. 8Samples were filtered through 0.5 µm Fluorpore membranes (Millipore) prior to injection of 10 µL into the HPLC system.In this work, HPLC analyses were performed using a Waters Alliance 2695 liquid chromatograph system with a photodiode array detector (PAD) 2996 and Empower Pro ® version 5.0 for data processing.Separation was performed on a Symmetry column (Waters, Milford, MA, USA) 250 mm long × 4.6 mm i.d.; 5 µm C 18 stationary phase) preceded by a guard column (20 mm long × 3.9 mm i.d.; 5 µm C 18 stationary phase).The mobile phase consisted of 0.2% formic acid (Merck, Darmstadt, Germany) in water (solvent A) and acetonitrile (Tedia Company, Fairfield, USA), (solvent B).The gradient profile used was: 0-8 min: 10 to 13% B; 8-25 min: 13 to 20% B; 25-40 min: 20 to 40% B; 40-45 min: 40-60% B. All the analyses were performed at a flow rate of 1.0 mL min -1 , at temperature of 40 °C and the detection was performed in 350 nm.
Diosmin (Sigma, St. Louis, MO, USA) was utilized for building the analytical curve at concentrations of 5, 10, 15, 25, 50, 100, 200, 300 and 400 mg L -1 in DMSO (J.T. Baker, Phillipsburg, NJ, USA) and was analyzed in the same chromatographic conditions used to separation of flavonoids.The results were calculated as mg total flavonoids (expressed as diosmin)/mL juice.All sugarcane samples were prepared and analysed in triplicate.
HPLC microfractionation
HPLC microfractionation was performed by a highloading procedure 14 involving the injection of concentrated leaf and juice extracts into the HPLC analytical-scale system due to the low concentration of flavonoids in extracts obtained after the clean-up step, as previously described. 8The methanol fraction (3 mL) containing the flavonoids obtained by SPE was evaporated on a rotary evaporator to reduce it to about ¼ of its original volume, and filtered through 0.5 µm Fluorpore membranes (Millipore) prior to injection (10 µL) into the HPLC system.The chromatographic conditions were the same as those described for the quantitative analysis of juice flavonoids.Fractions were collected manually every 5 min (5 mL) in Eppendorf tubes, after which all the fractions were evaporated to dryness using a rotatory evaporator prior to the TLC analysis.
TLC analyses of extracts and fractions
TLC analyses were performed on 0.20 mm-thick silica gel 60 TLC aluminum plates (Merck, Darmstadt, Germany) using ethyl acetate/formic acid/water (6:1:1 v/v) 15 as mobile phase.Each of the dried fractions obtained after HPLC microfractionation was dissolved in 300 µL methanol.Rutin hydrate (95% pure; Sigma, St. Louis, MO, USA was utilized as standard (100 µg mL -1 in methanol).For the analysis of leaves fractions, 5 µL of the samples and 2 µL of the rutin standard were placed on a silica TLC plate using graduated micropipettes Blaubrand (Brand, Wertheim, Germany) while 8 µL of sample and 3 µL of rutin standard were used for the analysis of juice fractions.After elution, the plates were dried at room temperature and sprayed with detection reagents, -carotene or 2,2-diphenyl-1-picrylhydrazil radical (DPPH). 13TLC plates were sprayed with an 0.02 mg mL -1 -carotene solution in dichloromethane and then exposed to ambient light until the -carotene was bleached, indicating the presence of antioxidant compounds. 13DPPH radical scavenging activity test was done by spraying a solution of 0.2% in methanol onto the TLC plate, which was allowed to stand at room temperature until yellow spots appeared, indicating the presence of active compounds. 13he limit of detection (LOD) of -carotene and DPPH reagents was determined by diluting the standard solution of rutin with methanol (100 µg mL -1 in methanol), in order to obtain solutions with a final concentration of 25, 50 and 75 µg mL -1 . 2 µL of each standard solution of rutin were placed on a silica TLC plate using graduated micropipettes and analyzed by TLC following the above described procedure.The LOD of -carotene and DPPH reagents was considered to be the smallest concentration of the rutin solution leading to a visible spot. 16The LOD of each reagent was determined in triplicate.
DPPH spectrophotometric assay for evaluation of antioxidant activity
A procedure slightly modified from the method reported by Brand-Williams et al. 17,18 was followed to evaluate the antioxidant capacity.Sugarcane juice (100 mL) was mixed with methanol (10 mL) and sonicated (5 min, ca. 25 o C), after which it was centrifuged (10000 rpm, 20 min, 25 ºC) and the resulting supernatant utilized for the antioxidant assay.Aliquots of sample extract (0.1 mL) in different concentrations (from 35.0 to 195.0 g L -1 ) were added to 3.9 mL of a DPPH solution (0.025 g L -1 in methanol).The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 1 h.The decrease in absorbance of the resulting solution was monitored at 515 nm after 1 h of reaction, using a Perkin Elmer UV-Vis model Cary 5G spectrophotometer.The blank solution consisted of 0.1 mL of methanol and 3.9 mL of a DPPH radical solution.A fresh DPPH radical stock solution was prepared each day.The percentage of scavenged DPPH was calculated from equation 1, 19 DPPH scavenging % = (Ao-A S /Ao) x 100 (1) where Ao is absorbance of the blank and A S is absorbance of the sample at 515 nm.The percentage of scavenged DPPH was then plotted against the sample concentration to calculate graphically the amount of antioxidant required to decrease the initial DPPH concentration by 50% (EC 50 ), expressed in terms of concentration (g L -1 ).Rutin in a concentration ranging from 0.06 to 1.00 g L -1 was used as the standard, following the same procedure described for the sugarcane extracts.
HPLC analyses
The flavonoid peaks in the samples studied here were identified by comparing the HPLC retention time and the UV-Vis/PAD spectra (Figure 1 and Table 1) against the data described by Colombo et al., 6,7 obtained in the same chromatographic conditions and confirmed by LChyphenated techniques (HPLC-UV-MS).The UV spectra of flavonoids (see Supplementary Information) are a very helpful feature for the unequivocal identification of flavonoid peaks, with two characteristic absorption bands (Band II, max between 240 and 280 nm and Band I, 300 to 380 nm). 20n addition to analyzing the antioxidant activity of sugarcane juice, the total flavonoids in the juice were also quantified, since previous quantitative data on sugarcane juice flavonoids were obtained only for commercial samples purchased from street vendors. 8No relevant difference was found here in the structure of the juice compounds, but a smaller amount of total flavonoids was observed (Table 2).
The differences between the quantitative data obtained in this study and in the former one suggest variations in sugarcane juice flavonoid content due to factors such as sugarcane cultivars, growth conditions etc., which are unknown for commercial juice samples.All the peaks exhibiting UV spectra characteristic of flavonoids and with an area greater than LOQ (0.83 mg diosmin L -1 ; LOQ value calculated taking into account a signal-tonoise ratio of 10:1) were considered in the determination of total flavonoids (Table 2).A calibration curve was built using diosmin as the standard, and the detector's response at 350 nm was linear from 5 to 400 mg diosmin L -1 .The regression equation was found to be y = 1.26.10 4 x -6.64.10 4 (r = 0.9999), with a variation coefficient below 2% for the triplicate analysis.Table 2 shows the individual content of the flavones that exhibited radical scavenging activity.
HPLC microfractionation of the main compounds with radical scavenging activity and evaluation of the antioxidant activity (EC 50 ) of sugarcane juice
Preliminary TLC tests showed low sensitivity of -carotene to detect antioxidant compounds, and the LOD of -carotene proved to be 100 mg L -1 , while the LOD of DPPH was 75 mg L -1 using rutin as the standard (see also Material and Methods -TLC).Therefore, further TLC tests of sugarcane samples were carried out using DPPH reagent.However, even using DPPH reagent, the injected samples had to be overloaded for the effective detection of compounds with radical scavenging activity by TLC, due to the small amount obtained in each fraction.A total of ten fractions from each extract were collected and the radical scavenging activity was detected in the regions highlighted in the chromatograms of Figure 1.The major compound found in the region with the most intense spot showing radical scavenging activity (t R between 10-15 min and area corresponding to 31.93% of the flavonoid peaks, Figure 1a) was flavone luteolin-8-C-(rhamnosylglusoside) (1), which corresponds to 23% of total leaves flavonoids and is therefore probably related to the overall antioxidant properties of the leaves extract.
On the other hand, TLC data is a screening and qualitative step, that needs to be supported by testing in the actual substrate, 21 so a quantitative assay of the overall activity of sugarcane juice was also done.The antioxidant activity of sugarcane juice was evaluated in terms of hydrogen-donating or radical-scavenging ability, using the DPPH method developed by Brand-Williams et al. 17,18 with a few modifications and rutin as a reference antioxidant compound.The percentage of antioxidant activity corresponds to the amount of DPPH consumed by the antioxidant compound, and the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% is called efficient concentration (EC 50 ).The higher the consumption of DPPH by a given sample, the lower the EC 50 value and the higher its antioxidant ability.
Sugarcane juice showed EC 50 = 100.2± 2.6 g L -1 , indicating antioxidant activity, although it was lower than rutin (EC 50 = 0.172 ± 0.43 g L -1 ).The difference between the activity of sugarcane juice and rutin must take into account the higher purity of the reference compound (rutin, 95% purity) while the sugarcane juice analyzed is a crude extract containing other compounds that are not necessarily proton and electron donors, e.g., sugars, mixed with the active compounds.Since the total flavonoids content corresponds to about 0.11% (m/m) of the juice (0.241 ± 0.001 mg flavonoids mL -1 juice, Table 2), the value of EC 50 of sugarcane juice suggests a direct correlation between the (low) antioxidant activity of sugarcane juice and the
Conclusions
The overall findings reported here on the antioxidant (radical scavenging) activity of sugarcane juice and the flavones obtained by HPLC microfractionation, allied to in vivo data, 10 may justify further studies of the potential of sugarcane juice as a natural source of antioxidant compounds.However, the low EC 50 value of sugarcane juice also indicates the need for detailed nutritional or physiological studies about the consumption of sugarcane juice and its effects on human health.HPLC microfractionation offers the significant advantage of being performed on an analytical-scale without requiring any additional optimization of chromatographic conditions, allowing the chromatographic data obtained here (retention time, UV/PAD spectra) to be combined with data obtained under similar chromatographic conditions. 6,7These results encourage the use of HPLC microfractionation as a complementary tool in the study of bioactive compounds in other Brazilian food or medicinal plants.
Figure 1 .
Figure 1.HPLC profile (detection at 350 nm) of Saccharum officinarum L. extracts (a) leaves; (b) sugarcane juice.The regions highlighted in the chromatograms showed radical scavenging activity (DPPH test).Chromatographic conditions as described in Materials and Methods.For identification of the peaks, see Table 1.
Table 1 .
1. Flavonoids identified in Saccharum officinarum L. extracts and their respective UV/PAD data
Table 2 .
Quantitative data about sugarcane juice flavonoids with radical scavenging activity (DPPH test) n = 3; data are expressed as mg diosmin/mL juice.sugarcaneflavonoidscontent.This content is low, but it includes compounds with radical scavenging activity (see Table2) as demonstrated by the microfractionation monitored by TLC and DPPH reagent. a | v3-fos-license |
2018-04-03T00:25:44.637Z | 2018-03-22T00:00:00.000 | 4856330 | {
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} | pes2o/s2orc | Encapsulation of Crabtree's Catalyst in Sulfonated MIL‐101(Cr): Enhancement of Stability and Selectivity between Competing Reaction Pathways by the MOF Chemical Microenvironment
Abstract Crabtree's catalyst was encapsulated inside the pores of the sulfonated MIL‐101(Cr) metal–organic framework (MOF) by cation exchange. This hybrid catalyst is active for the heterogeneous hydrogenation of non‐functionalized alkenes either in solution or in the gas phase. Moreover, encapsulation inside a well‐defined hydrophilic microenvironment enhances catalyst stability and selectivity to hydrogenation over isomerization for substrates bearing ligating functionalities. Accordingly, the encapsulated catalyst significantly outperforms its homogeneous counterpart in the hydrogenation of olefinic alcohols in terms of overall conversion and selectivity, with the chemical microenvironment of the MOF host favouring one out of two competing reaction pathways.
Response factors were determined after calibration with the commercially available compounds. All samples were filtered via a 0.2 μm syringe filter (Acrodisc® GHP) before injection. Table S6 lists all retention times for alkenes and olefinic alcohols.
ESI-MS data were collected on a Bruker MicroTOF interfaced with a glovebox. [4]
This was verified by 1 H NMR spectroscopy in solution after digestion ( Figure S10). Characterization of 2@1-SO3Na was carried out with the aid of ICP-OES (Table S3), PXRD (Le Bail fit is shown in Figure S7), N2 adsorption-desorption at 77K ( Figure S9), NMR spectroscopy ( Figure 2c, Figures S14-S15) and SEM imaging ( Figure S12). All analytical data concur that 2 is encapsulated intact inside the pores of
Catalysis
Heterogeneous hydrogenation of alkenes with 2@1-SO3Na: Alkenes were purified as described in materials, methods and instrumentation. Stock solutions were prepared for each substrate in CH2Cl2. Reactions were carried out in J-Young tubes. For hydrogenations at 1000 ppm loading (0.1 mol %), a 50 mL tube was used. For lower loadings, larger J-Young tubes were used with a volume of 100 mL (100 ppm loading) or 300 mL (< 100 ppm loading) in order to increase the amount of available H2. The following procedure describes hydrogenation of 1-octene (4) at 1000 ppm loading. Nominal concentrations and amounts of reagents were adjusted accordingly for other substrates and loadings (see Table 1 in the manuscript). 3 h at 300 rpm after which the tube was depressurized by immersion in liquid N2 for 10 min and slow evacuation of the overhead space for 1 minute to remove any H2 excess. Finally, the tube was left to thaw back to ambient temperature and backfilled with N2.
The product distribution was determined by GC (refer to general methods for more details).
The supernatant was filtered via a syringe filter (0.2 μm, Acrodisc® GHP) and then 200 μL were diluted to 1 mL with CH2Cl2. Retention times are provided in Table S6. Repeated reactions delivered a product distribution that was reproducible to within ± 5 %.
Leaching test: A heterogeneous hydrogenation reaction was set up with 2@1-SO3Na as the catalyst and 1-octene as the substrate (100 ppm loading, [1-octene] = 1.0 M, V = 4.0 mL). After 3 h, the supernatant was filtered off via a filter-cannula and the filtrate was collected in a J-Young tube and transferred inside a N2 purged glove-bag. The supernatant was filtered once more in the glove bag using a 0.2 μm syringe filter (Acrodisc® GHP) and subsequently an S12 aliquot of 100 μL was diluted to 1 mL with CH2Cl2 and analyzed by GC (35% conversion exclusively to n-octane, TON = 3500). The remaining filtrate was then re-pressurized with H2 and left to react overnight. Aliquots (100 μL) were collected after 3 h and 21 h and analyzed by GC. No more conversion was observed, revealing that turnover stopped once the heterogeneous catalyst was separated from the reaction mixture ( Figure S21). (97% conversion selectively to n-octane for the 2 nd cycle). The whole process was repeated once more. Conversion for the 3 rd cycle was 82% ( Figure S22).
Heterogeneous hydrogenation of olefinic alcohols with 2@1-SO3Na:
Olefinic alcohols were purified as described in materials, methods and instrumentation. The According to NMR analysis, all reactions were mass-balanced within ± 7%. For homoallylic alcohol 11a, product distribution and conversion was also determined by GC (Table S6 and Figure S33). Results lie in very good agreement with NMR and reaction is mass-balanced within ± 5%.
Homogeneous hydrogenation with Crabtree's catalyst:
Homogeneous reactions were always run "side by side" with their heterogeneous counterparts. Table S6 lists the retention times. When olefinic alcohols were employed as substrates, product distribution was determined by 1 H NMR, as described above for the heterogeneous system. Figure S32a). The same experiment S14 was performed with the heterogeneous catalyst 2@1-SO3Na. Addition of butanal also had a negligible effect on final product distribution ( Figure S32b).
Gas phase hydrogenation of 1-butene:
Gas/solid hydrogenation of 1-butene was carried out according to the literature procedure. [6] A high pressure NMR tube (volume ≈ 1.8 mL) was loaded with finely powdered catalyst (~0.5 mg) inside a glovebox. Subsequently, the high pressure NMR tube was transferred to a schlenk line and the argon atmosphere was replaced with 1-butene (1 atm). The tube was sealed and frozen in liquid nitrogen, hydrogen (1 atm) was introduced at 77 K. As a timer was simultaneously started, the tube was quickly sealed, thawed and transferred to a NMR spectrometer. Experimental results (TONs, TOFs and recycling) are shown in Figure S23.
Gas phase chemical shifts of butenes and butane are as follows: Figure S3 for 2-PF6, Figure S16 S21 Figure S1. Synthesis of 1-SO3H. [13] Interconnection windows with openings of ~1.1 nm and 1.3 nm, after correcting for van der Waals radii. [17] b) Model for the isostructural sulfonated analogue 1-SO3H. Dimensions of both windows do not change since the sulfonate groups are pointing towards the interior of the pores.
S22 Figure S3. Single crystal structure of 2-PF6 (Crabtree's catalyst) [15] and dimensions of the cation 2 (1.16 x 1.13 x 1.19 nm, V = 1.56 nm 3 ) by enclosure in a rectangular box, defined by tangents to its van der Waals surface (OLEX2 program). [12] The cuboid can be inscribed in a sphere with a diameter of ~ 2.0 nm. Therefore, only 1 cation can fit in the small mesopores (d Figure S8. TGA graphs of 1-SO3H (green) and 1-SO3Na (red). The latter shows a higher inorganic/organic ratio due to encapsulation of Na + cations. Weight loss at 275 °C corresponds to water trapped within the pores in the as synthesized MOFs and lies in good agreement with elemental analysis (Table S1). (Table S2). conversion with only 61% selectivity to hydrogenation and formation of n-pentanol (12b, red squares). Ill-defined condensation products are also detected, particularly after 24 h (magenta triangles). Yield and selectivity (in parenthesis) for each product are shown in the inset.
Mesitylene (*) is used as a standard to measure mass-balance (>95%). Addition of butanal does not significantly change relative concentration of reagents for either system within experimental error (see also Table S7).
S49 Figure S33. Product distribution based on GC for the hydrogenation of but-3-en-1-ol with 2@1-SO3Na as the catalyst. Total amount of substrate and products remains constant within experimental error of the GC analysis method (± 5%). Calibration curves for but-3-en-1-ol (top left), butanol (top right), crotyl alcohol (bottom left) and butanal (bottom right) are also shown. | v3-fos-license |
2018-04-03T04:03:09.586Z | 1971-07-25T00:00:00.000 | 37398283 | {
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} | pes2o/s2orc | Ferredoxin-dependent Phenylpyruvate Synthesis by Cell-free Preparations of Photosynthetic Bacteria*
SUMMARY A reductive synthesis of phenylpyruvate from phenyl-acetyl coenzyme A and bicarbonate was obtained in cell-free extracts from the photosynthetic bacterium Chromatium. This phenylpyruvate synthase activity-which appears to be distinct from pyruvate synthase-depends on the reducing power of ferredoxin and requires thiamine pyrophosphate as a coenzyme. Of several ferredoxins assayed, the native ferredoxin from Chromatium was found to be the most effective for phenylpyruvate synthesis. The enzyme extract also catalyzed the conversion of phenylpyruvate to phenylalanine (in the presence of an amino group donor) and the activation of phenylacetate to the phenylacetyl-CoA derivative. Thus, a net synthesis of phenylalanine from phenylacetate and CO2 was accomplished. Closely associated with the phenylpyruvate synthase reaction was an exchange reaction between 14C-bicarbonate and phenylpyruvate. The exchange reaction required thiamine pyrophosphate and CoA as cofactors. Apart from Chromatium, phenylpyruvate and phenylpyruvate-bicarbonate were also found in cell-free extracts from the green photosynthetic bacteria
This phenylpyruvate
synthase activity-which appears to be distinct from pyruvate synthase-depends on the reducing power of ferredoxin and requires thiamine pyrophosphate as a coenzyme.
Of several ferredoxins assayed, the native ferredoxin from Chromatium was found to be the most effective for phenylpyruvate synthesis. The enzyme extract also catalyzed the conversion of phenylpyruvate to phenylalanine (in the presence of an amino group donor) and the activation of phenylacetate to the phenylacetyl-CoA derivative. Thus, a net synthesis of phenylalanine from phenylacetate and CO2 was accomplished. Closely associated with the phenylpyruvate synthase reaction was an exchange reaction between 14C-bicarbonate and phenylpyruvate.
The exchange reaction required thiamine pyrophosphate and CoA as cofactors. Apart from Chromatium, phenylpyruvate synthase and phenylpyruvate-bicarbonate exchange activities were also found in cell-free extracts from the green photosynthetic bacteria Chlorobium thiosulfatophilum and Chloropseudomonas ethylicum.
Previous work in tllis laboratory has revealed a direct l)articil)ation oi" reduced fcrrcdosin in CO2 fixation reactions in bacterial l~l~otosyr~thesis. These rc~action~, driven by the strongly rcducing l)ot,ential of ferrcdosill-about equal to that of molecular hydrogen (1, 2)-involve a reductive carbosylation of an acyl coellzyme :1 derivative to fo1~1 the corresponding cr-keto acid (Aqualion I).
Bachofen, and Arnon in Chromatium (3). Pyruvate s)-ntliaae is now known to be present, in various types of phot,osynthetic (4-G) and nonphotosynthetic anaerobic bacteria ('i-12). h second reaction of this type is the reductive carboxylation of succinyl-CoA to form a-ketoglutarate, which has been found in certain photosynthetic bacteria (4, 6, 13) and has also been 01). served recently in an anaerobic rumen bacterium (14). Another fcrredoxin-dependent reductive carbosylation reaction is the synthesis of oc-ketohutyrate from COY and propionyl-CoR discovered recently by Iluchanan (15). l'yruvate synthase and a-ketoglutarate synthase arc key enzymes in the reductive carboxylic acid cycle (16, 17) , a new cyclic pat.liway for CO* fis:Ltion in bacterial photosynthesis. 21 ~o~~n~~on feature of the three fcrrcdoxin-dependent reductive curboxylation reactions is that the a-keto acids formed are rclatcd to the biosynthesis of amino acids which, in photosynthetic bacteria, constitute the tllain soluble products of COs fixation (18)(19)(20)(21)(22).
METHOlE
Chrornatiun~, strain I), was grown in 13-liter Pyres bottles either in the bicarbon:tte-rn:rlate medium ol Arnon, I&, and Anderson (21) or in the same medium modified by replacing malat e with sodium thiosulfate (final concentration 0.012 11). C. thiosulfatophilum, strnill Tassajara, was grown as described by Evans and Buchanan (5) in :I medium free of acetate. C. et/+ CWL, kindly supplied by Dr. J. M. Olson of the Brookhavcn National Laboratory, was cultured as described bp Evans (6). The harvested cells were stored at -20" prior to use.
The enzyme preparation was freshly made (at 4" under argon or osygen-free ST) for each experiment from 10 g of cell llaste Iesue of July 25, 1971 U. Gehriny and D. I. krnon that, was thawed, suspended ill 10 ml of 0.02 AI IIEPIW buffer, pI-I 7.7 (cont.aining 100 pmoles of dit,hiothreitol), and disrupted by sonication for 2 min with a Ih~nson sonifier (power setting 6). Buffer solutions were rendered anaerobic by bubbling argon gas.
The sonified cell suspension W:M caentrifuged for 10 min at 30,000 x g and the precipitat,e ws discarded.
Ferredosin was removed from the supernatant fluid by passing it t,hrough a DEAE-cellulose cohm~n (2 x 3 cm) c>quilibrated with 0.02 M IIEPES buffer, pII 7.7. -111 effluent volume about equal to the volume applied was collected and centrifuged for 2 hours at about 100,000 x g, and the l"ccil)itate was discarded.
To remove possible interfering subst,rates, the supernatant fluid was passed through a Sephades G-25 column (2 x 36 cm) equilibrated with 0.02 M IIEPES buffer, pII 7.7. The excluded protein fraction was used as the enzyme 1)rcparation.
The protein content was estimated by a modified Biuret method (25) in which a 7 : 2 mixture of acetoize-nlethanol was used instead of trichloroacetic acid for the precipitation of protein. The ferredorins used were isolated in pure form from Clostridium pasteurianum (26)) Chromatium (27)) C. thiosulfatophilum (28), and spinach leaves (1). Ferrcdosin from Azotobacter vinelandii (29) was a gift from I)r. I>. C'. Yoch of this laboratory. Protein concentrations of forredosin solutions were measurctl 1)) the Folin procedure (30) wit,h crysit~lline bovine serum albumin as standard.
Labeled phenylalanine was identified and separated from phenylpyruvate by the thin layer chromatography method (slightly modified) described by Buchanan (15). The identity of the labeled compound with suthcntic phenylalanine was cstablished by the coincidence of the radioactive spot with the spot of authentic phenylalanine as stained with ninhydrin. For the quantitative estimation of phenylnlnninc the radioactive areas of duplicate chromatograms were scral)ctl from the plates, and the material was mixed with scintillation fluid (37) and counted in a scintillat,ion counter.
When phenylacetyl-CoA was generated in the reaction mixture, the rate of phenylpyruvate synthesis was about the same, whether all of the ATP was added initially or whether it was being generated by the creatine phosphate-phosphokinase couple (Treatments 3 and 5, Table TI). In contrast to the pyruvnte synthase (3) and oc-ketobutyrate synthase (15) systems, the addition of semicarbazide as a car-bony1 trapping agent did not increase the synthesis of phenylpyruvate.
The formation of phenylp~ruvate, as measured under the coildit,ions described in Table I, proceeded linearly for about 70 min and Ivas proportional to the amount of enzyme preparation added Table I and for the exchange reaction were as given for the complete treatment in Table VI. up to about 12 mg of protein per 3 ml of reaction mixture. The optimum pII range for phenylpyruvate synthesis was 8.1 to 8.6 (Fig. l), whereas the optimum for the phenylpyruvate-bicarbonate cschange, which is discussed below, was ~1% 7.2.
A comparison of the relative effectiveness of different ferre- doxins in the phenylpyruvate synthase reaction showed that the ferredoxins from the two photosynthetic bacteria Chromati~m and C. thiosuZjutophiZum were the most effective (Table III). Clostridium ferredoxin was considerably less active, and the effectiveness of the recently isolated new type of ferredoxin from A. &elan&i was intermediate between that of Chromatium and Clostridium.
Spinach ferredoxin, known to be a poor substitute for bacterial ferredosins in the phosphoroclastic cleavage of pyruvate and in the nitrogenase system of C. pasteurianum, wus also virtually without effect in the phenylpyruvate synthase reaction.
The concentration effect of ferredoxin is shown in li'ig. 2. About 180 pg (per 3 ml) of Chromatium ferredoxin saturated the phenylpyruvate syuthase system; Clostridium ferredoxin failed to saturate, even at a concentration of 400 pg per 3 ml. A Lincweaver-Burk plot of the data showed the same Jnlax for both ferredoxins and gave an apparent Km of 5 x 1OP M for Chromafium ferredoxin and 8 x 1OP M for Clostridium ferredosin.
The experiments described so far were carried out with enzyme preparations from Chromatium cells grown autotrophically in a bicarbonate-thiosulfate medium. Table IV shows that comparable levels of phenylpyruvate synthase activity were also present in extracts of cells grown in a malate medium-an observatioc Issue of July 25, 1971 U. Gehring and D. I. dmon t.hat is compatible with the report of Allison and Robinson (23) of equal uptake of 1%~phenylacetate by Chromnti7~m cells grown in either a bicarbonate-thiosulfate or a malate medium. Phenylpyruvate synthase appears to be a constitutive rather than an inducible enzyme of Chromatium cells. Its format:ion was not affected by the addition of phcnylacetate (1 mu) to the culture medium.
S!/nf/~esis of Phenylalanine jrom Phenylacefate--Tlie addition of glutamate or glutamine to the reaction mixture did not alter substantially the total Y-bicarbonate fised but changed drastically the final products formed from pheaylacetnte (Table V).
-\ sharp decrease in 14C-pllen311pyruvatc was accompanied by a marked increase in 14C-pheaylalaaine.
These results show a net cell-free synthesis of phenylalanine from phenylacetate and COZ.
In the presence of either glutamate or glutamine there was also some incorporation of 14C-bicarbonate that could not be accounted for by the methods used.
l'henylpyruvate-Bicarbonate Exchange-Enzyme preparations from Chromatium cells were found to catalyze a 14C exchange reaction between '%-bicarbonate and phenylpyruvate. Incorporation of *4C into phenylpyruvate occurred only when thiamine pyrophosphate and catalytic amounts of CoA were present in the reaction mixture (Table VI).
Coh could not be replaced by any of the other sulfhydryl compounds tested. However, a vigorous exchange occurred without added Co.4 when the system was illuminated in the presence of chloroplasts, i.e. under conditions that could have brought about the photoreduction of any ferredosin present,. Although no ferredosin was added in this case, it seems likely that the &ivation of the eschangc reaction by illumination was due t,o the reduction of trace amounts of ferredosin that remained in the enzyme preparation after it was passed through the DESE-cellulose column.
The possibility that reduced ferredosin did indeed replace CoA was strengt'hencd by the observed stimulation of the exchange reaction upon adding ferredosin to the illuminated reaction mixture. aidded NADFI or N;XJ)PI-I was wholly ineffective as a substitute for CoA but some exchange activity occurred in the presence of sodium dithionite, possibly due to the reduction of traces of ferredosin that remained in the reaction mixture.
The pH curve for t.he COB-catalyzed exchange react.ion had a relatively flat drop on the acid side of the optimum pH of 7.2 ( Fig. 1)-a distinctly different pH profile from that of the phenyl-pyru\ratc synthase reaction.
At their rcspectire pl I optima, t.he rate of the exchange react,ion was about three times greater than the rate of phenylpyruvate synthesis.
Phenylpyruvate Synthase T'ersus P~r7~vafe Synthnse-I'hcnylpyruvate s)-nthase appears to be distinct from pyruvate synthase. This view is supported by (a) the presence of the pyruvate but not the phenylpgruvate enzyme in C. pasteurianum (see below), (b) differences in stability of phenylpyruvate synthase and pyruvate synthase in Chromatium cells stored at -20" (after 4 months of storage, some cells had no phenylpyruvate synthase activity while retaining a high level of pyruvate synthase activity), and (c) differential requirements of thiamine pyrophosphatc.
Phenylpyruvate synthesis by cell-free extracts of Chromatium had a distinct requirement for thiamine pyrophosphate (Table I). By contrast, pyruvate synt,hnse of Chromatium shows such a requirement. only after the endogenous thiamine pyrophosphate is removed by special treatment,s (38).
The replacements of CoA incllldsd 10 pmoles of dithiothrcitol or 20 Hmoles each of one of the compounds listed. Table I). These cell-free extracts also showed phenylpyruvate-bicarbonate exchange activity. Thus, the phenylpyruvate-bicarbonate exchange activity appears to be RIways closely associated with phenylpyruvate synthase. Neither phenylpyruvate synthase nor phenylpyruvate-bicarbonate eschange activity was detected in cell-free extracts of the nonphotosynthetic anaerobe C. pasfeurianum-an organism that contains an active pyruvate synthase system (7). | v3-fos-license |
2020-10-28T19:12:47.069Z | 2020-10-09T00:00:00.000 | 225169766 | {
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} | pes2o/s2orc | Data of the release properties of astaxanthin-loaded zein/calcium alginate composite microparticles in fatty food simulant system at 4 °C and 25 °C
Recently, we demonstrated the characteristics and molecular interactions of Astaxanthin (Asta), extracted from shrimp (Litopenaeus vannamei) by-products to zein/calcium alginate (CA) (named as Asta-loaded zein/CA composite microparticles). The encapsulation efficiency of Asta-zein/CA composite microparticles obtained from freeze dried, 40 °C or 50 °C oven dried was across 80% [1]. In this data, we investigted the release properties of Asta-loaded zein/CA composite microparticles in simulating fatty food system (95% ethanol solution) at 4 °C and 25 °C. At different points of time, the cumulative release percentages of Asta from the tested composite microparticles were calculated. The release kinetics of Asta from the composite microparticles was investigated using Zero order, First order, Higuchi and Rigter-Peppas models. We observed all of the tested composited microparticles displayed an initial burst effect followed by subsequent attenuating release in 4 °C and 25 °C fatty food simulant system. At 4 °C fatty food system, the Asta released from 40 °C oven dried and 50 °C oven dried composite microparticles fit best with First-order and Ritger-Peppas models, respectively. At 25 °C fatty food system, all of these tested composite microparticles fit best with Higuchi model. Our results indicate the prepared composite microparticles are expected to be used as a delivery carrier for restrained release of antioxidant Asta in fatty foods, such as in natural vegetable oils or fried foods.
all of these tested composite microparticles fit best with Higuchi model. Our results indicate the prepared composite microparticles are expected to be used as a delivery carrier for restrained release of antioxidant Asta in fatty foods, such as in natural vegetable oils or fried foods.
© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ) Table Subject Food Science Specific subject area Food microparticles and release property Type of data Table Figure How data were acquired Under a fatty food simulant system (95% ethanol solution), real time measuring of the Asta cumulative release from the tested composite microparticles were performed at 4 °C and 25 °C fatty food simulant system. Data format Raw Analyzed Parameters for data collection Asta-loaded zein/CA composite microparticles (freeze dried, 40 °C oven dried and 50 °C oven dried) were added into the fatty food simulant system and stored at 4 °C and 25 °C in the darkness. At time intervals (0-23 h), the cumulative release percentage of Asta was measured. Description of data collection At different points of time, the released Asta content was determined by measuring the absorbance at 477 nm [1] , and then the Asta cumulative release percentages of all tested composite microparticles were calculated. All measurements were performed in triplicates. Based on the results of Asta cumulative release percentages, the release kinetics were obtained by fitting Zero order, First order, Higuchi and Rigter-Peppas models associated with diffusion. Data
Value of the Data
• These data provide useful insights into the release properties of Asta-loaded zein/CA composite microparticles in fatty food simulant system. • Food scientist and nutritionist can benefit from these data.
• These data can provide useful information on diffusion mechanism of Asta-loaded zein/CA composite microparticles in fatty food system when stored at 4 °C and room temperatures. • These data will provide enhanced understanding of potential applications of Asta-loaded zein/CA composite microparticles in natural vegetable oils or fried foods.
Data Description
Detail raw data of the cumulative release percentages of Asta from the Asta-loaded zein/CA composite microparticles at different time points in fatty food simulant system at 4 °C and 25 °C were summarized in Table 1 and Table 2 . Data obtained from Table 1 to Table 2 were evaluated for the average cumulative release percentages of Asta from all tested composite microparticels when incubated in fatty food simulant system at 4 °C ( Table 3 ) and 25 °C ( Table 4 ), respectively. Note: Time in brackets were actural incubation time. Raw data in brackets were deleted in calculating the mean value and standard deviation due to high deviation. Table 3 shows an initial burst effect followed by subsequent slower release for all of these composite microparticles. For freeze dried composite microparticles, 97.70% of Asta was released during 20 min of burst effect stage. The cumulative release of Asta for the 40 °C oven dried composite microparticles was 88.87% at the end of its burst effect at 5 h. The 50 °C oven dried composite microparticles showed lower Asta cumulative release percentage than the other composite microparticles in the 4 °C fatty food simulant system. Table 4 shows that the freeze dried composite microparticles showed higher Asta release percentage than the 40 °C oven dried and 50 °C oven dried ones in 25 °C fatty food simulant system under same incubation time. A lower Asta release percentage was found in the 50 °C ven dried composite microparticles.
Based on the data of Table 3 and Table 4 , the release kinetics of Asta from all tested composite microparticles were measured to fit Zero order, First order, Higuchi and Rigter-Peppas models. Fig. 1 . describes the Zero order model (m t /m i = at + b ) prediction for the Asta cumulative release from freeze dried, 40 °C oven dried and 50 °C oven dried composite microparticles when Note: Time in brackets were actural incubation time. Raw data in brackets were deleted in calculating the mean value and standard deviation due to high deviation. incubated in fatty food simulant system under 4 °C and 25 °C. The compsotie microparticles showed higher release kinetics parameters ( R 2 ) in 25 °C fatty food simulant system than the corresponding ones in 4 °C fatty food simulant system. Fig. 2 . describes the First order model (Ln(1-m t /m i ) = at + b ) fitting for the Asta cumulative release from freeze dried, 40 °C oven dried and 50 °C oven dried composite microparticles when incubated in fatty food simulant system under 4 °C and 25 °C. The 40 °C oven dried and 50 °C oven dried composite microparticles in 4 °C fatty food simulant system showed higher release kinetic parameters ( R 2 > 0.93) than other microparticles. In 25 °C fatty food simulant system, no enough data were obtained in the free dried composite, due to the average cumulative release percentages across 100% at 0.67 h and 1.0 h of incubation. Fig. 3 . shows the Higuchi model (m t /m i = at 1/2 + b ) fitting for the Asta cumulative release from freeze dried, 40 °C oven dried and 50 °C oven dried composite microparticles when incubated in 4 °C or 25 °C fatty food simulant system. Except for the 40 °C oven dried composite microparticles incubated at 4 °C fatty food simulant system, the Asta release kinetics parameters ( R 2 ) were across 0.92 for the other microparticles in Higuchi model. Fig. 4 . describes the Rigter-Peppas model (Ln(m t /m i ) = aLnt + b ) fitting for the Asta cumulative release from freeze dried, 40 °C oven dried and 50 °C oven dried composite microparticles in fatty food simulant system. In 4 °C fatty food simulant system, the release kinetics parameters ( R 2 ) were over 0.90 for the 40 °C oven dried and 50 °C oven dried composite microparticles. In 25 °C fatty food simulant system, the Asta cumulative release of 40 °C oven dried composite microparticles fit better with the Rigter-Peppas model ( R 2 = 0.9166).
Materials and methods
Free Asta standard ( ≥ 98%, HPLC) was purchased from Aladdin Industrial Corporation (Shanghai, China). Asta-loaded zein/CA astaxanthin composite microparticles used in this data article (freeze dried, 40 °C oven dried and 50 °C oven dried) were prepared described in our cosubmitted related research article [1] .
Asta content determination
The Asta extracts have specific absorbance at 477 nm [2] . An Asta standard calibration curve ( y = 0.0946x + 0.0108, R 2 = 0.9926) was determined by measuring the absorbance (y) of Asta ethanol solutions (x) (concentration from 0 to 10 μg/mL) at 477 nm using a 1510micro-plate reader (Thermo Fisher Scientific Oy, Vantaa, Finland). The Asta content ( μg) of tested solution was quantified by multiply the Asta concentration ( μg/mL), determined by measuring absorbance at 477 nm, with the solution volume (mL).
Release property in fatty food simulant system
The oxidative products of natural vegetable oil formed during storage have specific absorbance at 390 nm to 550 nm, which could affect the Asta quantification by spectrophotometry at 477 nm. Therefore, the 95% ethanol solution (v/v) was used to stimulate the fatty food system [3] . Approximately 50 mg of the composite microparticles were blended with 10 mL of 95% ethanol solution in a 25 mL weighing bottle, and kept at 4 °C and 25 °C in darkness. At different time intervals, i.e. 0 to 23 h for 4 °C treated groups, and 0 to 7 h for 25 °C treated groups, 1 mL of solution was pipetted out and replaced with 1 mL of fresh 95% ethanol solution to remain constant volume. After centrifuged at 50 0 0 × g for 10 min, the resulted supernatants were collected and measured the absorbance of 477 nm, followed by calculation of the cumulative content of Asta released in 10 mL of fatty food system. The cumulative release percentage of Asta at different time points was determined as follows: Cumulative release / % = ( m t / m i ) × 100 m i : the initial Asta content ( μg) in the composite microparticles; m t : the cumulative Asta content ( μg) at each time point. All measurements were performed in triplicates. Data were expressed as mean ± standard deviation.
Release kinetics of Asta from the composite microparticles
After determining the cumulative release percentage of Asta at different time points, the release kinetics of Asta from the composite microparticles were investigated by fitting four models, i.e. Zero order, First order, Higuchi and Rigter-Peppas. | v3-fos-license |
2019-04-02T13:14:53.385Z | 2011-01-01T00:00:00.000 | 90166725 | {
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} | pes2o/s2orc | EXPRESSION OF RECOMBINANT HUMAN ARGININOSUCCINATE SYNTHETASE IN ESCHERICHIA COLI
Escherichia coli strain BL21(DE3)/pET42a/ASS – an efficient producer of recombinant human argininosuccinate synthetase (rhASS) – was constructed, and preparations of purified rhASS were obtained using His-tag affinity chromatography. The effect of specific inhibitor, a -methyl-DL-aspartate, and nitric oxide donor, sodium nitroprusside, on the ASS specific activity was evaluated with purified rhASS protein and in mouse liver lysates. The developed expression platform is a useful tool in search for new ASS inhibitors efficient under in vitro and in vivo conditions.
INTRODUCTION
Argininosuccinate synthetase (ASS; L-citrulline, L-aspartate ligase; EC 6.3.4.5) plays an important physiological role as a rate-limiting enzyme of urea cycle and biosynthesis of arginine, a semi-essential amino acid in humans [12]. Consequently, ASS affects arginine-dependent synthesis of nitric oxide, polyamines, proline and glutamate [14]. The inherited deficiency in ASS causes citrullinemia, an autosomal recessive disorder, characterized by the elevated levels of blood citrulline and ammonia, which often results in hyperammonemic coma and early neonatal death [11]. In turn, some types of tumors, such as melanomas, renal and prostate carcinomas, mesotheliomas, some hepatocellular carcinomas exhibit downregulation of ASS expression leading to the inability to synthesize endogenous arginine, and to intrinsic dependence of such tumors on extracellular arginine supply (known as "arginine auxotrophy") [6,7]. Enzymatic arginine deprivation is a novel approach to target proliferation of tumors which lack ASS expression [8]. Several phase I/II trials with arginine-degrading pegylated bacterial arginine deiminase and human arginase I have shown clinical benefits in suppressing tumor growth and low toxicity in patients with ASS-negative tumors [5,6].
It was also observed that arginine deprivation can trigger upregulation of ASS expression in certain tumors, which, in turn, can lead to in vivo drug resis tance [6,8,12]. In such cases, application of low molecular weight ASS inhibitors may be desirable to Biol
SDS-PAGE and Western blot analysis.
For Western blot analysis proteins were separated on 10% SDS-PAGE [15], and transferred onto a polyvinylidene difluoride membrane (Millipore) [19]. The membranes were blocked in 5% non-fat dried milk in PBS containing 0.05% Tween-20 and immunoblotted with primary monoclonal antibodies to ASS (BD Transduction Laboratories) or to His-Tag (Millipore). Secondary goat horseradish peroxidase-conjugated anti-mouse antibody (Millipore) and an enhanced chemiluminescence's system ("Amersham Biosciences", USA) were used for the detection of immunoreactive protein bands.
Assay for enzymatic activity of argininosuccinate synthetase. ASS activity was assessed based on the accumulation of the product pyrophosphate as inorganic phosphate [13], following cleavage with pyrophosphatase (Sigma). ASS (1-2 μg of purified protein) was added to the reaction buffer (20 mM Tris-HCl (pH 8.0), 2 mM ATP, 2 mM citrulline, 2 mM aspartic acid, 6 mM MgCl 2 , 20 mM KCl, and 0.1 units of pyrophosphatase) to a final volume of 100 µl. The samples were incubated at 37°C in 96-well plates, and the reactions were terminated after 30 min by the addition of an equal volume of molybdate buffer (10 mM ascorbic acid, 2.5 mM ammonium molybdate, 2% sulfuric acid) and further incubation at room temperature for 30 min. In the case of mouse liver lysate, the enzymatic reaction was carried out in Eppendorf microtubes (5-10 µg of protein in a probe), and 100 µl of 10% trichloroacetic acid was added to each one, mixed and centrifuged for 10 min at 12,000 g. 100 µl of supernatants were placed in 96-well plates with further adding of molybdate buffer as described above.
Accumulation of phosphate was determined by measuring the absorbance at 630 nm on a plate reader (BioTek, Winooski, Vermont, USA), and its concentration was determined by comparison with inorganic phosphate (Pi) standards. Net ASS activity was calculated by subtracting the value at the reaction condition without substrates from that with substrates [10,13].
Inhibition of the ASS catalytic activity by a-methyl-DL-aspartate (MDLA) or sodium nitroprusside (SNP) was examined as follows. Increasing amounts of inhibitors were added to ASS fraction in reaction buffer as described in Fig. 4 legends. After incubation at 37°C for 60 min, the enzyme activity in the mixtures was estimated by the addition of substrate mixture and further incubation at 37°C for 30 min [17]; finally, the concentration of generated Pi was measured as described above. Specific activity of ASS was expressed as μmol Pi/min per mg protein.
Statistics. Each experiment was performed at least three times. Significant difference from the control was compared using Student's t-test.
RESULTS AND DISCUSSION
Expression and purification of rhASS protein. The human ASS gene comprises 16 exons which span 63 kilobases and are mapped to chromosome 9 [22]. Based on cDNA cloning, the predicted protein product consists of 412 amino acids with molecular weight of 46.4 kDa. To aid in downstream rhASS purification, we expressed rhASS in E. coli as a fusion protein with both His-and GST-N-terminal tags.
Ni-NTA agarose was used for rhASS (His) 6 -tag affinity purification as described in Materials and Methods. It was shown that 72 kDa dominant band was withdrawn from cell lysates after treatment with Ni-NTA agarose (Fig. 2, A, lines 4, 5), and its main fraction was eluted with 250 mM imidazole (Fig. 2, B). The purity of the eluted protein was approximately 90% after dialysis and concentration with centrifugal filter devices (Millipore) (Fig. 2, B, lane 3). The yield of 5-10 mg of the partially purified rhASS was obtained per liter of culture broth. Utilization of GSH-sepharose for affinity purification of rhASS via binding to GST-tag produced a lower yield (see Fig. 3
, B).
To confirm the identity of the expressed recombinant protein, Western blot analysis was carried out using two different commercial monoclonal antibodies, anti-ASS and anti-His-Tag. With anti-ASS antibody, we revealed the immunoreactive band at 72 kDa in the induced E. coli cells, transformed with pET-42a/ASS plasmid, only (Fig. 3, A, lane 4). The lysate of human hepatocellular carcinoma HepG2 cell line was loaded as a positive control (Fig. 3, A, lane 2).
With anti-His-Tag antibody, we observed no immunoreactive signal in untransformed E. coli cells, regardless of IPTG induction (Fig. 3, B, lanes 1, 2), whereas it was present in the lysates of pET-42a/ASS transformant and significantly upregulated upon IPTG treatment (Fig. 3, B, lanes 3 and 4, respectively).
We also confirmed that rhASS can be purified by affinity binding to either Ni-NTA agarose or GSH-sepharose, with higher efficacy in the former (Fig. 3, B, lanes 5, 6, respectively).
Thus, based on our Western blot analysis with anti-ASS and anti-His-Tag monoclonal antibodies, we confirmed expression of rhASS in the constructed E. coli producer. However, heterogenity of the recombinant protein product was also evident, probably due to the inefficient transcription of the unmodified heterologous gene in E. coli host Fig. 3, B). Our attempts to overcome this problem by manipulating with media and cultivation conditions, as well as by expressing pET-42a/ASS in alternative E. coli host strains were not successful (data not shown). Inhibitory effect of MDLA and SNP on ASS activity. In order to verify, whether the expressed His (6) -ASS fusion protein is functional, we assayed its catalytic activity. After detailed analysis of the literature data, we found that for the measurement of ASS specific activity different methods were applied. In particular, for investigation of citrullinemia type I genetic variants by in vitro studies, C. Berning et al. [1] determined acti vity of wild-type and mutant ASS proteins in nickel agarose elution samples, by measuring the formation of argininosuccinate from aspartate, citrulline, and ATP using newly developed assay tandem mass spectrometry. Another accepted method to assay ASS acti vity is based on the conversion of radiolabeled aspartate to argininosuccinate [18,22]. In the work of G. Hao and co-authors [13], ASS activity was assessed based on the accumulation of its product, pyrophosphate. However, the ASS activity was represented as percentage of a control value. This method, with modifications described in Materials and Methods section, was utilized in current study. We found that purified preparations of rhASS exhibit specific activity around 30 µmol×min -1 ×mg -1 protein, whereas the ASS activity in mouse liver lysate, used as a positive control, was almost twice as high. A preheated sample (100°C, 10 min) of rhASS protein was used as a negative control and exhibited no detectable ASS activity.
An aspartate analogue, a-methyl-DL-aspartic acid (MDLA), has been previously shown to be a specific inhibitor of ASS from a variety of tissues [9,18]. For instance, to elucidate the effect of the down-regulation of ASS activity on nitric oxide (NO) production in bovine aortic endothelial cells, MDLA was used at concentration of 10 mM [18] or 8 mM [9]. Experimental procedure of Guerreiro et al [10], in which 1 mM MDLA was added into culture medium of human embryonic kidney 293 cells, or administered to spontaneous hypertensive rats at 1 mmol/kg, revealed that ASS is a functional target for a snake venom anti-hypertensive peptide. Therefore, MDLA is an ASS inhibitor in low mM range, which, however, may be too high to be used in humans as auxiliary agent of anticancer enzymotherapy (see Introduction).
Although arginine deprivation therapy, as an anticancer strategy, has been investigated for several decades [12], it is only recently was shown encouraging activity in patients with specific tumor types [21]. However, prolonged arginine starvation in human may cause vasoconstriction and thrombosis due to the deficit of arginine derivative, nitric oxide (NO), as vasodilator and disaggregant [4]. This problem can be overcome via supplementation with exogenous NO-donors in vivo, which could diminish some side effects of arginine deprivation. Sodium nitroprusside (SNP) is one of the most widely used agents for the management of hypertensive emergencies, and has been in clinical use for several decades [20]. Using the BRENDA database of the comprehensive enzyme information system (http://www.brenda-enzymes.org), nitric oxide was also found among the list of potential in vivo ASS inhibitors. Therefore, in this work we evaluated the effect of MDLA, an aspartate analogue, and SNP, an exogenous NO donor, on ASS activity in purified rhASS protein and in mouse liver lysates.
We observed that both analyzed ASS inhibitors elicited concentration-dependent inhibition of ASS (Fig. 4, A, B). The ASS enzymatic activity was completely blocked at concentration of MDLA 4 mM and higher, similarly for the purified rhASS and in mouse liver lysate (Fig. 4, A). SNP, in a range of 1-5 mM, was more effective on purified protein in comparison with mouse liver lysate (Fig. 4, B). Inhibitory concentration index (IC 50 ) toward ASS for MDLA was around 2.5 mM, for SNP -1.5 mM. The observations on SNP inhibitory effect (Fig. 4, B) are consistent with a mechanism, whereby NO donors reversibly inactivate human ASS by S-nitrosylation of its single cysteine residue [13]. It was previously shown in our laboratory that 0.1-0.2 mM SNP releases exogenous NO at the level which is close to NO physiological range and may be potentially used as an adjuvant compensatory agent upon arginine deprivation-based therapy for some types of tumors [3]. Therefore, we decided to examine the cumulative effect of SNP at 0.2 mM concentration and low dose of MDLA on ASS activity.
We observed that the combinational treatment of hrASS protein with NO donor and MDLA in low mM range revealed the opposite effect on its activity (Fig. 4, C) as compared to a separate administration of these inhibitors in a range of 1-5 mM (Fig. 4, A, B). Apparently, 0.2 mM SNP compensates the inhibitory influence of the aspartate analogue. The mechanism of this phenomenon requires further investigation.
Taken together, the (His) 6 -and GST-tagged recombinant human ASS was expressed in the bacterial system. It was confirmed that (His) 6 -tag does not disturb the ASS enzymatic activity, but allows easy one-step purification procedure by Ni-chelating chromatography. The inhibitory effect of a-methyl-DL-aspartate, as well as NO donor, SNP, on the enzyme's activity of mouse liver lysate and purified protein was confirmed. The observed IC 50 concentration ranges for effective inhibition by MDLA and SNP were found to be in low millimolar range (Fig. 4, A, B), a relatively high level from physiological and pharmacological points of view. At lower physiological concentrations, these inhibitors do not provide a desirable ASS inhibition. Nevertheless, the developed scheme will be used in our next studies for in vitro search for more effective ASS inhibitors as components of combinational antitumor enzymotherapy. | v3-fos-license |
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} | pes2o/s2orc | Substrate Recognition by Oligosaccharyltransferase STUDIES ON GLYCOSYLATION OF MODIFIED ASN-X-THR/SER 'fRIPEPTIDES*
The minimum primary structural requirement for N-glycosylation of proteins is the sequence -Asn-X-Thr/Ser-. In the present study, NHz-terminal deriva- tives of Asn-Leu-Thr-NHz and peptides with asparagine replacements have been tested as substrates or inhibitors of N-glycosylation. The glycosylation of a known acceptor, N"-[3H]Ac-Asn-Leu-Thr-NHCH3, was optimized in chicken oviduct microsomes. The reaction was shown to be dependent upon Mn2+ and linear for 10 min at 30 "C; the apparent K , for the peptide was found to be 10 NM. N"-Acyl derivatives of Asn-Leu-Thr-NHz (N-acetyl, or N-t-butoxycarbonyl) inhibited the glycosylation of N"-r3H] Ac-Asn-Leu-Thr-NHCH3 in a dose-dependent manner; additional experiments demonstrated that these com- pounds were alternative substrates rather than true inhibitors. The benzoyl and octanoyl derivatives were 10 times as effective as N"-Ac-Asn-Leu-Thr-NH, in inhibiting glycosylation. In contrast, peptides containing asparagine modifications or substitutions were neither substrates
The minimum primary structural requirement for N-glycosylation of proteins is the sequence -Asn-X-Thr/Ser-. In the present study, NHz-terminal derivatives of Asn-Leu-Thr-NHz and peptides with asparagine replacements have been tested as substrates or inhibitors of N-glycosylation. The glycosylation of a known acceptor, N"-[3H]Ac-Asn-Leu-Thr-NHCH3, was optimized in chicken oviduct microsomes. The reaction was shown to be dependent upon Mn2+ and linear for 10 min at 30 "C; the apparent K , for the peptide was found to be 10 NM. N"-Acyl derivatives of Asn-Leu- Thr-NHz (N-acetyl, N-benzoyl, N-octanoyl, or N-tbutoxycarbonyl) inhibited the glycosylation of N"-r3H] Ac-Asn-Leu-Thr-NHCH3 in a dose-dependent manner; additional experiments demonstrated that these compounds were alternative substrates rather than true inhibitors. The benzoyl and octanoyl derivatives were 10 times as effective as N"-Ac-Asn-Leu-Thr-NH, in inhibiting glycosylation. In contrast, peptides containing asparagine modifications or substitutions were neither substrates nor inhibitors of N-glycosylation. They did not compete for glycosylation of 3H-peptide at 100fold greater concentrations, and did not deplete endogenous pools of oligosaccharide-lipid. Thus, the asparagine side chain is an absolute requirement for recognition by the transferase. The majority of the glycosylated product (61%), but only l% of the unglycosylated peptide, remained associated with the microsomes after high speed centrifugation.
A large 41amino acid residue acceptor peptide, cu-lac17-ae, was a poor substitute for glycosylation unless detergent was added to the microsomes. In contrast, glycosylation of tripeptide acceptors was not stimulated by detergent. Both of these findings suggest that the tripeptides are freely permeable to the microsomal membrane and support the earlier conclusion that glycosylation of proteins occurs at the luminal face of the microsomes.
Several lines of evidence indicate that formation of Nglycosidically linked glycoproteins is a co-translational event * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. involving transfer of oligosaccharide from oligosaccharylpyrophosphoryldolichol to nascent polypeptide acceptors attached to membrane-bound polysomes (1,2). The N-glycosylation reaction is catalyzed by an integral membrane protein of the endoplasmic reticulum, oligosaccharyltransferase (3), and the minimum primary structural determinant for the acceptor substrate is the amino acid sequence -Am-X-Thr/ Ser-(4). Transfer of oligosaccharide to peptide is thought to take place at the luminal face of the endoplasmic reticulum; i n vitro topology experiments have shown that the newly synthesized dolichol-linked oligosaccharide faces the lumen (5, 61, and that the glycosylated product formed by transfer of oligosaccharide to endogenous acceptors or translated polypeptide is sequestered within the endoplasmic reticulum (7).
Other features of the polypeptide backbone must also play a role in recognition of substrate by oligosaccharyltransferase because many potential glycosylation sites within proteins are not glycosylated in vivo (8). One factor involved in recognition of glycosylatable sequences by oligosaccharyltransferase appears to be protein folding. Intact proteins containing potentially glycosylatable -Am-X-Thr/Ser-sites are not glycosylated in vitro; however, these same proteins are acceptors after denaturation by reduction and alkylation (8). Small peptides containing proline in the X amino acid position are not acceptors, whereas replacement of proline with many different amino acids results in active acceptor substrates (9, 10). Furthermore, small cyclic peptides are poorer acceptors than their linear counterparts, presumably due to conformational limitations in their cyclic backbone (11). Some penta-, hexa-, and heptapeptides, but not tripeptide substrates, have circular dichroism profiles which are altered by the presence of dimethyl sulfoxide or phospholipid. Addition of dimethyl sulfoxide to the larger peptides makes them better substrates, whereas tripeptide acceptor activity is unaffected (12, 13). Other factors involved in recognition by enzyme, such as the role of polypeptide interaction with membrane or the effect of amino acid side chain analogs within -Am-X-Thr/Ser-, have not been studied in great detail. Substitution of asparagine with glutamine and serine or threonine with homoserine, 0-methylthreonine, or P-hydroxynorvaline appears to destroy acceptor activity in vitro (14-16).
In the present study, we have developed a sensitive competition assay to measure the ability of various NHz-terminal and asparagine derivatives of the tripeptide -Am-Leu-Thr-to act as substrates for or inhibitors of oligosaccharyltransferase in intact hen oviduct microsomes. The results of studies on the NH2-terminal derivatives indicate that a variety of chemical modifications are permitted in this region of the acceptor without loss of acceptor activity; in fact, activities increase as hydrophobicity of the acceptors increase. In contrast, the asparagine derivatives were found to be inactive as acceptors and inhibitors. Therefore, the asparagine side chain appears to be an absolute requirement for recognition by oligosaccharyltransferase.
Methods
Preparation of 3H-labeled Peptide-Five mg of Asn-Leu-Thr, dissolved in 200 p1 of sodium borate, pH 10.0, containing 2% (v/v) dimethyl sulfoxide was N-acetylated by treatment with 25 mCi of [3H]acetic anhydride (10 Ci/mmol) at room temperature. After 1 h, nonradioactive acetic anhydride was added in five equal aliquots, with each aliquot containing a 10-fold molar excess over primary amino groups. The pH of the reaction was maintained between 9 and 10 by addition of NaOH. Following acetylation, the reaction mixture was treated with 1 M hydroxylamine hydrochloride for 1 h at pH 10.0 to reverse any 0-acetylation. The reaction components were then separated by gel filtration chromatography on Sephadex (2-10 in 0.1 M NH4HC03, pH 7.8. The 3H-labeled component (representing approximately 25% of the starting radioactivity) that eluted immediately after Vo was recovered, lyophilized, and treated with 1 M methylamine HCI, pH 4.7, in the presence of 0.25 M l-ethyl-3-(3-dimethylamino-propy1)carbodiimide HCI for 8 h a t room temperature. The reaction products were then separated by gel filtration on a Sephadex G-10 column. Material in the 3H-labeled peak was recovered, lyophilized, dissolved in a small volume of HzO, and applied to a mixed bed ion exchange column to eliminate any peptide that was charged and therefore not blocked a t both the NH, and COOH termini. Approximately 95% of the 3H-labeled peptide did not bind to the column. The specific activity of the purified, blocked peptide was approximately 4.0 X 10' cpmlpmol.
Synthesis of Tripeptide Substrates for Oligosaccharyltransferase-The synthesis of potential substrates was accomplished using various protected amino acids and either the mixed anhydride procedure (17) or the p-nitrophenyl ester method (18) for peptide bond formation. In general, the yield obtained for peptide bond formation was between 60 and 80%. The acyl substrates were prepared by reacting a suitable p-nitrophenyl ester with the a-amino group of the corresponding tripeptide. All substrates exhibited the expected NMR resonances in dimethyl sulfoxide-& solution ( 6 values are reported with respect to trimethylsilyl derivatives), were homogeneous on thin layers of silica using butano1:acetic:acidwater (4:1:5 (v/v/v), upper layer) as the eluent, and were at least 96% pure as determined using reversed phase high performance liquid chromatography on a CIS pBondapak column (Waters Associates), with CH30H:HZ0:CF&OOH as the mobile phase. The physical constants for all substrates used in this study are summarized in Table I. Specific details for Nu-Ac-Asn-Leu-Thr-NHZ and N"-Boc-Asn(N8-Me)-Leu-Thr-NH2 are given below.' N"-Boc-Leu-Thr-NHz-To a stirred solution of Boc-Leu (2.77 g, 12 mmol) in tetrahydrofuran (25 ml) a t -15 'C, N-methylmorpholine (1.32 ml, 12 mmol) was added followed by isobutyl chloroformate (1.56 ml, 12 mmol). Stirring was continued for 7 min and a precooled (-15 " c ) solution of Thr.NH,.HCl (1.85 g; 12 mmol) and NMM (1.32 ml, 12 mmol) in DMF (14 mi) was added. After stirring for 30 min a t -15 "C and 2 h a t room temperature, the solvent was evapo-' The abbreviations used are: Ac, acetyl; Abu, L-a-aminobutyric acid; Nva, L-norvaline; DMF, dimethylformamide; NMM, N-methylmorpholine; ONp, p-nitrophenyl ester; alac17.58, tryptic peptide containing residues 17-58 of a-lactalbumin; Bz, benzoyl; Oc, octanoyl; BOC, tert-butoxycarbonyl; Endo H, endo-@-N-acetylglucosaminidase H; NP-40, Nonidet P-40, Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. (0.93 g, 2.8 mmol) was dissolved in CHZClZ-trifluoroacetic acid (15 ml; 1:1, v/v) and allowed to stand at room temperature for 30 min. The solvent was then evaporated in uacuo and the residue was precipitated by the addition of ether. The trifluoroacetate salt was isolated and dried. It was coupled with Boc-Asn-ONp (0.99 g, 2.8 mmol) in the presence of NMM (0.31 ml, 2.8 mM) in DMF (5 ml). After a reaction period of 24 h, the solvent was evaporated in Vacuo and the residue was dissolved in aqueous citric acid (5%, 25 ml). The solution was extracted with three 25-ml portions of ethyl acetate to remove the unreacted active ester and p-nitrophenol.
N"-Boc-Asn(NB-Me)-OH-To a solution of Boc-Asn(N@-Me)-OBzl (504 mg, 1.5 mmol) in methanol (4 ml), paladium black (-100 mg) and formic acid (go%, 0.3 ml) were added. The resulting mixture was stirred at room temperature and after 30 min the catalyst was removed by filtration through Celite and the filtrate was evaporated to dryness. The residue was crystallized from tetrahydrofuran-petroleum ether.
N"-Boc-Asn(NB-Me)-Leu-Thr-NH2-To a solution
of Nu-Boc-Asn(NO-Me)-OH (246 mg, 1 mmol), 1-hydroxybenzotriazole (153 mg, 1 mmol), trifluoroacetic acid-Leu-Thr-NHz (345 mg, 1 mmol), and NMM (0.11 ml, 1 mmol) in DMF (3 ml) at 0-5 "C, dicyclohexylcarbodiimide (206 mg, 1 mmol) was added and the mixture was stirred for 4 h. The solution was then allowed to come to room temperature and the reaction continued for 20 h. The dicyclohexylurea was then filtered off, the solvent was evaporated in vacuo, and the residue was worked up in the same manner described for Boc-Asn-Leu-Thr-NHZ. . The S-carboxymethylated protein was digested with 10 mg of trypsin by two additions of the L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated enzyme at 3-h intervals and the reaction was allowed to proceed overnight in 0.2 M NH4HC03, 2 M urea at 37 "C. The digestion products were separated by gel filtration chromatography on Sephadex G-50 (2.5 X 85 cm) in 0.1 M NH4HC03. The first major product absorbing a t 280 nm and eluting after V, of the column was recovered, lyophilized, and rechromatographed on Sephadex G-50, thereby yielding a homogeneous component with an apparent molecular weight expected for the 41-residue peptide containing residues 17 through 58 of a-lactalbumin (20). The overall yield of peptide was 40 mg. The acceptor activity of this material was demonstrated by incubation with microsomes in the presence of radioactive sugar nucleotides followed by separation of the glycosylated peptide from free sugar nucleotides by gel filtration chromatography on Sephadex G-50 as previously described (21). The glycosylatable site within a-lacl-i-ss is a t Asn residue 45 in the sequence -Asn-45-Gln-Ser-.
Oviduct Microsome Preparation-The magnum portion of freshly killed laying hens was cleared of connective tissue, minced, homogenized, and centrifuged as described by Pless and Lennarz (22). Before use, microsomes were stored a t -70 "C a t a concentration of approximately 30 mg of protein/ml.
Oligosaccharyltransferase Assays Using Endogenous Oligosacchar-3 0 p~ N"[3H]Ac-Asn-Leu-Thr-NHCH3 (5 X lo5 cpm), 10 mM MnCL, ide Lipid-Standard reaction mixtures (50 p1 final volume) contained and approximately 300 pg of microsomal protein in 50 mM Tris-HC1, pH 7.4, containing 140 mM sucrose, 25 mM NaCl, and 1 mM EDTA. Reactions were started by addition of microsomes and proceeded at 30 "C for 5 or 10 min before termination by addition of 50 p1 of 20% trichloracetic acid. After 5 min on ice, samples were centrifuged in a Beckman microfuge 11 at setting 10 for 5 min; 75 pl of the supernatant was removed, backwashed three times with 1 ml of ether, and gassed with Nz to remove residual ether. The sample was then analyzed by gel filtration, affinity chromatography, or paper chromatography as described below. Any additions to the reaction mixture, such as dimethyl sulfoxide or detergent, were made prior to microsome addition.
-CH-0-). glucose were included and reactions were allowed to proceed for 15 or 30 min. For preparation of doubly labeled glycosylated product, 1 pM GDP-[14C]mannose (400,000 cpm/assay) was utilized. For preparation of glycopeptides formed by transfer of ['4C]Man-oligosaccharide to nonradioactive peptides, the assay conditions were the same as those for doubly labeled peptide except that the nonradioactive peptide was present instead of N"-[3H]Ac-Asn-Leu-Thr-NHCH3.
Competition Experiments-Competition experiments utilized endogenous oligosaccharide-lipid and were done in an manner analogous to those described above except that the indicated amounts of competingpeptides were included in the reaction mixtures before addition of microsomes.
Endogenous Oligosaccharide-Lipid Depletion Experiments-Assay conditions were the same as above except that N"-[3H]Ac-Asn-Leu-Thr-NH-CH3 was replaced by nonradioactive peptides and the reaction was allowed to proceed for 15 min before 100-fold dilution into cold oviduct buffer and immediate centrifugation at 39,000 X g for 15 min. The pellets were then washed with another 5 ml of oviduct buffer, centrifuged, and resuspended in a minimum volume (150 p l ) before addition of sugar nucleotide precursors and N"-[3H]Ac-Asn-Leu-Thr-NHCH3. After 30 min at 30 "C, assays were terminated with trichloroacetic acid and prepared as described above.
Chromatographic Procedures-Descending paper chromatography was performed in 1-butano1:acetic acidwater (12:3:5, v/v/v) on Whatman 3 " paper for 16 h. Bio-Gel P-4, 200-400 mesh, gel filtration chromatography was carried out using a column (1.6 X 110 cm) equilibrated in 0.1 M ammonium bicarbonate. Fractions of 1.4 ml were collected at a flow rate of approximately 8 ml/h. Concanavalin A affinity chromatography was done by a batchwise procedure. Samples were added to 50 p1 of concanavalin A-agarose beads prewashed in 20 mM Hepes, pH 7.5, containing 1 mM CaC1, and 1 mM MnCl,. After 1 h, the beads were washed six times with buffer and the glycosylated peptide was eluted overnight by incubation with 0.5 ml of the same buffer containing 0.5 M a-methylmannoside.
Glycosylation of N"-pH]Ac-Asn-Leu-Thr-NHCH3: Product
Characterization and Optimization of Conditions-In previous studies, the enzymatic glycosylation in hen oviduct microsomes of endogenous membrane proteins and exogenously added proteins and peptides, was quantitated by the incorporation of radioactive sugars into nonradioactive acceptors (4, 7). In this study, we have utilized nonradioactive endogenous pools of oligosaccharide-lipid present in hen oviduct microsomes as one substrate; the other substrate was exogenously added N"-[3H]Ac-Asn-Leu-Thr-NHCH3. There were a number of technical reasons for using labeled peptide instead of labeled oligosaccharide-lipid to measure the formation of glycopeptide. First, introduction of label in peptide eliminated the requirement for and variability in de novo synthesis of the oligosaccharide-lipid during transferase assays. Second, the presence of label in the peptide acceptor permitted the development of a competition assay for measurement of acceptor and inhibitor activities of a variety of unlabeled tripeptide derivatives of Asn-Leu-Thr-NH2. Third, it allowed one to measure the apparent K,,, for the acceptor. Finally, it facilitated determination of the topological orientation of the glycopeptide product within the microsomes. N"-['H]Ac-Asn-Leu-Thr-NHCH3 was chosen as the labeled acceptor because it was found to be a good substrate in a previous study (4).
The formation of glycosylated N"-[3H]Ac-Asn-Leu-Thr-NHCH3 was routinely measured by paper chromatography. Authentic standards of peptide substrate and glycopeptide product migrated near the solvent front or remained at the origin, respectively (Fig. 1, A and B). Glycopeptide formation, measured as radioactivity remaining at the origin, was time dependent (Fig. 1, C and D ) and was linear for a t least 10 min a t 30 "C (Fig. 2). As expected, treatment of the glycosylated peptide product shown in Fig. 1D with Endo H resulted in the elimination of radioactivity at the origin (Fig.lE). The recovery of radioactivity from paper was typically 35% of that applied. Unless microsomes had been predepleted of endogenous substrate, the production of glycopeptide did not require addition of exogenous oligosaccharide-lipid donor to microsomes (see below).
The 3H-glycopeptide product bound to concanavalin Aagarose and eluted from the lectin in the presence of 0.5 M amethylmannoside. Chromatography of the glycosylated product on a Bio-Gel P-4 column showed that a 3H-labeledproduct with increased molecular weight had been formed. This compound contained 14C and 3H label when 14C-sugar nucleotides were included in the reaction mixture along with 3H-peptide (Fig. 3A). As shown in Fig. 3B, treatment of the doubly labeled material with Endo H caused the 3H-component to elute at a position of apparent molecular weight close to that of unglycosylated peptide, which is consistent with cleavage of the original 3H-glycopeptide to a peptide containing one GlcNAc residue. The 14C-labeled product released by Endo H eluted in the position expected for oligosaccharide. Another 3Hproduct with a molecular weight greater than peptide was also formed during the glycosylation reaction (Fig. 3A). The structure of this product is unknown, but it was not labeled with either [14C]mannose or N-[14C]acetylglucosamine and it constituted only a small percentage of the initial 3H-peptide. This product did not bind to concanavalin A-agarose and it migrated near the solvent front on descending paper chromatography. In most experiments, glycosylation of peptides was confirmed by independent measurement of 3H-glycopeptide formation by paper chromatography, concanavalin A-agarose binding, and gel filtration on Bio-Gel P-4.
During preparation of hen oviduct microsomes, a preincubation with Mn2+ was done to increase the activity of the oligosaccharyltransferase (22). Mn2+ was then washed from microsomes by high speed centrifugation before storage of the
TABLE I1
Divalent metal requirement for N-glycosylation activity Frozen microsomes were thawed, suspended in oviduct buffer containing 20 mM EDTA, collected by centrifugation at 39,000 X g for 15 min, and then washed once in buffer containing 1 mM EDTA by resuspension and centrifugation. Incubation was carried out for 5 min at 30 "C with 3H-peptide in the presence or absence of the various metals. http://www.jbc.org/ Downloaded from microsomal pellet at -70 "C. Upon thawing the microsomes, the addition of 10 mM Mn2+ yielded transferase activity which was approximately 2-fold higher than that observed in the absence of added metal (Table 11). The presence of EDTA lowered activity considerably, indicating that the enzyme does require a divalent metal. Incubation with Mg2+ or Ca2+ rather than Mn2+ did not increase enzyme activity above the control, whereas incubation with Zn2+ or Cu2+ abolished activity. Taken together, these results indicate that oligosaccharyltransferase has a marked preference for Mn2+. Apparently, once bound, this Mn2+ is difficult to remove, even in the presence of EDTA.
The addition of 0.25 mM dithiothreitol, 5% dimethyl sulfoxide (v/v), or NP-40 up to 0.1% (v/v) to incubation mixtures did not affect glycosylation of N"-[3H]Ac-Asn-Leu-Thr-NHCH,. The rate of glycopeptide formation was dependent upon peptide concentration. The Lineweaver-Burk plot shown in Fig. 4 indicated an apparent K,,, for the peptide of 10 @M; the V,,, was found to be 3.1 pmol/mg of protein/min. Typically, 30 FM peptide was added to the reaction mixtures; as much as 20% of the peptide substrate was glycosylated in experiments containing large quantities of microsomes.
Use of a Competition Assay to Measure Acceptor or Inhibitor Activities of Unlabeled Peptides-Structures of peptides containing asparagine replacements or N"-acyl derivatives of Asn-Leu-Thr-NH, are shown in Table 111. To evaluate their ability to act as acceptors or inhibitors, an in vitro glycosylation competition assay was developed. In this assay, the glycosylation of IV-[3H]Ac-Asn-Leu-Thr-NHCH3 was measured in the presence of up to 100-fold molar excess of unlabeled competitor peptides. As shown in Fig. 5, a number of analogs inhibited the glycosylation of 3H-acceptor in a dosedependent manner. At a molar ratio of 1:1, glycosylation of the "-peptide is inhibited by nearly 50% by Ne-Ac-Asn-Leu-Thr-NHz, indicating that this compound and N"-[3H]Ac-Asn-Leu-Thr-NHCH3 are equally effective acceptors. Unblocked tripeptides have previously been shown to be inactive as acceptors in hen oviduct microsomes (4). Consistent with this finding, Asn-Leu-Thr-NHz was found to have little inhibitory effect on glycosylation of N"-[3H]Ac-Asn-Leu-Thr-NHCH3.
In contrast to the W-acyl derivatives, peptides with aspar-
FIG. 4. Lineweaver-Burk plot for glycosylation of LV"-[~H]
Ac-Am-Leu-Thr-NHCH3. Standard reaction mixtures containing 3, 10, 30, 100, or 300 PM N"-[3H]Ac-Asn-Leu-Thr-NHCH3 were incubated for 10 min at 30 "C and then analyzed for glycopeptide production by paper chromatography. "All NH2-terminal modifications were synthesized starting with the Asn-Leu-Thr-NH2 as described under "Experimental Procedures." bThe asparagine modifications were synthesized using Leu-Thr-NH, as starting material as described under "Experimental Procedures." agine replaced by other amino acids did not inhibit production of glycosylated 3H-peptide, even at the highest concentration tested (Fig. 5 ) . Therefore, the asparagine replacements are at best very poor acceptors for or very poor inhibitors of oligosaccharyltransferase.
Studies to Determine the Acceptor or Inhibitor Properties of Unlabeled Peptides-Experiments were performed to determine whether the peptide derivatives that inhibited the glycosylation of N"-[3H]Ac-Asn-Leu-Thr-NH2 are acceptor substrates for oligosaccharyltransferase or are true inhibitors of this enzyme. Acceptor activity was evaluated by measuring the extent of depletion of endogenous oligosaccharide-lipid that occurred upon preincubation of peptide derivatives with oviduct microsomes (Table IV). Preincubation of microsomes with the known acceptor N"-Ac-Am-Leu-Thr-NH2 depleted endogenous oligosaccharide-lipid and thereby prevented the subsequent glycosylation of 3H-peptide added after removal of the unlabeled peptide (Table IV, entry 1). Addition of fresh sugar nucleotide precursors for synthesis of oligosaccharidelipid resulted in 3H-glycopeptide production, thereby indicating that Ne-Ac-Asn-Leu-Thr-NH, does not act as an irreversible inhibitor of oligosaccharyltransferase. As expected, preincubation of microsomes in the absence of any peptide derivative did not lead to the depletion of endogenous oligosaccharide-lipid because fresh sugar nucleotides were not required for formation of 3H-labeled glycopeptide (Table IV, entry 2). Preincubation with the peptide containing an octanoyl group in place of an acetyl group, a compound that competed efficiently for glycosylation of "-peptide in the Ac-Asn-Leu-Thr-NHCH3 and the indicated amounts of unlabeled tripeptide derivatives were incubated for 5 min and the supernatants were analyzed for 3H-glycopeptide production by paper chromatography or binding to concanavalin Aagarose. Abbreviations for the peptide derivatives are listed in Table 111.
TABLE IV
Endogenous oligosaccharide-lipid depletion Thawed microsomes were preincubated with the indicated tripeptide derivatives (30 p~) for 15 min at 30 "C, washed twice with oviduct buffer to remove modified peptides, and then incubated with 3H-acceptor (30 p~) in the absence or presence of sugar nucleotides as described under "Experimental Procedures." 3H-Glycopeptide formation in the absence of added sugar nucleotides serves as a measure of endogenous oligosaccharide-lipid remaining in microsomes after preincubation with various tripeptides. 3H-Glycopeptide production in the presence of sugar nucleotide serves to indicate that the abolishment of glycopeptide formation is reversible by synthesis of new oligosaccharide-lipid from precursor substrates. Values shown are the average of duplicates. (Table IV, entry 3). Similar results were obtained for all the NHz-terminal derivatives (data not shown). Based upon these results, we conclude that the NHz-terminal modified peptides are acceptors and not inhibitors of oligosaccharyltransferase. In contrast, preincubation with a peptide containing N-methylasparagine in place of asparagine did not deplete endogenous pools of microsomal oligosaccharide-lipid (Table IV, entry 4). Analogous results were found with the other asparagine derivatives (data not shown). Therefore, compounds containing asparagine replacements are neither acceptors nor inhibitors of N-glycosylation. To confirm the above conclusions, various peptide derivatives were incubated with microsomes in the presence of 14Csugar nucleotides and the formation of glycosylated products was directly established by gel filtration on a Bio-Gel P-4 column. Incubation with W-Ac-Asn-and N"-Boc-Asn-peptides led to the formation of 14C-labeled products with apparent molecular weights virtually identical with that of glycosylated N"-[3H]Ac-Asn-Leu-Thr-NHCH3. Treatment with Endo H caused the radioactivity to elute on Bio-Gel P-4 at the position where that oligosaccharide elutes. In contrast, incubation with Asn-Leu-Thr-NHz or any of the peptides containing asparagine replacements did not lead to formation of a high molecular weight, 14C-labeled product. These observations support the conclusion that the Ne-Ac-Asn-and W -Boc-Asn-peptides are acceptors for oligosaccharyltransferase, whereas asparagine derivatives are not.
Additional evidence supports the conclusion that the N"-Oc-Asn-and N"-Bz-Asn-derivatives are very efficient acceptors. First, it was found that competition for glycosylation could be overcome by addition of increasing amounts 3Hacceptor to a fixed amount of octanoyl peptide (data not shown). This finding rules out the possibility that some contamination in the unlabeled peptide preparation causes either nonspecific or irreversible inactivation of oligosaccharyltransferase. Second, the Ne-Oc-Asn-peptide depleted more endogenous oligosaccharide-lipid when microsomes were incubated with limiting quantities of peptide than did N"-Ac-Asn-Leu-Thr-NH2 as measured by an oligosaccharide-lipid depletion experiment (Table V). Thus, the apparent K , for this peptide must be lower than the apparent K, = 10 FM measured for Ne-[3H]Ac-Asn-Leu-Thr-NHCH3. Similar results were obtained with W-Bz-Asn-peptide. The results of all of these experiments conclusively establish that these peptides are very effective acceptors.
Topological Aspects of Peptide Glycosylation-To better understand the process resulting in glycosylation of these peptides, the topological orientation of the glycopeptide product and the oligosaccharyltransferase in microsomes was examined. After reaction with 3H-peptide, the microsomes were diluted with buffer and the soluble components were separated from membrane-associated or entrapped components by high speed centrifugation The percentage of the total unglycosylated substrate and the glycopeptide product associated with the membrane pellet was then quantitated by paper chromatography. The results, shown in Table VI, indicate that most of the glycopeptide product (61%) and very little of the N"-Oc-Asn-L.eu-Thr-NH2 and N"-Ac-Asn-Leu-Thr-NH2 The rate of peptide glycosylation under conditions of increasing concentrations of N"-Ac-Asn-or N"-Oc-Asn-peptides was compared by preincubation of microsomes with peptides for 5 min, followed by centrifugation to separate microsomes from unlabeled peptide substrate. Subsequent quantitation of the amount of endogenous oligosaccharide-lipid remaining was accomplished by measuring glycopeptide formation during a second incubation for 30 min with 3H-peptide.
3H-Glycopeptide formed after Preincubation Peptide concentration dur-with ing Preincubation
N"-Oc-Am-Leu-N"-Ac-Asn-Leu-Thr-NH, Thr-NH, After incubation of 3H-acceptor peptide (lo6 cpm) with 6 mg of microsomal membranes in a total volume of 300 p1 for 15 min a t 30 "C, two 15-pl portions were removed and treated with 10% trichloroacetic acid, and the total amount of 3H-peptide and 3H-glycopeptide was quantitated by paper chromatography. Another two 100-pl portions of the assay were diluted into 5 ml of cold oviduct buffer and spun at 39,000 X g for 15 min. The pellet was then treated with 10% trichloroacetic acid, and the 3H-peptide and 3H-glycopeptide associated with the membranes were determined by paper chromatography as above. unglycosylated substrate (1%) remained associated with membrane. These results suggest that glycosylated product is located on the luminal side of oviduct microsomes. Support for the theory that transferase activity is present at the inner face of the endoplasmic reticulum was derived from a competition experiment utilizing a large molecular weight acceptor, the tryptic peptide encompassing residues 17 through 58 of a-lactalbumin. As seen in Fig. 6, the large peptide acceptor competes very poorly for 3H-acceptor glycosylation unless detergent is added. In contrast, the competition by N"-Ac-Asn-Leu-Thr-NHz is virtually independent of detergent concentration. These results suggest that the membranes are freely permeable to these small peptides but not to larger acceptors such as the a-lactalbumin fragment. Only under conditions in which the microsomes are made permeable can the large peptide interact with oligosaccharyltransferase and therefore act effectively as a competitor. The simplest explanation for these findings is that oligosaccharyltransferase is oriented at the luminal face of the endoplasmic reticulum, as previously proposed (7).
DISCUSSION
In earlier studies, it was established that not only unfolded proteins (8) but also peptide fragments (21,23,24) containing the sequence -Am-X-Ser/Thr-could serve as substrates for glycosylation in vitro. Subsequently, this laboratory showed that simple tripeptides of the type -Asn-X-Ser/Thr-were active acceptors if the NH, and COOH termini were blocked (4). These observations have been confirmed and extended by others (13,15,24,25). The finding that simple tripeptides are substrates for oligosaccharyltransferase raises the possibility that analogs of the tripeptide can be prepared that will serve as reversible or irreversible inhibitors of this enzyme. Indeed, one such inhibitor that acts irreversibly has been reported recently (26). Such inhibitors may be of great utility in studying the regulation of the dolichol-linked pathway and the role of glycosylation in general, and would not be expected to concomitantly inhibit protein synthesis.
As a first step in the development of inhibitors of oligosaccharyltransferase, we have synthesized several tripeptide derivatives of Asn-Leu-Thr-NHz containing different derivatives at the NH2 terminus or asparagine substitutions or modifications, and have measured their ability to act as substrates for or inhibitors of oligosaccharyltransferase. At the outset, we developed a simple and rapid assay to test peptides as possible alternative substrates or inhibitors. N"-[3H]Ac-Asn-Leu-Thr-NHCH3 was synthesized and conditions were optimized for its glycosylation by the oligosaccharyltransferase in hen oviduct microsomes. The initial screen for evaluating whether various labeled compounds were or were not acceptors or inhibitors involved a direct competition experiment with N"-[3H]Ac-Asn-Leu-Thr-NHCH3. The experiment was designed so that co-incubation of the 3H-acceptor peptide with tripeptide derivatives that were either acceptors or inhibitors would block formation of glycopeptide either by competition for endogenous oligosaccharide-lipid or by actual inhibition of oligosaccharyltransferase. Tripeptide derivatives which were not substrates or inhibitors would have no effect upon 'H-glycopeptide formation.
All the NHz-terminal derivatives of Asn-Leu-Thr-NH, that were tested inhibited 3H-glycopeptide production in a dosedependent manner. They were subsequently shown to be acceptors for oligosaccharyltransferase because they either served as acceptors of radioactive oligosaccharide and/or they depleted pools of endogenous oligosaccharide from oviduct microsomes. None of the peptides were irreversible inhibitors as they were readily washed from microsomes and did not inhibit subsequent glycosylation of 'H-acceptor. As expected, the NO-Ac-Asn-compound inhibited glycosylation by 50% at a concentration equivalent to that of the 'H-peptide, whereas the nonacylated peptide had no inhibitory activity. TWO of the hydrophobic NH,-terminal derivatives tested, W-Oc-Asn-Leu-Thr-NHn and P-Bz-Asn-Leu-Thr-NH2, inhibited glycosylation of the "-peptide by approximately 50% a t a concentration 10-fold below that of the "-substrate. The P -Boc-Asn derivative was slightly poorer as a substrate than the 'H-peptide, as measured by the competition assay. The decreased acceptor activity of this compound may be due to some hydrolysis of the NH2-terminal protecting group by enzymes in oviduct microsomes. Alternatively, it could be because the urethane linkage at the NHz terminus is sterically and electronically different from the simple amide bond in the other acceptors. In any case, the fact that the N"-Boc-Asn derivative is an acceptor implies that oligosaccharyltransferase does not require an amide bond at the NH2-terminal position and that other derivatives that neutralize the positive charge can be used. A similar observation has been reported using dinitrophenylated and dansylated tetrapeptide substrates which contain carbon-nitrogen linkages rather than the typical amide bond (26).
In contrast, a methylamide derivative of asparagine, which contains a single methyl substitution for hydrogen in the asparagine side chain, was neither an acceptor substrate nor an inhibitor of oligosaccharyltransferase. It did not inhibit in the competition assay, nor did it deplete endogenous oligosaccharide-lipid or accept radioactive oligosaccharide. Furthermore, when the carboxyamide group of the side chain was replaced by a H or a CH, group, the resulting peptide was not an inhibitor. These results suggest that recognition by oligosaccharyltransferase is extremely dependent upon the precise structure of the asparagine side chain and that modifications in this side chain abolish binding to the active site. Based upon previous work demonstrating that tripeptides alone contain all the necessary information for glycosylation, it has been suggested that accessibility of this sequence to oligosaccharyltransferase is the major factor controlling glycosylation of -Am-X-Thr/Ser-sites i n vivo (4). Several other results point also to this conclusion. First, the oligosaccharide chains of N-linked glycoproteins generally are found a t pturns (27). Second, the presence of proline in the X amino acid position of peptide substrates results in a loss in acceptor activity (9, 10). Third, only denatured, and therefore unstructured, proteins are glycosylatable in vitro (8). Fourth, disulfide bonds limit glycosylation of intermediate size peptides i n vivo (11). Finally, dimethyl sulfoxide and phospholipid alter the circular dichroism profiles of certain peptide acceptors as well as increase their activity as substrates (12,13).
The increased acceptor activity observed with Ne-Oc-Asnand N"-Bz-Asn-peptides, which were insoluble in water in the absence of dimethyl sulfoxide, suggests that peptide hydrophobicity increases acceptor activity. A role for hydrophobicity is consistent with the biology of the process of glycosylation. I n vivo, the nascent chains attached to membranebound polysomes (1, 2) are glycosylated by the integral membrane protein, oligosaccharyltransferase. Glycosylation is believed to occur at or near the luminal face of the endoplasmic reticulum where the oligosaccharide-lipid is synthesized ( 5 , 6), while the protein is passing through the membrane on its way to the lumen. The findings of the current study indicate that glycosylation of small peptide acceptors in oviduct microsomes occurs by a similar intraluminal mechanism. The majority of the 3H-glycopeptide produced remains associated with the microsomes, whereas virtually all of the unglycosylated peptide substrate is found in the supernatant after the microsomes are washed. Furthermore, the presence of the nonionic detergent NP740 has no stimulatory effect on the acceptor activity of the %-peptide. In contrast, a large peptide acceptor peptide containing 41 amino acid residues is a very poor acceptor unless NP-40 is added. These results indicate that the small peptides can freely enter and exit from the microsomes and that their glycosylation, like the glycosylation of nascent chains, occurs at or near the luminal face.
The finding that a variety of substitutions are permissible at the NH2-terminal end of the peptide and that hydrophobic substitutions increase the efficiency of the peptide as a substrate for oligosaccharyltransferase indicates that this would be a suitable site for introduction of chemically reactive or photoreactive groups designed to generate an irreversible inhibitor of oligosaccharyltransferase. Furthermore, the hydrophobic nature of such compounds raises the possibility that they could be delivered to cells either directly or indirectly via liposomes. If this is feasible, such peptides could be valuable tools to study the consequences of i n vivo inhibition of oligosaccharide transfer to protein as well as the mechanism of N-glycosylation. In fact, we have recently found that the tripeptide substrates described in this report inhibit the cotranslational glycosylation of ovalbumin and vesicular stomatitis virus G protein i n vitro (28). | v3-fos-license |
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} | pes2o/s2orc | Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells
The in vitro methods currently used to screen bioactive compounds focus on the use of a single model of oxidative stress. However, this simplistic view may lead to conflicting results. The aim of this study was to evaluate the antioxidant properties of two natural extracts (a mix of red wine polyphenols (RWPs) and epigallocatechin gallate (EGCG)) with three models of oxidative stress induced with hydrogen peroxide (H2O2), a mixture of hypoxanthine and xanthine oxidase (HX/XO), or streptozotocin (STZ) in RINm5F beta cells. We employed multiple approaches to validate their potential as therapeutic treatment options, including cell viability, reactive oxygen species production, and antioxidant enzymes expression. All three oxidative stresses induced a decrease in cell viability and an increase in apoptosis, whereas the level of ROS production was variable depending on the type of stress. The highest level of ROS was found for the HX/XO-induced stress, an increase that was reflected by higher expression antioxidant enzymes. Further, both antioxidant compounds presented beneficial effects during oxidative stress, but EGCG appeared to be a more efficient antioxidant. These data indicate that the efficiency of natural antioxidants is dependent on both the nature of the compound and the type of oxidative stress generated.
Introduction
Oxidative stress can be defined as an imbalance between proand antioxidants and is often associated with free radical [1] overproduction and/or defective physiological defence mechanisms resulting in the cell being overwhelmed with oxidizing radicals [2]. This phenomenon involves reactive oxygen species [3], such as superoxide anion (O 2 − ) [4], hydroxyl radical (OH • ) [1], singlet oxygen ( 1 O 2 ), and hydrogen peroxide (H 2 O 2 ) [5]. High concentrations of ROS can cause lipid peroxidation, protein oxidation or denaturation, nuclear acid oxidation, and many other macromolecular changes that can lead to serious cellular damage [6]. Such ROS-related damage has been identified to occur in numerous diseases, including metabolic syndrome, diabetes, multiple types of cancer, Alzheimer's disease, and cardiovascular diseases.
Further, obesity, hyperglycemia, and hyperlipidemia have also been shown to promote oxidative stress through elevated ROS production [7], which is likely due to the higher occurrence of mitochondrial dysfunction and superoxide production that has been associated with fat accumulation [8]. Under normal conditions, enzymatic defence mechanisms [9], such 2 Evidence-Based Complementary and Alternative Medicine as scavenging by superoxide dismutase [10] and glutathione peroxidase, are active in most types of cells to degrade ROS and prevent cellular damage. However, the antioxidant defence system functioning in insulin producing beta cells, which have been linked to both diabetes and obesity, is known to be very weak [1,11,12], making these beta cells highly sensitive to oxidative stress, which can lead to cell death and disease [5]. Notably, the prevention of ROS-related beta cell destruction using antioxidant compounds has been identified to be an effective strategy to delay the onset of diabetes [10,13,14].
In fact, several dietary plants that have pharmacological properties shown to prevent apoptosis induced by oxidative stress are under investigation as treatment options for diabetes [9,15]. Some of these plants appear to utilize antioxidant mechanisms related to their rich flavonoid (polyphenols family) content. The unique chemical structures and redox properties of these polyphenols [16] allow them to scavenge free radicals as well as chelate transition metals and inhibit prooxidant enzymes, such as inducible nitric oxide synthase (iNOS) in macrophages [17]. For example, tea catechins, especially epigallocatechin gallate (EGCG), appear to have antiobesity and antidiabetic properties [11,18], and the beneficial effects of red wine polyphenols (RWPs) in diabetics have been widely documented [19]. RWPs are qualitatively and quantitatively rich in polyphenols, particularly anthocyanins, flavonol, and stilbene. In general, polyphenols are characterized by antioxidant activity and in vitro studies have shown that they act as radical peroxyl scavengers [20]. However, most of these in vitro studies were performed using a single model of stress, such as hypoxanthine/xanthine oxidase (HX/XO) [21] or H 2 O 2 [22,23], whereby HX/XO was a direct supplier in O 2 − , while H 2 O 2 activated NADPH oxidase or NOS which produce O 2 − . In diabetes, in addition to O 2 −• , generated by chronic hyperglycemia [4], other types of ROS are produced during insulin resistance and hyperinsulinism development [24]. Therefore, a single model of oxidative stress does not reflect the full complexity of this disease. In more relevant studies, oxidative stress was induced by multiple mechanisms using cytokines [25], alloxan [26], or streptozotocin [9,27]. Notably, STZ is an NO donor and induces the formation of several kinds of ROS (e.g., O 2 −• , H 2 O 2 , OH • , and peroxynitrite; Szkudelski, 2001) as well as DNA alkylation and tricarboxylic citric acid (TCA) cycle inhibition, all of which lead to cell damage and death. Thus, STZ can be used to induce multiple levels of oxidative stress in order to more appropriately mimic that which occurs during diabetes in vivo.
Obviously the oxidative stress observed during diabetes is complex, and the screening of antioxidant compounds cannot be reduced to the use of a single chemical stress. It is therefore important to validate the antioxidant properties of each antioxidant treatment compound, such as the RWPs, using both simple (single) and complex (multiple) oxidative mechanisms. The aim of this study was to create three in vitro models of oxidative stress (one with single radicals produced by HX/XO and two with more complex oxidative reactions using H 2 O 2 and STZ) in order to assess the antioxidant efficiencies of a RWP extract and a purified extract of EGCG.
A stock solution of this RWP extract was prepared by diluting 10 mg/mL in a 1 : 1 mixture of distilled water and 100% ethanol. The EGCG extract was a pure form of green tea Teavigo (DSM Nutritional Product, Gland, Switzerland). To confirm its purity, Teavigo was analysed using chromatographic separation on a octadecylsilyl silica gel LC column (l: 0.125 mm; d: 4; 0 mm; Thermo Scientific, France) with spherical particles. Mobile phase consisted of water : formic acid (0.1%, phase A) and methanol : formic acid (0.1%, phase B). A split system was used allowing the HPLC eluate to enter the MS detector at a flow rate of 0.2 mL/min. The injection volume was 20 L. UV spectral data were acquired at 275 nm (the chromatogram was presented in Supplementary Material available online at http://dx.doi.org/10.1155/2015/859048). The extract was prepared at a 10 mg/mL stock solution concentration which was then diluted in 1× phosphate-buffered saline (PBS), pH 7.4 (Gibco, Invitrogen) as previously described [18]. Cells were cultured for 48 hours before all treatments. The antioxidant compounds (200 to 1000 pg/mM for the EGCG and 100 to 1000 g/mL for the RWPs) were added to cells seeded in 96-well treated microplates (BD Falcon, Franklin Lakes, USA) at 30,000 cells/well and incubated for 1 hour. The toxicity of the EGCG and RWP extracts was then assessed as described below.
Evidence-Based Complementary and Alternative Medicine 3
To evaluate the toxicity of the EGCG and RWP antioxidants alone, various concentrations (EGCG at 200, 500, and 1000 g/mL; RWPs at 10, 50, 100, 150, and 200 g/mL) were added to stressed and unstressed cells and incubated for 1 hour.
MTS Assay.
Cell viability was assessed by measuring the mitochondrial activity with the CellTiter 96 AQueous One Solution Cell Proliferation Assay from Promega Corporation (Madison, USA). After treatment, 100 L of culture medium containing 20 L of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo xymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added. Cells were incubated for 2 hours at 37 ∘ C, in 5% CO 2, and the absorbance was measured at 490 nm with a Metertech 960 microplate reader (Metertech Inc., Taipei, Taiwan). The quantity of the formazan product was directly proportional to the mitochondrial activity related to number of living cells. Results are expressed as the percentage of cell viability compared to the appropriate negative controls.
Caspase 8.
Oxidative stress often affects the cells very quickly, making it difficult to identify the primary apoptotic effects of the ROS. Therefore, the expression and/or activity of initiator caspases, such as caspase 8 [31], are often used to study apoptosis in this context as they reflect the initial effects of oxidative stress. Here, the activity of caspase 8 was determined by flow cytometry using fluorescent inhibitor of activated caspase 8 (caspase 8 FAM; Millipore, Guava Technologies, Hayward, CA). Briefly, cells were seeded in 96well treated microplates (BD Falcon, Franklin Lakes, USA) at 30,000 cells/well. After treatment, fresh medium supplemented with 10 L of caspase inhibitor was added. After mixing, plates were incubated for 1 hour at 37 ∘ C and centrifuged for 5 minutes at 300 ×g. Cells were then resuspended and 200 L of 7-aminoactinomycin D (7 AAD) was added and incubated for 10 minutes at room temperature in the dark before analysis. A Guava Easycyte microcapillary flow cytometer (Millipore) was used with laser excitation at 488 nm in order to detect caspase 8 FAM emission at 517 nm with carboxyfluorescein [6] according to the manufacturer's instructions. For each assay, 200 cellular events were collected. Results were analysed with CytoSoft software (Guava Technologies Inc., Hayward, CA, USA) and are expressed as the percentage of stained cells.
ROS Production.
Cells were seeded in 24-well treated microplates (BD Falcon, Franklin Lakes, USA) at 500,000 cells/well. After treatment, 500 L of fresh medium supplemented with 2 ,7 -dichlorofluorescein diacetate (DCFH-DA; Sigma) at final concentration of 5 M [32] was added. DCFH-DA is deacetylated by a membrane esterase, forming DCFH, which is then transformed by intracellular H 2 O 2 to a fluorescent molecule [28]. Cells were incubated for 1 hour and then trypsinized. After centrifugation during 10 minutes (500 ×g), pellets were dissolved with 500 L PBS and 200 L of the cell solution was transferred to 96-well treated microplates. The suspended cells were then treated with 10 L of propidium iodide [11] for 15 minutes and assayed for fluorescence by flow cytometry at 530 nm. Assays were analyzed with CytoSoft software and the results are expressed as a percentage of ROS production (DCF-labelled cells) compared to the appropriate negative controls.
A 50 g aliquot of the total protein was then separated on 4-12% Bis-Tris Criterion XT Precast Gels (Bio-Rad), transferred to 0.45 m nitrocellulose membranes (Bio-Rad), and detected with the following primary antibodies: anti-catalase produced in mouse (Sigma) or anti-MnSOD produced in rabbit (Sigma), each diluted to 1/1000 e , as well as a 1/5000 e dilution of anti--actin monoclonal antibody produced in mouse. All antibodies were diluted in blocking buffer from the WesternBreeze Chemiluminescent Kit (Invitrogen, Grand Island, USA) overnight at 4 ∘ C. Secondary antibody solution (anti-mouse (1/2000) or anti-rabbit (1/4000) coupled to alkaline phosphatase) was incubated with the membranes for 30 minutes with continuous rotation as described in the kit. Membranes were then exposed with a Bio-Rad Chemi-Doc XRS System for 600 seconds, and the captured images were analysed using Quantity One software. Expression of CAT and MnSOD proteins was normalized to the quantity of -actin and expressed as a percentage compared to the appropriate negative controls.
Statistical Analysis.
Samples were assayed at least three times for each test and the results are given as the mean ± standard error (SEM). Data were analysed with one-way analysis of variance [34] when designated using the Statistica program (Statsoft©, Créteil, France). Treatment differences were subjected to Fischer's test with a 95% significance ( < 0.05) threshold.
Cellular Effects of Various Oxidative Stressors.
We first screened the cell viability following treatment with multiple concentrations of each oxidative molecule to determine the concentration of each that induced RINm5F cell death in at least 50% of the culture (Figure 1). H 2 O 2 appears to induce a significant decrease in cell viability at concentrations above 25 mol/mL ( < 0.01), with the lowest number of viable cells reaching 21.4 ± 0.5% at the 40 mol/mL concentration (Figure 1(a)). Further, a ratio of 0.25 mmol/L of HX and 10 mU/mL of XO was sufficient to induce a significant loss of cell viability, resulting in only 45.9 ± 9.2% of the cells being viable at this ratio ( < 0.01) (Figure 1(b)). Finally, even though the 10 mmol/L concentration of STZ induced a significant decrease in cell viability, leaving only 76.5 ± 0.7% of the cells ( < 0.01), a more marked reduction of cell viability similar to that of the other oxidants was observed at 25 mmol/L, which resulted in only 32 ± 1.6% of the cells remaining alive in the culture ( < 0.01) (Figure 1(c)). Concentrations that induced a loss greater than 50% (25 mol/L of H 2 O 2 , a ratio of 0.25 mmol/L of HX and 10 mU/mL of XO, and 25 mmol/L of STZ) were the sole concentrations used in the subsequent analyses to study the antioxidant properties of the EGCG and RWP extracts.
Antioxidant Toxicity Assessment.
The potential toxicity of each antioxidant extract on the unstressed RINm5F cells was evaluated by measuring the cell viability in the presence of different concentrations of EGCG and RWPs (Figure 2). RWPs appear to have no effect on cell viability until reaching a concentration of 500 g/mL, at which point the number of viable cells drops to 92.4 ± 14.7% (not significant). A dosedependent decreasing trend in cell viability was also observed around this concentration; however, this decrease was not significant until the RWP concentration reached 1000 g/mL ( < 0.05) (Figure 2(a)). In contrast, EGCG was observed to induce a significant increase in cell viability at the 500 g/mL (152 ± 26%, < 0.01) and 1000 g/mL (233.5 ± 13.0%, < 0.01) concentrations (Figure 2(b)).
Effects of EGCG and RWPs during H 2 O 2 -Induced Oxidative Stress.
RWPs were shown to reduce the loss of RINm5F cell viability induced by H 2 O 2 oxidative stress (Figure 3(a)) in a dose-dependent manner starting at a concentration of 50 g/mL (32.4 ± 1.8%, < 0.01) and reaching a maximum level at 200 g/mL (73.9 ± 3.5%, < 0.01), the highest concentration used in this study. Further, 500 g/mL of EGCG was sufficient to significantly improve cell viability after oxidative stress (82.3 ± 6.2%, < 0.01) (Figure 3(b)). This effect again appeared to be dose-dependent, with the cell viability being completely restored and enhanced at an EGCG concentration of 1000 g/mL (110 ± 8.9%, < 0.01).
The antioxidant properties of RWPs and EGCG were also confirmed by measuring apoptosis through the evaluation caspase 8 expression using 150 g/mL of RWPS and 500 g/mL of EGCG. Both antioxidants appear to reduce the significant increase in caspase 8 activation observed during H 2 O 2 -induced oxidative stress back to levels similar to the unstressed control cells ( < 0.01). Surprisingly, the significant increase in ROS production observed during H 2 O 2 oxidative stress was not reduced by the RWP extract; it actually appeared to induce an increase in ROS production, going from 11.1±2.1% to 19.2±1.9% ( < 0.01). On the other hand, EGCG extract significantly reduced ROS production during oxidative stress. In terms of antioxidant enzyme expression during H 2 O 2 -induced oxidative stress, MnSOD (Figure 3(e)) and CAT protein expression (Figure 3(f)) was comparable to that of the unstressed control cells. Further, MnSOD protein expression was significantly reduced by RWPs during oxidative stress ( < 0.05), while CAT protein expression was reduced using EGCG ( < 0.01).
Effects of EGCG and RWPs on HX/XO-Induced
Oxidative Stress. The significant decrease RINm5F cell viability observed during HX/XO-induced oxidative stress was significantly increased when 200 g/mL of RWP was added, but the percentage of viable cells did not exceed 40.2±1.9% ( < 0.01) (Figure 4(a)). The efficiency of EGCG to improve the viability of stress cells was similar to that observed for RWPs at the 200 g/mL concentration (43.1±3.8%), although this was not significant (Figure 4(b)). However, at higher concentrations EGCG was able to significantly increase cell viability from 16.5 ± 2.1% in the stressed control cells to 88.5 ± 7.4% at 500 g/mL ( < 0.01) and over 100% at the 1000 g/mL concentration.
Further, the 150 g/mL and 500 g/mL concentrations of RWPs and EGCG, respectively, were then used to study their antioxidant properties during HX/XO-induced oxidative stress. It appears that these concentrations of RWPs and EGCG significantly reduce caspase 8 cleavage during HX/XO-induced oxidative stress ( < 0.05 and < 0.01, resp.; Figure 4(c)), bringing the caspase 8 activity back down to levels similar to the unstressed control cells. Moreover, both antioxidants significantly reduced the ROS production as well (Figure 4(d)). Notably, only EGCG was observed to increase the protein expression of MnSOD (Figure 4(e)) and CAT (Figure 4(f)) during HX/XO-induced oxidative stress.
Effects of EGCG and RWPs on STZ-Induced Oxidative
Stress. The significant decrease in cell viability during STZinduced oxidative stress was slightly opposed by 200 g/mL of RWPs, which significantly increased the number of viable cells to a mere 50.6±0.5% ( < 0.01). Similarly, a 1000 g/mL concentration of EGCG was needed to significantly increase the cell viability during STZ-induced oxidative stress (60 ± 14%, < 0.05) (Figure 5(b)). For further investigation of the antioxidant properties of the RWPs and EGCG extracts during this type of oxidative stress, we focused on the 200 g/ mL and 1000 g/mL concentrations of RWPs and EGCG, respectively.
Notably, caspase 8 activation was not observed to increase during STZ-induced oxidative stress; the addition of either of the antioxidants did not significantly change these levels compared to the unstressed or stressed control cells (Figure 5(c)). However, the production of ROS was significantly increased during STZ-induced stress and only EGCG treatment caused a significant reduction of this induced ROS production ( < 0.05; Figure 5
Discussion
In the present study, we sought to accurately determine the antioxidant properties of two specific natural compounds, RWPs and EGCG. In doing so, we have demonstrated that antioxidant properties depend not only on the nature of the compound itself, but also on the type of oxidative stress induced. These data show, for the first time, that using one model of oxidative stress is not sufficient to make meaningful conclusions concerning the action of an antioxidant, particularly in regard to disease-related oxidative stress. Complex mechanisms related to glucose autoxidation and hyperinsulinism, which increase ROS production, have been identified in diabetes [5]. Further, the increased level of ROS has been associated with beta cell apoptosis, which, in time, induces insulin dependence [34,35]. In order to decrease beta cell oxidative stress, nutraceutical approaches have been developed focusing on the screening of plant extract [36][37][38]. For the initial screening, the efficiency of the bioactive compounds in the plant is currently validated in vitro using a single type of oxidative stress [27,[39][40][41]. Here, in order to provide a comprehensive overview of ROS generated in diabetes, we have utilized three models of oxidative stress to screen our bioactive compounds. Not surprisingly, the level of cell death induced by each type of oxidative stress studied was similar. However, the level Evidence-Based Complementary and Alternative Medicine 9 of ROS produced in the cells was found to be higher in the HX/XO model compared to the others. In fact, our data show that there is no correlation between ROS production and the level of cell death, indicating that cell viability alone is not an accurate marker of the oxidative stress response in a cell or tissue. This is in contrast to several previous studies that have linked oxidative stress to cell viability [3,26,28]. Notably, the conclusions in these studies were all based on single models of oxidative stress. We chose to measure ROS production in parallel with other markers of the cellular stress response, such as changes in SOD and CAT expression as well as the caspase 8 activity in the cells, in order to evaluate the full effect of the oxidant. According to these results, the cellular defence mechanisms, characterised by SOD and CAT expression, were more activated by the HX/XO-induced stress, which is correlated to the higher level of ROS. Thus, we suggest that antioxidant enzymes likely play a crucial role in cell protection following the introduction of oxidative stress, particularly because the cell viability was comparable to the other conditions as previously described [21,40]. This phenomenon in the HX/XO experiments could also be explained by the type of radical generated. In fact, the stress induced by H 2 O 2 and STZ was more complex (ROS generation via NADPH oxidase activation and/or NO synthase) compared to that generated by HX/XO (direct ROS provider), which may result in the latter inducing the cell's response to stress earlier or more efficiently. Additional work is necessary to further elucidate the differing effects caused by the various free radicals.
Using the three stress models outlined here in conjunction with parallel investigations of ROS generation and cellular defence mechanisms to screen bioactive compounds also provided more detailed information on the cellular stress response. For example, in these experiments, EGCG appeared to be a better antioxidant for the three types of different stresses induced, with a decrease in ROS production and an activation of MnSOD and CAT expression, whereas RWPs were shown to be efficient only on the strongest stress (HX/XO) and even then only had a limited influence on antioxidant enzyme expression. Thus, the use of multiple types of free radical stress indicates that EGCG is likely the most efficient scavenger [26] and pharmacological treatment [37] investigated in this study. This is corroborated by a previous study that demonstrated the neuroprotective effects of EGCG involving inhibition of the Fenton reaction and upregulation of several antioxidant enzymes, such as superoxide dismutase and catalase, resulting in the attenuation of oxidative stress [42]. In contrast, the antioxidant properties of RWPs seem to be solely related to scavenging during severe changes in oxidative stress. Moreover, our study suggests that RWPs may be both antioxidant and prooxidant because we observed an increase of ROS production when RPWs were added during H 2 O 2 oxidative stress. It is well known [43] that polyphenols extract could autoxidize and produce more hydrogen peroxide. In our study, we have demonstrated in this condition that RWPs have no impact on SOD or CAT protein expression but other protective mechanisms could be activated like the stimulation of CAT and SOD activity or a direct action glutathione or thioredoxin. Notably, while we sought to use the same concentration of compound (pure or extract) for both RWPs and EGCG, the composition of these compounds made this impractical. Using HPLC [29], it appears that the raw RWP extract also contains several flavonoids (epicatechin, catechin, and gallic acid) but is found at concentrations that are 100 times less than that of the pure EGCG used in this study. This composition could explain the relatively low efficiency of RWPs shown here. In contrast, the pure EGCG extract was able to activate antioxidant pathways through several mechanisms, which could be due to the high concentration of the flavonoids present.
Regardless of the composition of the compounds, if only one oxidative stress model had been used or only one ROS or cell death assay had been performed, then it is unlikely that the complex nature of these antioxidants would have been uncovered. Therefore, we believe that the sole use of one model or one assay to validate the efficiency of bioactive compounds in the current literature [30,32] greatly limits the scope and conclusions of these studies. Further, a more precise determination of cell death, ideally using additional apoptosis signalling pathways, like additional caspase activation, antiapoptotic Bcl-xL, and proapoptotic Bax expression [27,37], as well as further analysis of oxidation-related enzymes could also be utilized to expand our current analysis. Additional work is necessary to determine the full functionality of our multitype oxidative stress model in testing the antioxidant properties of other compounds.
Conclusion
Here, we have demonstrated that the level of ROS generated by different oxidative stresses was variable and a drug screening using a single kind of stress can introduce bias in the estimation of the antioxidant properties of a compound. Therefore, a combination of several stresses and several cellular and molecular approaches would provide a more accurate experimental text to determine if the compound is an efficient antioxidant. This in vitro model also more accurately mimics the in vivo biological situation, which will help avoid unnecessary spending of money and time when transitioning a bioactive antioxidant treatment from the context of cell culture to clinical validation. Taken together, this study provides a much needed method for the comprehensive assessment of antioxidants for the treatment of oxidative stressrelated diseases. | v3-fos-license |
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} | pes2o/s2orc | Tartaric Acid-Modified Holarrhena antidysenterica and Citrullus colocynthisBiowaste for Efficient Eradication of Crystal Violet Dye from Water
Two novel adsorbents Holarrhena antidysenterica (HA) and Citrullus colocynthis (CC) were collected from native Pakistan and treated with tartaric acid. +e adsorbents were characterized by Fourier-transform infrared spectroscopy (FT-IR) and scanning electron microscopy, and their adsorptive behavior was studied against model cationic dye crystal violet (CV). Role of biosorbent dose, time of contact, temperature studies, agitation rates, and solution pHwas investigated. Optimum conditions obtained for the removal of CV dye for H. antidysenterica-tartaric acid modified (HA-TA) were as follows: 0.8 g adsorbent dose, 35 minutes contact time, 5.0 pH, 40°C temperature, and 150 rpm agitation rates as compared to H. antidysenterica that gave 1.4 g adsorbent dose, 40 minutes time of contact, 6.0 pH, 50°C temperature, and 150 rpm agitation speed. C. colocynthis-tartaric acid modified (CC-TA) removed CV dye at 0.6 g adsorbent dose, 30 minutes contact interval, 4.0 pH, 40°C temperature, and 125 rpm agitation speed in contrast to C. colocynthis which gave 0.8 g adsorbent dose, 40 minutes time of contact, 6.0 pH, 50°C temperature, and 125 rpm agitation speed, respectively. Isothermal studies for both raw and modified biosorbents were compliant with the Langmuir model indicating monolayer, chemisorption. +e maximum Langmuir capacities were up to 128.20mg/g, 136.98mg/g, 144.92mg/g, and 166.66mg/g for HA, CC, HA-TA, and CC-TA. Pseudo-second-order kinetic model well fitted the dye removal data. +e rate-determining steps involved both surface and intraparticle diffusion mechanisms. Adsorption of dye molecules on active surfaces was governed by electrostatic attractions and chelating abilities. +ermodynamics research revealed the spontaneous and exothermic nature of the reaction. +e adsorbents serve promising candidates for the effective removal of hazardous dyes from aqueous solutions.
Introduction
Environment around us is constantly being damaged, due to the incessant release of waste substances, even at minute amounts. Multiple industries such as textile, paints, and plastics contain dyes in their wastewaters. Dyes are chemical compounds that have aromatic rings and high degree of saturation which adhere to any fiber or material and impart characteristic color to it [1][2][3][4]. When discharged into water, they are difficult to degrade and biological entities suffer severe damage due to their structure, such as high mitotic index and chromosomal and nuclear aberrations [5][6][7][8][9].
us, the treatment of wastewaters has been major concerns for centuries. Traditional approaches such as coagulation, sedimentation, ion-exchange, flocculation, reverse osmosis, electrochemical operation, and biological treatments [10] have the delimitations of high energy consumption, expensive equipment, incomplete ion removal, and toxic sludge production [11], and none of them provide complete color removal from wastewater, so constant efforts are made in continuation of the newest approach [12]. Adsorption therefore is a far superior method to all these methods Pakistan, modify with tartaric acid to enhance their sorption capacity, increasing shelf life (b) to characterize the raw and treated adsorbents by different techniques such as FTIR and SEM (c) test for removing CV dye from wastewaters by various adsorption parameters such as dose, agitation speed, time of contact, pH, and temperature studies, along with statistical modelling of isothermal, kinetics, and thermodynamics (d) new theoretical results contributing towards a deeper understanding of the mechanism involved for the adsorption of crystal violet dye on the adsorbents, further enhancing the worth of this work.
Preparation of the Biosorbents.
Biosorbents HA and CC were acquired from the regional marketplace of Lahore, Pakistan. ey were washed first with tap water and then by distilled water. Left over moisture was removed by sun drying. e final adsorbent was grinded into 60 mesh size by using an electric grinder. For chemical modification, the obtained powder was soaked in 10% tartaric acid aqueous solution and then filtered and dried again prior to usage.
Selection of Chemical Treatment Applied for Medication of
Biosorbents. Chemical modification enhances biosorption capacity and increases stability of biosorbents by reducing moisture contents, retarding biological decomposition due to fungal or bacterial attack [39]. Functionality of biosorbent is also enhanced due to uniform distribution of ions. Especially acid modifications yield increased protonation [40,41] and ester linkages [42] while basic treatment provided high micropore percentages [43]. Rapid achievement of equilibrium is attained [44]. To check the best performance, 0.1 M hydrochloric acid, 0.1 M sodium hydroxide, methanol, ethanol, iso-propanol, iso-butanol, acetone, urea [45], thio-urea [46], citric acid [47,48], tartaric acid [49], and ethylene diamine tetra acetic acid (EDTA) [50] were used. Tartaric acid gave the maximum biosorption capacity, so it was selected for further studies, as shown in Figure 1. 5 g of HA and CC was selected and added in aqueous solutions of 25 mL of abovementioned solvents and kept in glass beakers for 4 hours approximately. e containers were completely covered by aluminum sheets. ese solutions were subjected to filtration. After drying, 0.5 g of the resultant biosorbent residue was added to 25 ml of 10 mg/L CV dye solution, for 15 minutes at 100 rpm. Filtration of the resultant solutions helped in determining the final dye concentrations [51].
Batch Adsorption Studies.
Dye standard solutions (5-25 mg/L) were prepared from stock solutions (1000 mg/ L). Batch wise experiments comprised the study of time effects, adsorbent dose, pH studies, temperatures, and agitation rates. Dye concentration was monitored in filtrates using a UV/Vis spectrophotometer at λ max : 590 nm.
For contact time studies, 5 minutes to one hour with a difference of 5 minutes each was selected. 0.4 g of adsorbents was added to 25 mg/L dye solution, revolving at 100 rpm at ambient temperatures. Adsorbent dose was studied by selecting a range of 0.2 g to 2 g of adsorbents, added to 25 mg/L dye solutions, and agitated at 100 rpm at room temperature for 35 minutes.
For pH effects, range of 1-10 was selected. 0.4 g of adsorbents was added to 25 mg/L dye solution and agitated at 100 rpm at room temperature for half hour.
Temperature studies were done at 20-80°C range with 10°C interval. 0.4 g of adsorbents was added to 25 mg/L dye solution and rotated at 100 rpm at room temperature for half hour.
25 rpm to 200 rpm range with 25 rpm interval was selected for agitation speeds study. 0.4 g of adsorbents was added to 25 mg/L dye solution and rotated for different speeds at ambient temperatures for 30 minutes.
For isothermal studies, 10-40 mg/L concentrations of CV dye along with optimum parameter conditions were used. For kinetic studies, 25 mg/L dye solutions was selected at 25°C with all optimized conditions. ermodynamic studies were employed utilizing raw and modified forms with a temperature range of 283-323 K. e experiments were repeated, and percentage adsorption of dye was studied as follows: where C o (mg/L) is the initial CV dye concentration and C e (mg/L) is the adsorption at equilibrium. e amount of dye adsorbed per unit mass is given as follows: where q (mg/g) is the quantity of dye adsorbed by the adsorbent, V (L) is volume of dye solution, and m (g) is the mass of biowaste used [52,53].
Results and Discussion
3.1. FTIR Analysis before and after Adsorption. FTIR spectra for the CV dye adsorption on untreated HA (Figure 2(a)) were obtained, and it ensures the dominance of hydroxyl groups along with carboxylic moieties, on the sites of adsorbent. e peaks at 3904.4 cm −1 to 3482.5 cm −1 are responsible for the presence of -OH (bending), physically adsorbed water molecules [54]. Also, 3011.9 cm −1 represents carboxylic acid moieties. Bands at 2921.8 cm −1 and 2852.1 cm −1 represent asymmetric and symmetric C-H groups, and 2366.2 cm −1 shows CO 2 from the atmosphere. 1740.4 cm −1 shows aldehyde stretch. 1636.3 cm −1 peaks depict doubly bonded carbons of alkene moieties, whereas 1457.6 cm −1 shows aromaticity. Also, 1168.3 cm −1 represents C-N groups. When CV dye on the acid-treated HA was seen ( Figure 2(b)), bands from 3919.5 cm −1 till 3502.5 cm −1 represent the free hydroxyl moieties. Decreased bands were observed at 2922.5 cm −1 and 2852.3 cm −1 for C-H functional groups, indicating the reaction has been occurred. 2359.9 cm −1 shows single bonded alkanes. Alkyne functional groups were depicted by stretching at 1989 cm −1 , while 1789 cm −1 represents alkenes. 1559.2 cm −1 shows aromaticity, while 1718.5 cm −1 represents aldehydic groups. 1363.5 cm −1 represents bending of alkanes.
CC when reacts with CV ( Figure 2(c)) displays wide peaks from 3841 cm −1 to 3328 cm −1 that are responsible for the presence of hydroxyl groups indicating the reaction of the dye with the adsorbent. Stretching at 2183 cm −1 represents double bonds of alkynes. 1636 cm −1 indicates the presence of doubly bonded carbons of alkenes, and peaks at 1419 cm −1 represent C�C of aromatic rings, indicting the presence of CV dye within the adsorbent surfaces. 1705 cm −1 displays -C�O stretch of aldehydes, while 1363 cm −1 shows the presence of -N�O groups. Band at 1029 cm −1 contributes to C-N groups of aliphatic amines. Acid-modified CC adsorbent when reacts with CV dye (Figure 2(d)) displays reduced consecutive peaks from 3902 cm −1 to 3447 cm −1 , showing the presence of freed OH groups which is due to the reaction between dye and adsorbent [19]. Peak at 2132 cm −1 contributes towards unsaturated -C�C bonds of alkyne moieties. 1653 cm −1 stretching responds towards the doubly bonded carbon groups, and 1314.1 cm −1 contributes towards the nitrile stretching CN of aromatic amines. 1363 cm −1 stretch depicts -N�O groups, and 1028 cm −1 confirms the presence of -CN stretch of aliphatic amines. After dye adsorption, bands intensity decreases and elongated bands of -OH widens, confirming the reaction of CV dye with the adsorbents. e presence of oxygen containing functional moieties provides more adsorption surfaces and thus helps in the achievement of greater adsorption of dyes. e nitrogen groups facilitate the eradication of dyes and organic pollutants.
Tartaric acid modification resulted in conversion of round structures to flaky, crumpled, chiseled ones containing numerous voids/cages/channels that provide additional surface sites for CV adsorption (Figure 3(b)). CC displayed large flaky structures with channels between them (Figure 3(c)). Acidic modification resulted in numerous spherical, vertical, agglomerated compact structures, offering many potential sites for dye adsorption (Figure 3(d)). e presence of granular structures of acid moieties, clustered well on surface, is evident of the successful preparation of the adsorbent.
Factors Affecting Adsorption of Dyes on the Biosorbents.
Different factors such as adsorption dosage, pH effect, time of contact, temperature, and agitation rates were employed for the determination of equilibrium parameters for the eradication of CV on to the raw and modified adsorbents.
Impact of Biosorbent Dose.
Biosorbent dose is a significant criterion towards the achievement of biosorption efficiency. At low concentration, maximum adsorption is achieved due to availability of many adsorption sites, but at higher concentration, less saturation of surfaces leads towards a lower dye removal. 0.2-2g dose of adsorbents was selected. For HA, 96% dye removal was observed at 1.4 g after which the dye establishes equilibrium with the adsorbent. e modified HA develops equilibrium at 0.8 g removing 97% of the dye. Figure 4(a) displays the % age removal of the CV dye with the raw and tartaric acidmodified adsorbents. Acid treatment incorporates the addition of oxygen containing functional moieties that chelate the dye molecules increasing the rates of adsorption. CC adsorbent removed CV dye up to 97% at 0.8 g, whereas the acid treated yielded 97.5% dye removal at 0.6 g. Decrease in adsorption after equilibrium is due to decline in amount biosorbed by unit mass of the sorbent, leaving vacant adsorption sites [55].
Impact of Time of Contact.
Time of contact helps in rate determining steps and possible mechanism involved. Adsorption raised up to a certain time until equilibrium is achieved [56]. e decrease in adsorption is due to the repulsion among the adsorbed ions towards the unadsorbed ones. e experiment was allowed to run for a 60-minute time period with a difference of 5 minutes (Figure 4(b)). HA removed 97% CV dye at 40 minutes as compared to tartaric acid-modified form which gave 97% dye removal at 35 minutes. A steady decrease in rate of adsorption was observed after reaching plateau (saturation point) due to complete saturated adsorption surfaces. CC observed 86% dye removal at 40 minutes which raised to 98% at 30 min for the modified form. Modification helped in availability of more active sites which increased the biosorption efficiencies. is increase may be attributed to (a) dye molecules crossing the outer boundary layer and (b) diffusion of CV towards the internal exfoliated porous surface (intraparticle diffusion) along with reduction in viscosities and lower complexation [57][58][59]; however, accumulation of dye fragments over the period of time halts the adsorption at the later stages [37].
Impact of pH.
e effect of pH on solution holds prime importance as the molecules are self-ionizable. Point of zero charge pHpzc is the isoelectronic point where the negative and positive surfaces become equivalent [60,61]. Negative charged surface is formed if the pH is higher than pHpzc due to deprotonation of functional moieties like OHand COO-groups; thus, cationic dyes adsorption occurs; however, if the pH is less than pHpzc, surface becomes positively charged because of protonation [10]. Hence, anionic dyes adsorption occurs. Initial pH is taken at the x- Journal of Chemistry axis, and difference between initial and final (ΔpH) is taken as the y-axis to find the value of pHpzc, as shown in Figure 4(c). It was determined by an electrochemical method [62], where 50 mL of (0.05 M) sodium chloride was added to a series of beakers, pH was adjusted from 2 to 10 by adding appropriate amounts of hydrochloric acid (0.1 M) and sodium hydroxide (0.1 M), followed by addition of 40 mg adsorbent.
e prepared solutions were kept at constant agitation at room temperature for 48 hours [60]. e pHpzc for HA is 6, whereas CC is 5 that infers to a negative surface above this pH and below a positive one.
As seen in Figure 4(d), adsorption capacity was found 97% at pH 6 for HA, whereas CC showed 86% dye removal at pH 6. is is because the interactions between the charged dye ions and adsorbent surfaces control the adsorption capacities. CV + is a cationic dye. As the number of -OH ions increases, solution pH and adsorption capacities of the dye tend to increase. Low eradication of dye in the acidic circumstances was assigned towards the struggle for potential sites, among the cationic part of the dye and the H + ions, therefore contributing an immense feature in the electrostatic mechanism of adsorption method [60,63]. When surface ionized density is low, it provides repulsion in the dye particles and adsorbent sites, thus offering adsorption in acidic environment [64]. Here, CV and excess H + ions compete for adsorption sites, thus lowering adsorption capacity in the basic medium; also, adsorbent surface hydrolysis creates positive charged sites, thus rendering the favorable adsorption of the dye in an acidic medium. Tartaric acid-modified HA gave 98% dye adsorption at pH 5 as compared to modified CC which gave 97% at pH 4.
Impact of Temperature.
Temperature impacts on the biosorption procedure imply whether it is an exothermic or endothermic one [27]. e study was done for the adsorptive removal of CV dye on raw and tartaric acid-treated adsorbents and observed the adsorption decreases along with the increase in temperature, indicating the process might be an exothermic one. is increase is attributed to the increase in movement of the CV dye particles with the rise in kinetic energy and enhanced rates of intraparticle diffusion. Furthermore, increasing temperature brings swelling effect which increases further penetration of the dye molecules. A range of 30°C-80°C was selected for the temperature studies, as seen in Figure 4(e); 91% adsorption at 50°C was observed for HA which increased to 98% at 40°C for treated HA, while CC displayed 94% dye removal at 50°C that raised to 98.7% at 40°C for modified CC.
Impact of Agitation Speed.
e distribution of solutes among the bulk and external boundary films formation is determined by rotating speed. CV reduces the resistance of the external boundary sheet resulting in favored correlation of dyes to that of the biosorbent, thus rising movement. Better adsorption of the dye molecules resulted in the increased external mass transfer coefficient [65]. Agitation speed with a range of 20-200 rpm was checked for CV dye removal on raw and tartaric acid-modified forms, and 89% adsorption efficiency was observed at 150 rpm for HA that elevated to 93% for modified HA, whereas CC gave 84% dye removal at 125 rpm that increased to 96% for modified CC, as shown in Figure 4(f ).
Isothermal Studies.
A relationship between adsorbate and sorbent was established to fully comprehend the environment of adsorption process and reaction mechanism by utilizing isothermal models. ese are Langmuir, Freundlich, Temkin, and the D-R isotherms. e relationships among the equilibrium bulk concentrations and masses of adsorbate per unit weight of adsorbent is given by the Langmuir model. Also, it says that adsorption takes place homogeneously on the adsorption sites [66,67]. e linear model of the Langmuir isotherm is given as follows: where C e is the liquid phased concentrations of the CV dye at the equilibrium stage (mg·L −1 ), q max is the monolayer sorption capacity of the adsorbents (mg g −1 ), q e is the concentration of dye adsorbed on the biosorbent at equilibrium (mg·g −1 ), and b represents energy of biosorption (L·g −1 ). A plot of 1/C e vs. 1/q e gives a straight line for this isotherm (Figure 5(a)). e dimensionless equilibrium parameter R L which is also used for evaluating the possibility of the biosorption process is obtained by employing the underlying equation: where C o is the highest initially dye concentrations (mg·g −1 ) and K L is the Langmuir adsorption constant (L·mg −1 ).
As seen in Table 1, Langmuir removal capacities q max for biosorption of CV dye on the raw and modified biosorbents displayed higher values for the modified form as compared to the raw ones, indicating that the acid-treated biosorbents display better adsorption capacity for CV dye due to the availability of more binding sites of their masses and also increase in functional groups, as depicted by the FTIR spectrum which favors the adsorption of dye. Q max was found to be highest for CC-TA among the four adsorbents. R 2 values were highest in Langmuir as compared to Freundlich, Temkin, and D-R isotherms indicating homogeneous, chemisorption with no side reactions [66]. Positive b values are clearly indicative of the appreciable affinities of the biosorbents towards the removal of CV dye. R L values were less than 1, displaying a favorable adsorption procedure.
Heterogeneous surfaces in multilayer adsorption along with nonuniform distribution of heat of surface adsorption are presumed by Freundlich [68]. Its linear form is represented as follows: where K F is the Freundlich constant which relates towards biosorption capacity and n is the biosorption strength factor, in which 1-10 value is indicative of favorable biosorption, n > 2, n < 1, and n � 0-1 are representative of better, average, and good adsorptions [69], and 1/n is the constant related to intensity of biosorption or heterogeneity factor. 1/n < 1 displays Freundlich isotherm, whereas 1/n > 1 indicates cooperative biosorption [66]. Graphical plots of log qe vs. log Ce give a straight line from which values of n and K F are determined from the slope and intercept of the graphical plot ( Figure 5(b)). When checked for Freundlich Isotherm as in Table 1, it was dawned upon that R 2 values were lesser [70]. Linear form of this isotherm is given as where A T �equilibrium binding constant related to maximum binding energy (L·g −1 ) and B T � Temkin isotherm constant relating to adsorption heat (J·mol −1 ) [71]. Physical or chemical interactions among the dyes and adsorbent are given by B T values, where <8 shows physical nature while >8 is representative of chemical interactions [72,73]. Less than 8 values of B T , as shown in Table 1, depicts the weak and physical forces between the dye molecule and the adsorbents. Graph between q and ln C e gives the desired results ( Figure 5(c)). Tartaric acid offered strong binding forces for the dye molecules exhibiting an increase in the B T for the modified forms in comparison to raw biosorbents. Unsatisfactory fitting of the Temkin model is observed due to less R 2 values than the Langmuir and Freundlich ones.
Dubinin-Radushkevich isothermic model [74] represents biosorption mechanism with Gaussian energy distributions onto heterogeneous surfaces. It is only appropriate for intermediate ranges of biosorbate concentration. e model describes pore filling mechanism and consists of semiempirical equation that qualitatively describes the biosorption of gases and vapours on microporous sorbents [75]. It assumes a multilayer character for physical adsorption involving Van der Waal's forces. Linearly, it is shown as following equations: q e is the CV adsorbed on adsorbents, C e is the concentration of CV at equilibrium, q m (mg·g −1 ) is the Dubinin-Radushkevich constant related to degree of adsorption. B D (mol 2 ·kJ −2 ) is the free energies of CV dye that travel from infinite distance towards the sorbent, and ε is the Polanyi potential. Mean free energy, E (kJ/mol), is calculated as follows: Value of E also inferences about adsorption mechanism where E < 8 kJ·mol −1 represents physisorption, while 8 < E < 16 kJ·mol −1 shows ion-exchange and E > 16 kJ·mol −1 displays chemisorption process [76].
In Table 1, R 2 values were found to be less than those of Langmuir isotherm, and values of q m were found to be increasing for the modified forms which might be because of the increase in possible spots as a result of acid treatment. E values gave results lesser than 8 inferring the biosorption procedure to be physiosorption. Figure 5(d) displays the comparative adsorption for D-R isotherm.
Kinetic Study.
e rate and mechanism of the reaction can be studied from kinetic modelling to ensure a competent and swift biosorption procedure for commercial purposes. For this purpose, popular kinetic models like Elovich, Lagergren pseudo-first-order kinetics, and Lagergren pseudo-second-order kinetics are employed. Quantitative checking can also be done by using percent relative deviation (P), as follows: where q e(exp) is the experimented biosorption capability (mg/ g), q e(cal) is the biosorption capacities obtained after calculations by utilizing kinetic models(mg/g), and N is the total number of observations [77]. e Elovich model is based on the principal that adsorption increases exponentially with adsorption sites implying multilayered adsorption [78]. Chemisorption of gas onto solids was first described by this equation [79]. Linear form of the Elovich equation is shown as follows [80]: where qt (mg/g) is the concentration of dye adsorbed at any time t, α (g/mg min) is the initial rate of sorption, and b (g/ mg) is linked to number of potential sites covering the surfaces. Higher α values represent chemisorption. For this model, maximum adsorption capacity along with Elovich constants can be calculated from slope and intercept of graph of ln t versus qt. Table 2 represents the Elovich parameters obtained for raw and modified adsorbents, and Figure 6(a) displays graphs obtained after adsorption. Lagergren pseudo-first-order kinetic model was established upon the assumption that adsorption is equivalent to the amount of freely bonded surfaces [81,82].
It is shown as follows: ln q e − q t � ln q e − k 1 t(pseudo − first − order), (12) where K 1 (h −1 ) is the rate constant and q t (mg·g −1 ) represents the quantity of CV dye taken at any instant t while q e (mg g −1 ) shows the quantity of CV dye taken at the equilibrium stage. A larger variance (P) amidst the experimented and calculated q e values renders the first-order ineffective.
Negative P values show that q e calculated was higher than q e experimental. Moreover, lesser values of correlation coefficient R 2 implement the unsatisfactory fittings of the pseudo-first-order model, as evident in Table 2 and Figure 6(b).
Journal of Chemistry
Ho's model [83,84] is supported by the presumption that rate of reaction is equivalent to dye concentration, and the square of binding locations present on the biosorbent is shown as in the following equation: where k 2 (g g h −1 ) is the rate constant for pseudo-secondorder. Also, q e (mg/g) is the uptake capacity of CV dye at the equilibrium stage and q t (mg/g) is the uptake capacity of CV dye at a given instant t. e high value of R 2 and lower relative percentage deviation (P%) as compared to first-order kinetics indicates higher fitness of the pseudo-second-order model to the kinetics data. Moreover, q e(cal) is closest to that of q e(exp) , suggesting adsorption process to be chemisorption [85]. Initial rate of adsorption (h) (mg/g·min) is Product of uptake capacity and rate constant give the half-life (t 1/2 ) of the adsorption process, and the time in which process is halved, as given in the following equation: It was seen that h values of the HA-TA and CC-TA displayed an increment in the initial sorption rate than unmodified ones, while half-life of modified adsorbents was lower than untreated ones [43,86]. Table 2 represents the desired results along with Figure 6(c).
For understanding of the mechanism of the biosorption process, intraparticle diffusion model was studied [82,87], which is shown by Weber and Morris, as follows: e coefficient of intraparticle diffusion is represented by k id , and·C (mg g −1 ) is the boundary layer effect. If the plot of q t vs. t 1/2 is a straight line and pass through the origin, then the intraparticle diffusion is the mechanism; otherwise, some other mechanisms are involved such as film diffusion [88]. Dual nature was observed for the plots for diffusion mechanism with the (a) first to be a curved line contributing towards boundary layer (surface diffusion) (b) and the straight line in the later stages inferring towards intraparticle diffusion (Figures 5(d) and 5(e)). is leads towards complex mechanism of the CV dye adsorption [19], as clear from Table 2. Not even one of the graphical plots passed through the origin; hence, it is concluded intraparticle diffusion model was not the complete mechanism involved [89].
Boyd proposed another mechanism for predicting the mechanism of the biosorption process which is based upon the fractional achievement of equilibrium with variance of time [90]. e following equation shows where Boyd's constant is represented by B b and the fractional achievement of equilibrium q t /q e is shown by F at any instant t. is equation is simplified as follows: If a graph with a straight line passes through the origin, then the mechanism involved will be the Webber-Morris plot; otherwise, boundary layer diffusion will be the mechanism involved (Figures 5(f ) and 5(g)). R 2 was compared in Table 2 and was seen that the graphs exhibited not only intraparticle diffusion behavior but also film or boundary layer as the rate determining step for the removal of CV dye on the raw and tartaric acid-modified biosorbents [10].
Nonlinear Fashion of Equilibrium and Kinetic Modelling.
Nonlinear equations were employed to check the validation of the equilibrium data in the linear mode. Nonlinear equations of Langmuir, Freundlich, Temkin, and Dubnin-Radouskevich [91] are shown in the following equations: q e � q m exp −βε 2 .
Root mean square error (RMSE) values [92] were calculated using the following formula: Lesser value of RMSE as evident in Table 2 indicates the fitness of experimental to the calculated data.
From the alterations of linear equations, nonlinear equations of Elovich, pseudo-first-order, and pseudo-second-order forms [93] are obtained (equations (24)-(26)): where q t (mg/g) is the uptaking capacities of CV at any time t, q e (mg/g) is the equilibrium binding capacities, α (g/mg min) is the initial rate of adsorption, and b (g/mg) is the number of active sites. k 1 (h −1 ) and k 2 (g g h −1 ) are the firstand second-order rate constants, respectively. RMSE results were calculated to investigate the most fitted of the kinetic models, comparing the experimental vs. calculated values, by using the following equation: where q t(cal) (mg·g −1 ) is the calculated binding capacity, q t(exp) (mg·g −1 ) is the experimental binding capacity at any instant t, and N is the number of observations. e lowest RMSE values are seen for those of second order hence making it most appropriate ( Table 2).
3.7.
ermodynamic Studies. Temperature modifications affect the dye removal process as it instantly changes the kinetic energy involved in the procedure; increment in temperature results in raised diffusion in the lignocellulosic structures [94]. Vant Hoff's equation is given as follows: ΔG°� ΔH°− TΔS°.
(28)
A graphical plot was commenced between lnK D versus 1/ T ( Figure 6). Negative values of ΔH°and positive ΔS° [95] show exothermic reaction and increase in arbitrariness of the system [96,97], as clear from Table 3. Moreover, a spontaneous and favorable adsorption procedure at 283 K-323 K temperatures is shown by negative values of Gibbs energy (ΔG°) [98]. e magnitude of K d representing distribution coefficient [93] rises with temperature implying that temperature raise increases biosorption. More surface area, expansion of sizes of pores, and activated biosorbent surfaces are responsible for the greater sorption efficiency of the CV dye-modified adsorbents, as compared to raw ones [99][100][101].
Mechanism of Adsorption.
To understand the adsorption mechanism of the CV dye, the surface of adsorbent is considered which consists of cellulosic moieties mainly consisting of hydroxyl and carboxyl groups, as seen in the FT-IR analysis. When the adsorbent interacts with acidic groups of tartaric acid, it interacts with the functional groups, thus leading towards increment in acidity of the adsorbent which further helps to interact with the adsorption process. e cationic dye in solution dissociates into CV + and Cl − groups that adhere on the surface by interacting with hydrogen bonding with the carboxyl and hydroxyl functionalities ( Figure 7). Overall, the adsorption of CV on the adsorbents involves a monolayer procedure including spontaneous occupation of available active surfaces (bulk diffusion) followed by penetration of the adsorbate on the microspores from surface films (pore and intraparticle diffusion steps) and attaining equilibrium stage at the last step (Collins and Elijah, 2019 [102]). Although the Langmuir and pseudo-second-order imply this procedure to be a chemisorption but isotherms such as Temkin and D-R pose it to be a physical one, adsorption of CV dye on raw and modified novel Holarrhena antidysenterica and Citrullus colocynthis is concluded to be a physicochemical one and its maximum adsorption capacity is comparable with reported sorbing materials, as clear from Table 4.
Conclusion
us, utilization of H. antidysenterica and C. colocynthis for effective eradication of CV dye from wastewaters is considered very effective as it provides the maximum removal efficiencies both in raw and modified forms. Tartaric acid provides the availability of more acidic functional groups such as hydroxyl and carbonyl as seen by FT-IR and SEM analyses. Batch adsorption studies confirmed the modified forms to be better adsorbing than the nonmodified ones. Isothermal studies were performed using Langmuir, Freundlich, Temkin, and D-R models indicating the Langmuir isotherm to be most promising due to highest regression coefficients hence proving monolayer adsorption. q max (mg g −1 ) for the HA was 128.2051 mg·g −1 that increased up to 144.9275 mg·g −1 for the tartaric acid forms while CC gave 136.9863 mg·g −1 that raised to 166.6667 mg·g −1 for the modified ones. Pseudo-second-order kinetics were followed by all the adsorbents. ermodynamics proved the adsorption procedure to be an exothermic and spontaneous process due to negative values of ΔH and ΔG°. e mechanism involves both bulk and pore diffusion by the adhering dye molecules to the adsorbent via hydrogen bonding and electrostatic interactions.
us, it is a physicochemical biosorption procedure.
Data Availability
All data related to this work are presented in Results along with references.
Conflicts of Interest
e authors have no conflicts of interest regarding publication of this paper. | v3-fos-license |
2018-10-10T18:45:39.555Z | 2018-10-10T00:00:00.000 | 52948570 | {
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} | pes2o/s2orc | Mitochondrial Morphology, Function and Homeostasis Are Impaired by Expression of an N-terminal Calpain Cleavage Fragment of Ataxin-3
Alterations in mitochondrial morphology and function have been linked to neurodegenerative diseases, including Parkinson disease, Alzheimer disease and Huntington disease. Metabolic defects, resulting from dysfunctional mitochondria, have been reported in patients and respective animal models of all those diseases. Spinocerebellar Ataxia Type 3 (SCA3), another neurodegenerative disorder, also presents with metabolic defects and loss of body weight in early disease stages although the possible role of mitochondrial dysfunction in SCA3 pathology is still to be determined. Interestingly, the SCA3 disease protein ataxin-3, which is predominantly localized in cytoplasm and nucleus, has also been associated with mitochondria in both its mutant and wildtype form. This observation provides an interesting link to a potential mitochondrial involvement of mutant ataxin-3 in SCA3 pathogenesis. Furthermore, proteolytic cleavage of ataxin-3 has been shown to produce toxic fragments and even overexpression of artificially truncated forms of ataxin-3 resulted in mitochondria deficits. Therefore, we analyzed the repercussions of expressing a naturally occurring N-terminal cleavage fragment of ataxin-3 and the influence of an endogenous expression of the S256 cleavage fragment in vitro and in vivo. In our study, expression of a fragment derived from calpain cleavage induced mitochondrial fragmentation and cristae alterations leading to a significantly decreased capacity of mitochondrial respiration and contributing to an increased susceptibility to apoptosis. Furthermore, analyzing mitophagy revealed activation of autophagy in the early pathogenesis with reduced lysosomal activity. In conclusion, our findings indicate that cleavage of ataxin-3 by calpains results in fragments which interfere with mitochondrial function and mitochondrial degradation processes.
Alterations in mitochondrial morphology and function have been linked to neurodegenerative diseases, including Parkinson disease, Alzheimer disease and Huntington disease. Metabolic defects, resulting from dysfunctional mitochondria, have been reported in patients and respective animal models of all those diseases. Spinocerebellar Ataxia Type 3 (SCA3), another neurodegenerative disorder, also presents with metabolic defects and loss of body weight in early disease stages although the possible role of mitochondrial dysfunction in SCA3 pathology is still to be determined. Interestingly, the SCA3 disease protein ataxin-3, which is predominantly localized in cytoplasm and nucleus, has also been associated with mitochondria in both its mutant and wildtype form. This observation provides an interesting link to a potential mitochondrial involvement of mutant ataxin-3 in SCA3 pathogenesis. Furthermore, proteolytic cleavage of ataxin-3 has been shown to produce toxic fragments and even overexpression of artificially truncated forms of ataxin-3 resulted in mitochondria deficits. Therefore, we analyzed the repercussions of expressing a naturally occurring N-terminal cleavage fragment of ataxin-3 and the influence of an endogenous expression of the S256 cleavage fragment in vitro and in vivo. In our study, expression of a fragment derived from calpain cleavage induced mitochondrial fragmentation and cristae alterations leading to a significantly decreased capacity of mitochondrial respiration and contributing to an increased susceptibility to apoptosis. Furthermore, analyzing mitophagy revealed activation of autophagy in the early pathogenesis with reduced lysosomal activity. In conclusion, our findings indicate that cleavage of ataxin-3 by calpains results in fragments which interfere with mitochondrial function and mitochondrial degradation processes.
INTRODUCTION
Generation of free radicals and deficiencies in energy supply, calcium buffering, or regulation of apoptosis contribute to a progressive decline of the central nervous system in aging and neurodegeneration (Mandemakers et al., 2007). Mitochondrial dysfunction, which can account for all these problems, is commonly involved in neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD), and Huntington disease (HD) (reviewed in de Moura et al., 2010). However, only little is known about the impact of mitochondria on the pathogenesis of Spinocerebellar Ataxia Type 3 (SCA3). SCA3, also known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited late-onset progressive neurodegenerative disorder which is caused by an expanded CAG repeat and belongs to the group of polyglutamine repeat diseases (reviewed in Riess et al., 2008). The altered protein, ataxin-3, is expressed ubiquitously with strong expression in the central nervous system (Costa et al., 2004). Normally, ataxin-3 localizes in the cytoplasm but in SCA3 patients' aberrant ataxin-3 also aggregates in the nucleus of a specific subset of neurons. Both, wildtype and polyglutamine-expanded ataxin-3, are also associated with mitochondria (Pozzi et al., 2008;Kristensen et al., 2018). Whether ataxin-3 binds directly to mitochondria or indirectly through interaction partners remains elusive. One possible interaction partner linking ataxin-3 to mitochondria is Parkin, a PD-associated E3 ubiquitin ligase, which is recruited to dysfunctional mitochondria, ubiquitinates outer membrane proteins and targets mitochondria for degradation under stress conditions (Narendra et al., 2008;Durcan et al., 2011). Recent mass spectrometry analyses revealed several new mitochondrial proteins as confirmed or potential interaction partners of wildtype and/ or mutant ataxin-3, including cytochrome C oxidase subunit NDUFA4 (NDUFA4), succinate dehydrogenase (ubiquinone) iron-sulfur subunit (SDHB) and cytochrome C oxidase assembly factor 7 (COA7) (Kristensen et al., 2018). Mitochondrial DNA (mtDNA) deletions were frequently found in SCA3 transgenic mice as well as in most SCA3 patients and were more pronounced in the preclinical stage but not present in healthy individuals or SCA3 mutation carriers (Yu et al., 2009;Kazachkova et al., 2013;Ramos et al., 2015;Raposo et al., 2018). Additionally, the protein mitochondrial genome maintenance exonuclease 1 (MGME1) which is linked to mitochondrial DNA repair was found enriched in ataxin-3 overexpressing HEK293 cells by mass spectrometry analysis. Based on the same methodology, succinate dehydrogenase complex, subunit A (SDHA) and SDHB which are constitute parts of the complex-II of the electron transport chain, were identified as interaction partners of ataxin-3 and therefore, may explain the reduced complex-II activity in SCA3 patient lymphoblast cell lines and cerebellar granule cells from transgenic SCA3 mice (Laço et al., 2012;Kristensen et al., 2018).
It is unclear, if these described alterations are directly linked to full-length wildtype and/ or mutant ataxin-3 or to N-and C-terminal fragments generated by calpain cleavage during the pathogenesis. It was previously shown that the mitochondrial ubiquitin ligase (MITOL) promotes the degradation of N-terminally truncated polyglutamineexpanded ataxin-3 via the ubiquitin-proteasome pathway, attenuating mitochondrial accumulation of the pathogenic ataxin-3 (Sugiura et al., 2011). New insights into the influence of an N-terminally truncated polyglutamine-expanded ataxin-3 fragment to mitochondrial function and health indicate that this truncated form is directly causing increased mitochondrial fission, decreased mitochondrial membrane potential, increased reactive oxygen species and finally increased cell death rates (Hsu et al., 2017). Hsu and coworkers used an artificial N-terminally truncated polyglutamine-expanded ataxin-3 fragment beginning at amino acid position 163 which should represent a truncated fragment derived from cleavage at amino acid position 186 (Haacke et al., 2006). We have published a genetrap mouse model which expresses endogenously a C-terminally truncated mouse ataxin-3 fragment from amino acid 1 to amino acid 259 without harboring the polyglutamine tract (Hübener et al., 2011). Surprisingly, homozygous genetrap mice developed an SCA3 reminiscent neurological phenotype including tremor, weight loss, coordination and balance deficits subsequently leading to premature death which was associated with cytoplasmic aggregate formation, cell death and abnormal response to ER stress (Hübener et al., 2011). Furthermore, our recent study using mass spectrometry demonstrated that ataxin-3 is cleaved by calpains at four positions H187, D208, S256, and G259, and that cleavage at position D208 and S256 demonstrated the most likely pathogenic cleavage fragments in human disease material (Weber et al., 2017). Here, we used mouse embryonic fibroblasts (MEF) generated from the described genetrap mouse model and brain samples from homozygous genetrap mice to analyze the influence of an endogenously expressed N-terminal ataxin-3 fragment (Atx3 1−259 ) whose C-terminus is very closely located to an important calpain cleavage site (S256) to study mitochondrial function, biogenesis and mitochondrial mitophagy. The endogenous expression of the truncated N-terminal fragment (Atx3 1−259 ) in the murine background resulted in mitochondrial fragmentation and cristae alterations. This was accompanied by a significantly decreased capacity of mitochondrial respiration and decreased mitochondrial membrane potential contributing to an increased susceptibility to apoptosis. Our results indicate that cleavage of full-length ataxin-3 into N-and C-terminal fragments causes important molecular alterations and leads to dysfunctional mitochondria and biogenesis.
Ethics Statement
This study was carried out in strict accordance with the recommendations presented in the Guide for Care and Use of Laboratory Animals of the University of Tübingen, Germany. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tübingen, Germany.
Animal Model
The generation and characterization of the ataxin-3 C-terminally truncated (Atx3 1−259 ) mouse model used in this study have been described previously (Hübener et al., 2011(Hübener et al., , 2012. All animals were housed under standard conditions with a 12 h light-dark cycle, food and water ad libitum. For genotyping, primers and conditions were used as described in Hübener et al. (2011). All experiments with mouse brain samples were performed using wildtype, heterozygous and homozygous Atx3 1−259 mutant mice, experiments with mouse embryonic fibroblasts were only done using wildtype and homozygous Atx3 1−259 mutant genotypes.
Cell Culture and Transfection
Mouse embryonic fibroblasts (MEF) with wildtype or Atx3 1−259 homozygous mutant genotypes were generated from day 12.5 to day 14.5 mouse embryos as previously described (Hübener et al., 2011). Shortly, uterine horns were removed, and each embryo was separated from placenta and surrounding membranes. After removing head and internal organs (used for genotyping), the rest of the embryo was minced into small fragments that were seeded into 25 cm 2 cell culture dishes. All cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco R , Thermo Fisher Scientific) in the presence of 1% penicillin/streptomycin and 10% fetal bovine serum (both Invitrogen) at 37 • C and 5% CO 2 . For all analyses only MEF with a passage number lower than 10 were used. The passage number of wildtype and Atx3 1−259 MEF was identical for all experiments. To analyze endoplasmic reticulum stress, cells were treated with 1 µg/ml tunicamycin (Sigma-Aldrich) for 24 and 48 h.
Neuronal Differentiation of Induced Pluripotent Stem Cells
Human fibroblasts from a SCA3 patient (female, 70 CAG repeats in the expanded allele) and a sex-matched health subject were obtained by skin biopsy and reprogrammed to induced pluripotent stem cells (iPSCs) as described in Hayer et al. (2018). Differentiation of iPSCs to iPSC-derived cortical neurons (iCNs) was done as described previously (Shi et al., 2012;Weber et al., 2017).
Cell-Based Calpain Activation Assay
Activation of endogenous calpains in iCNs was performed as previously described (Weber et al., 2017). Briefly, calpains were activated by incubating cells with 1 µM of the Ca 2+ ionophore ionomycin (Sigma-Aldrich) and 5 mM CaCl 2 diluted in Opti MEM R I Reduced Serum Media (Gibco R , Thermo Fisher Scientific) for 1 h at 37 • C in 5% CO 2 . For negative controls, cells were pre-treated with 10 µM of the calpain inhibitor CI III (carbobenzoxy-valinyl-phenylalaninal) (Merck Millipore) in Opti MEM R I Reduced Serum Media.
Immunofluorescence Microscopy
Mitochondrial morphology and number of lysosomes were analyzed by live cell microscopy. For this, MEF were cultured in Lab-Tek R II chambered coverglasses (Nalge Nunc International). Lysosomes were stained with 50 nM LysoTracker R Red DND-99 and mitochondria with 200 mM MitoTracker R Green FM or MitoTracker R red FM (all Invitrogen) for 15 min at 37 • C. Analysis of the number of lysosomes and/ or mitochondrial morphology was performed using an inverted Zeiss Axiovert microscope (Zeiss Plan-Apochromat 63 x/1.4) with an incubation chamber temperature of 37 • C. Time lapse pictures were taken with an AxioCamMRm camera (Zeiss) in 5 s intervals for up to 5 min using AxioVision software (Zeiss). The series of pictures was saved uncompressed and analyzed with AxioVision software (Zeiss). Colocalization studies, MEF cells were incubated with 250 nm MitoTracker R red FM for 30 min and afterward fixed with 4% paraformaldehyde for 20 min. After blocking of fixed cells with 10% normal serum, cells were stained with cytochrome C (1:500, Cell Signaling) overnight and the secondary antibody anti-Cy2 (1:300, Jackson ImmunoResearch) for 2 h. DAPI (Invitrogen) was used to stain nuclei. Images were analyzed by Zeiss LSM 510 Confocal Microscope and Zeiss LSM Image Examiner LSM510.
Quantification of Mitochondrial Morphology
Fluorescence microscopy images were converted to grayscale, inverted to show mitochondria-specific fluorescence as black pixels and the threshold was adjusted to optimally resolve individual mitochondria. Using Image J 1.41o software with a plugin to analyze mitochondrial morphology (Dagda et al., 2009), each single mitochondrion was analyzed for morphological characteristics such as area, perimeter, circularity and major/ minor axis. On the basis of these parameters, the form factor [perimeter 2 /(4π × area)] and the aspect ratio (ratio between the major and minor axes of the ellipse equivalent to the object) were calculated representing mitochondrial elongation and interconnectivity (adapted from Krebiehl et al., 2010).
Electron Microscopy
Electron microscopy of MEF was performed as previously described (Lundkvist et al., 2004) and followed the description in Hübener et al. (2011).
Respirometry
Mitochondrial respiration was measured under continuous magnetic stirring either in intact or in homogenized MEF (wildtype and homozygous Atx3 1−259 cells) with a Clarktype oxygen electrode using a high resolution OROBOROS oxygraph (Haller et al., 1994). For homogenization, MEF were spun down and the pellet was gently homogenized with mild strength by hand for 1 min in cold HBSS buffer (3 × 10 6 cells/200 µl HBSS consistent of 132 mM NaCl, 5.4 mM KCl, 0.44 mM KH 2 PO 4 , 0.34 mM NaH 2 PO 4 , 0.49 mM MgCl 2 , 0.41 mM MgSO 4 , 10 mM HEPES, 1 mM CaCl 2 , 10 mM pyruvate, pH 7.3). Mitochondrial respiration of intact cells (final concentration 2 × 10 6 cells/ml) was measured at 37 • C in HBSS buffer. Oligomycin (5 µg/ ml), an inhibitor of F 0 F 1 ATPase and FCCP (100 nM), an uncoupler of oxidative phosphorylation were added in the course of the experiment (Gizatullina et al., 2003). Furthermore, mitochondrial respiration in cell homogenates (final concentration 3 × 10 6 cells/ ml) was measured at 30 • C in MMMPK buffer (containing 5 mM MgCl 2 , 120 mM mannitol, 40 mM MOPS, 5 mM KH 2 PO 4 , 60 mM KCl, pH 7.4) by means of multiple substrate inhibitors protocol as described previously (Deschauer et al., 2006) with 10 mM glutamate, 2 mM malate, 1.5 µM rotenone, 10 mM succinate, and 5 µM CAT. Compounds were added with the help of Hamilton syringes through a small hole in the cover of the chamber. The oxygen concentration in the air-saturated medium was determined to be 200 nmol O 2 /ml at 95 kPa barometric pressure. The weight-specific oxygen consumption was calculated from the time derivative of the oxygen concentration (DATGRAPH Analysis software, OROBOROS). Each experiment was repeated eight times independently.
Mitochondrial Membrane Potential Assessment by Fluorescence-Activated Cell Sorting
Mitochondrial membrane potential (MMP) of MEF generated from homozygous Atx3 1−259 or wildtype mice was determined by a FACS-based method. For this purpose, 150,000 cells per genotype were seeded as triplicates 48 h prior to performing the FACS measurements. As negative control, cells were treated with 0.5 µM staurosporine for 4 h. Cells were harvested with 2 mM EDTA in phosphate-buffered saline (PBS) and washed twice with PBS. To measure the MMP cells were stained with 200 nM tetramethylrhodamine methyl ester (TMRM, Invitrogen) in PBS for 30 min at 37 • C in the dark. After two more washing steps with PBS cells were counted in the fluorescence activated cell sorter (FACS). At least 50,000 cells per sample were analyzed on a CyAnTM ADP apparatus (Beckman Coulter) using the 488 nm argon laser and the PE emission filter (575 nm). Experiments were performed five independent times.
Quantitative Real-Time PCR
For the detection of differentially regulated genes, RNA was isolated from whole mouse brain using the RNeasy Midi Kit or from MEF using the RNeasy Mini Kit (both Qiagen). RNA quality was validated using an RNA 6000 NanoChip (Agilent Technologies) and complementary DNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was performed with the QuantiTect SYBR green PCR Kit (Qiagen) using the LightCycler R 480 system (Roche Applied Science).
Standard curves of each amplified gene were created to calculate PCR efficiency. Hydroxymethylbilane synthase (Hmbs), pyruvate dehydrogenase (lipoamide) beta (Pdhb), succinate dehydrogenase complex, subunit A (Sdha), TATA box binding protein (Tbp), and tyrosine 3-monooxygenase/tryptophan 5monooxygenase activation protein zeta polypeptide (Ywhaz) were analyzed as reference/ housekeeping genes and were used for normalization. The C P -values of the reference and the target genes were obtained with the LightCycler Software 1.5.0 SP3 (version 1.5.0.39). The relative expression levels of all genes were calculated using the mathematical model of Pfaffl (2001). Primer pairs used for polymerase chain reaction amplification are listed in Supplementary Table S1.
Detection of Cell Death Rate
Cell death was analyzed in MEF by the Cell Death ELISA (Roche Applied Science) assay, which determines histoneassociated DNA fragments, according to manufacturer's instruction.
Statistical Analyses
All data were analyzed using Prism 6.0 software, GraphPad. Standard two-way ANOVA (data not matched) to assess the effects of genotype and age and Bonferroni post hoc tests were conducted to compare individual genotype effects at individual ages. Data are presented as mean ± SEM. Differences were considered significant if p ≤ 0.05.
Proteolytic Cleavage of Ataxin-3 in Human Cortical Neurons Leads to Formation of a Specific N-terminal Fragment
As previously reported, ataxin-3 is cleaved by a group of Ca 2+ -activated endogenous proteases, called calpains. Cleavage at mainly two cleavage sites occasionally leads to the formation of several N-terminal and C-terminal fragments under physiological conditions (Weber et al., 2017). To reinvestigate and confirm this condition, iPSC-derived cortical neurons, generated from an SCA3 patient and a healthy control, were treated with the Ca 2+ -ionophore ionomycin to activate calpains by increasing the intracellular Ca 2+ levels. Calpain activation by ionomycin led to the reduction of wildtype and polyglutamine-expanded full-length ataxin-3 and the predominant accumulation of an N-terminal, polyglutaminenon-containing fragment ( Figure 1A), which has recently been identified as an N-terminal breakdown product resulting from cleavage at the amino acid position S256 (Weber et al., 2017). The fragmentation can be also found in whole brain lysates of wildtype mice and wildtype MEF under unstimulated baseline conditions (Figures 1B,C). Interestingly, this N-terminal fragment spanning from amino acid 1-256 (Atx3 1−256 ) ( Figure 1D) is only three amino acids shorter at its C-terminal end than the N-terminal fragment (Atx3 1−259 ) generated by the genetrap approach in our previously published mouse model ( Figure 1E). Also of note is, that whole brain lysates of homozygous Atx3 1−259 mice did not represent the cleavage fragment S256/G259 at around 35 kDa. However, the fusion protein of the respective cleavage fragment and β-galactosidase (generation construct of genetrap mice) can be detected at 110 kDa ( Figure 1B). Moreover, the N-terminus of ataxin-3 is highly conserved in Homo sapiens (reference isoform, NP_004984.2) and Mus musculus (isoform 2, NP_083981.2) featuring a completely identical amino acid sequence from amino acid position 1-181, thus including the catalytic Josephin domain as well as the two known nuclear export signals (NES) (compare Figures 1D,E). This indicates that the function of the Josephin domain as deubiquitinating enzyme and the calpain cleavage at aa S256 as well as the nuclear export signals (NES) at aa 77-99 and aa 141-158 in Mus musculus can be functional active like in humans. Therefore, our mouse model expressing endogenously the murine ataxin-3 fragment Atx3 1−259 is an excellent model to study the influence of the calpain cleavage at aa S256 and the resulting N-terminal fragment and therefore, the link to SCA3 neuropathology independently from the glutamine stretch in vivo.
Mitochondrial Fragmentation and Cristae Disruption Result in Complex-I and -II Dysfunction
Mitochondrial morphological changes were investigated in mouse embryonic fibroblasts (MEF) from wildtype and homozygous Atx3 1−259 mutant (Atx3 1−259 Homo) mice by live cell imaging. Significant differences in mitochondrial morphology were found in Atx3 1−259 homozygous MEF compared to wildtype MEF (Figures 2A-D). Mitochondria of Atx3 1−259 homozygous MEF displayed disintegration of the mitochondrial network and a reduced branching (Figures 2C,D) which eventually resulted in disruption of the tubular network compared to wildtype cells (Figures 2A,B). Using ImageJ software to quantify mitochondrial branching (form factor, Figure 2G, * p = 0.006) and length (aspect ratio, Figure 2H, * * p = 0.001), which both serve as morphological parameters, revealed significantly shorter and less branched mitochondria in Atx3 1−259 homozygous MEF compared to wildtype MEF. Similar results were obtained by transfecting wildtype MEF with an ataxin-3 specific fragment. The hereby obtained cells remodeled the genetrap mouse-derived Atx3 1−259 MEF without comprising the C-terminal β-galactosidase component but an EGFP tag. This experiment confirmed that ataxin-3 itself and not the remaining components of the β-galactosidase element influences mitochondrial morphology. Additionally, co-transfection of wildtype MEF with full-length ataxin-3 constructs expressing different polyglutamine lengths (15Q, 70Q, 147Q) revealed fragmented mitochondria in Atx3-fl-70Q and Atx3-fl-147Q compared to wildtype cells expressing a non-expanded ataxin-3 with 15 glutamines (Supplementary Figure S1). Electron microscopy showed that the observed increased fragmentation in Atx3 1−259 homozygous MEF was accompanied by a loss of the double-membrane structure that resulted in a derangement of the inner mitochondrial membrane cristae structure (Figures 2E,F).
As disruption of the tubular and dynamic mitochondrial network can result in loss of mitochondrial function, mitochondrial oxygen consumption as measure for mitochondrial respiration was investigated in wildtype and Atx3 1−259 homozygous MEF. Normal basal respiration was observed in both wildtype and Atx3 1−259 homozygous cells. Addition of oligomycin, an ATPase-synthase inhibitor, allowed measuring non-phosphorylating respiration (state-4 olig ), which is also similar in both genotypes. Titration with FCCP typically leads to uncoupled mitochondrial respiration followed by respiration inhibition at higher FCCP concentrations.
Stepwise addition of FCCP demonstrated significantly decreased FCCP-dependent respiration in intact Atx3 1−259 homozygous MEF ( Figure 2I). Statistically, the maximum of uncoupled rates (87.0 ± 23.8 nmol O 2 /min/2 × 10 6 cells) was already observed at 400 nM FCCP concentration and was about 35% lower in Atx3 1−259 homozygous MEF compared to wildtype MEF (Figure 2I and Supplementary Table S2). Hence, also the ratio of maximum uncoupled respiration and state-4 olig respiration, a measure for respiratory control (RC), was 25% decreased in Atx3 1−259 homozygous MEF compared to wildtype MEF (Supplementary Table S2). Inhibition of uncoupled respiration was observed in Atx3 1−259 homozygous MEF at a FCCP concentration of 600 nM compared to 800 nM in wildtype MEF (Supplementary Table S2). These results demonstrated that mitochondria of Atx3 1−259 homozygous MEF have a significantly lower respiratory capacity and are more prone to respiratory uncoupling.
FIGURE 1 | Representation of one of the known calpain cleavage sites at amino acid position S256 in ataxin-3 and the respective breakdown product detected in cortical neurons differentiated from patient and control-derived cell lines as well as in mouse brain and in murine embryonic fibroblasts. (A) Western blot analysis of ataxin-3 fragmentation in cortical neurons (iCNs) differentiated from patient-derived iPSCs revealed an N-terminal fragment resulting from calpain cleavage at amino acid S256 (center antibody) after ionomycin (IM) treatment. This effect was abolished by pretreating the cells with calpain inhibitor III (CI III). β-actin is shown as loading control. (B,C) Western blot analysis of ataxin-3 fragmentation pattern in wildtype MEF (C) and wildtype and Atx3 1-259 whole brain lysates reveals a breakdown product, derived from cleavage at amino acid position 256 of full-length wildtype ataxin-3 under non-stimulated conditions. In homozygous Atx3 1-259 whole brain lysates no respective cleavage fragment could be detected, most likely as the fusion protein (β-gal-Atx3 1-259 is a combination of a cleavage fragment at amino acid position 259 and β-galactosidase) is not further proteolytically processed. As loading control, α-tubulin is shown. (D) Schematic illustration of full-length human ataxin-3 (reference isoform, NP_004984.2, Atx3-fl) with a previously identified calpain cleavage site at amino acid position S256, and the localization of the nuclear export (NES) and nuclear import signal (NLS). Cleavage at amino acid S256 lead to an N-terminal fragment (bdp 1-256 ), which contains only NES but not the NLS and the polyglutamine tract. (E) Schematic illustration of the analyzed murine C-terminally truncated ataxin-3 at amino acid position G259 and the location of the identified calpain cleavage site S256. wt, wildtype; eg, endogenous; ex, expanded; fl, full-length; bdp, breakdown product; β-gal-Atx3 1-259 , fusion protein from β-galactosidase and the Atx3 1−259 (genetrap approach to generate the mice).
To address whether complex-I (Com-I) or complex-II (Com-II) of the respiratory chain are specifically affected in Atx3 1−259 homozygous MEF, cell homogenates were investigated by a special substrate inhibitor titration protocol (Deschauer et al., 2006). Hereby, maximum oxidative phosphorylation (state-3 pyr respiration) was observed in the presence of complex-Ispecific substrates (pyruvate, malate and ADP). Addition of cytochrome C during state-3 respiration subsequently allowed assessing the integrity of the outer mitochondrial membrane. Com-I-dependent state-3 respiration was then inhibited by 1.5 µM rotenone and succinate was added to adjust Com-II-dependent oxidative phosphorylation (state-3 suc ). The rate of state-4 cat respiration was measured after inhibiting the ADP/ATP-translocator by carboxyatractyloside. These above described measurements of mitochondrial respiration in cell homogenates represented significantly lower rates of Com-I and Com-II-dependent state-3 respiration in Atx3 1−259 homozygous MEF compared to wildtype MEF (Figure 2J, for Com-I p = 0.008; Com-II p = 0.033). The ratio of state-3 to state-4 respiration, called mitochondrial respiratory control index (RCI), which indicates the tightness of coupling between respiration and phosphorylation, was significantly lower in Atx3 1−259 homozygous MEF compared to wildtype controls (wildtype: RCI I = 4.3 and RCI II = 2.8; Atx3 1−259 Homo: RCI I = 3.6 and RCI II = 2.4). The detected significant reductions of the mass-independent ratios RCI and RC show that decreased capacities of oxidative phosphorylation and, hence, mitochondrial impairment are caused by functional changes and not simply by decreased number of mitochondria.
Abnormalities in Mitochondrial Morphology and Function Are Not Mainly Caused by Differential Expression of Fission and Fusion Proteins
To understand the molecular basis of the observed fragmentation, the levels of mitochondrial-shaping proteins were measured. No changes in pro-fusion (Mfn-1, Mfn-2, Opa1) FIGURE 2 | Fragmented mitochondria with disrupted cristae structure and decreased uncoupled respiration in an ataxin-3 mouse model expressing C-terminally truncated ataxin-3 at amino acid position G259 (Atx3 1−259 ). (A-D) Mitochondrial morphology in living Atx3 1−259 homozygous and wildtype MEF was analyzed by life cell imaging microscopy (Cell Observer Z1, Zeiss, Germany) at 37 • C using ApoTome R optical slices with 0.300-0.350 µm z-stacks. Mitochondria were stained with MitoTracker R green FM and analyzed for area, perimeter, major and minor axis and circularity using Image J 1.41o software. Immunofluorescence analyses revealed a disruption of the tubular and dynamic network of mitochondria in homozygous Atx3 1−259 MEF (C,D) compared to wildtype MEF (A,B). (E,F) Electron microscopy showed that increased fragmentation was accompanied by a derangement of mitochondrial cristae structure. (G,H) Quantification of mitochondrial morphological changes in homozygous Atx3 1−259 MEF revealed a significantly reduced degree of mitochondrial branching (form factor, G, * p = 0.006) and length (aspect ratio, H, * * p = 0.001) compared to wildtype MEF. Scale bar indicates 20 µm (immunofluorescence) or 0.5 µm (electron microscopy). Representative pictures from 3 independent experiments are shown, in total 45 cells were analyzed. (I) Mitochondrial oxygen consumption measured in freshly harvested homozygous Atx3 1−259 and wildtype MEF in Hank's medium containing 10 mM pyruvate. Normal endogenous respiration was observed in both wildtype and Atx3 1−259 MEF without addition of further supplements. Oligomycin-resistant respiration was followed in the presence of 5 µg/ml oligomycin. Stepwise addition of 100 nM FCCP revealed a one third lower activity and less stability against the uncoupler (FCCP) in homozygous Atx3 1−259 MEF. Starting from the fourth FCCP addition significantly different uncoupled respiration could be observed in Atx3 1−259 MEF compared to wildtype MEF ( 4 p = 0.044; 5 p = 0.026, 6 p = 0.024, 7 p = 0.018, 8 p = 0.012, 9 p = 0.011; n = 6). (J) Complex-I and Complex-II function was analyzed by respiratory measurements in cell homogenates of homozygous Atx3 1−259 and wildtype MEF using different substrate inhibitors. Measurements were performed for 3 × 10 6 cells/200 µl in MMMPK buffer with addition of 2 nM ADP, 8 µM cytochrome C (Cyt C), 1.5 µM rotenone (R), 10 mM succinate (S) and 5 µM carboxyatractilate (CAT). Blue line represents oxygen concentration in the oxygraph versus reaction time (left ordinate); red line indicates the first derivative of the oxygen time curve, directly indicating the rate of respiration (right ordinate). This analysis revealed significantly decreased complex-I and complex-II-dependent respiration rates in Atx3 1−259 homozygous MEF compared to wildtype controls. Typical respirograms from eight independent experiments are shown (Com-I p = 0.008; Com-II p = 0.033).
or pro-fission (Fis1, Dnm1l) protein levels were noticed in whole brain lysates of 12 months old phenotypical Atx3 1−259 homozygous mice (Figures 3A,B) or Atx3 1−259 homozygous MEF (Figures 3C,D). However, densitometric quantification revealed significantly increased protein levels of the pro-fission protein Dnm1l in Atx3 1−259 homozygous mice (p = 0.028, Figure 3B). Similar results were also found for mRNA levels of the fission and fusion genes in brain lysates of 12 months old phenotypical Atx3 1−259 homozygous mice compared to wildtype controls (Supplementary Figure S2A). Slight mRNA reduction was observed for the fusion proteins Mfn-2 and Opa1 and the fission protein Dnm1l in Atx3 1−259 homozygous MEF compared to wildtype (Supplementary Figure S2B). Moreover, no differences in protein levels of several subunits of the respiratory chain, including complex II-SDHB subunit 30 kDa, complex III subunit Core 2, complex IV subunit I and ATP synthase subunit α (complex V) were found in whole brain lysates of 3 and 12 months old Atx3 1−259 homozygous mice FIGURE 3 | Abnormalities in mitochondrial morphology are not mainly caused by differential expression of fission and fusion proteins. (A,C) Western blot analyses in whole brain lysates of 12 months old mice (3 animals per genotype; A,B) or MEF lysates (one sample per genotype; C,D) represent an equal expression of fission (Dnm1l and Fis1) and fusion proteins (Mfn-1, Mfn-2 and Opa1). β-actin is shown as loading control. (B,D) Densitometric quantification revealed significantly increased Dnm1l protein levels in mice homozygous for truncated Atx3 1-259 compared to wildtype (p = 0.028). Figures 3A-D), respectively. This indicates that the above described deficiencies in mitochondrial respiration are not caused by altered expression of respiratory chain proteins.
Changes in Mitochondrial Morphology and Function Are Linked to Reduced Mitochondrial Membrane Integrity and Higher Rates of Cell Death
Mitochondrial membrane potential (MMP), a measure for healthy and active mitochondria, was investigated by TMRM staining and subsequent fluorescence-activated cell sorting (FACS). The experiment revealed a significant reduction of MMP in Atx3 1−259 homozygous MEF compared to wildtype MEF (Figure 4A, * * p < 0.01), which is not caused by differential protein expression of members of the mitochondrial permeability transition pore, namely Ant2 and Vdac1 (Figures 4G,H). The changes in mitochondrial membrane potential may explain the significantly increased cell death rate observed in Atx3 1−259 homozygous MEF ( Figure 4B, * * p < 0.01). Similar results were found in whole brain lysates of phenotypic Atx3 1−259 homozygous mice, where the level of free cytochrome C, an indicator for mitochondrial-associated apoptosis, was significantly increased compared to age-matched wildtypes (Figures 4C,D, * p < 0.05). Co-immunofluorescence staining of cytochrome C and mitochondria in MEF demonstrated fragmented mitochondria in Atx3 1−259 homozygous MEF compared to wildtype MEF. Moreover, a clear colocalization of mitochondria and cytochrome C was detected in both genotypes (Figures 4E,F). FIGURE 4 | Reduced mitochondrial membrane integrity and higher rate of apoptosis were found in homozygous Atx3 1-259 MEF. (A) Mitochondrial membrane potential (MMP), a marker for mitochondrial membrane integrity, was determined in MEF by TMRM treatment for 30 min at 37 • C and subsequent FACS analyses. In five independent experiments a significant reduction of MMP was found in homozygous Atx3 1-259 MEF compared to wildtype cells ( * * p < 0.01). (B) Measuring the cell death rate using a cell death-detection kit revealed significantly more apoptosis in homozygous Atx3 1-259 MEF compared to wildtype MEF ( * * p < 0.01, n = 3). (C,D) As indicator for mitochondrial-associated apoptosis in whole brain lysates of heterozygous and homozygous Atx3 1-259 mice, the protein level of free cytochrome C was measured in three mice per genotype at the age of 12 months. Densitometric quantification revealed significantly more free cytochrome C in heterozygous and homozygous Atx3 1-259 mice compared to wildtype controls ( * p < 0.05). (E,F) Immunofluorescence staining of cytochrome C in MEF cells revealed a disrupted mitochondrial network in Atx3 1-259 homozygous MEF and a good overlap of cytochrome C with mitochondria in both genotypes. (G,H) Analyzing protein levels of members of the mitochondrial permeability transition pore (Ant2, Vdac1) in whole brain lysates of 3 mice per genotype also did not identify differences in protein expression at the age of 12 months, which was confirmed by densitometry quantification (G). β-actin is shown as loading control.
FIGURE 5 | Expression levels of the transcriptional coactivator PGC1α, a regulator of mitochondrial biogenesis, are slightly reduced in homozygous Atx3 1-259 mice. (A) mRNA expression levels of full-length PGC1α and N-terminally truncated PGC1α (NT-PGC1α) are reduced in homozygous Atx3 1-259 whole brain lysates at the age of 12 months. But statistical analyses revealed that only the differences in mRNA level of N-terminally truncated PGC1α reached significance (n = 3; wildtype to Atx3 1-258 _PGC1α p = 0.16; wildtype to Atx3 1-259 _NT-PGC1α * p = 0.032). (B) Protein analyses in the same mice (3 per genotype, 12 months of age) demonstrated less full-length PGC1α and similar levels of N-terminally truncated PGC1α in homozygous Atx3 1-259 mice compared to wildtype mice. (C,D) Densitometry quantification revealed no significant differences for the levels of full-length (p = 0.081) and N-terminally truncated PGC1α (p = 0.932) protein comparing wildtype and homozygous Atx3 1-259 mice. Significant differences were only found for full-length PGC1α protein levels in homozygous Atx3 1-259 mice compared to heterozygous Atx3 1-259 mice ( * * p = 0.0025, C).
Additionally, analyses of PGC1α, a master regulator of mitochondrial biogenesis, demonstrated slightly decreased mRNA and protein levels in whole brain lysates of Atx3 1−259 homozygous mice compared to wildtype controls (Figures 5A-D). It is controversially discussed in the field whether the full-length or the N-terminally truncated form of PGC1α represents the most promising factor to analyze mitochondrial biogenesis (Cui et al., 2006;Weydt et al., 2006;Johri et al., 2011). In our study, full-length PGC1α showed the strongest downregulation in Atx3 1−259 homozygous mice compared to wildtype animals. However, densitometric quantification did not reach statistical significance due to the high variability among wildtype animals (Figures 5A-D, p = 0.081).
Ataxin-3 Fragmentation Influences Autophagy
As described earlier, mitochondrial dynamics and mitochondrial membrane potential are linked to autophagy and lysosomal impairment. Depolarized mitochondria, which are a substrate for mitophagy, are a consequence of increased mitochondrial fission or decreased mitochondrial fusion. Indeed, mitochondria found in autophagosomes show decreased fusion capability and reduced MMP (Twig and Shirihai, 2011).
To analyze the impact of autophagy on the degradation of fragmented mitochondria found in Atx3 1−259 homozygous MEF, mRNA and protein expression levels of important autophagic genes/ proteins were measured in MEF and whole brain lysates. mRNA levels of Atg5, Becn1 and LC3 (Map1lc3a) were significantly higher in whole brain lysates of Atx3 1−259 homozygous mice compared to wildtype controls at the age of 3 months but not at the age of 12 months ( Figure 6A, * p < 0.05, * * p < 0.01). On protein level, the amount of LC3 II was slightly but not significantly increased in Atx3 1−259 homozygous mice at the age of 3 months, and was not affected in 12 months old animals (Figures 6C,D, p = 0.107). Furthermore, levels of the autophagy receptor protein p62/SQSTM1 in whole brain lysates were not altered between genotypes at 3 and 12 months (Supplementary Figure S4). Similarly, Atg5 protein levels (shown as Atg5-Atg12 conjugate) were slightly higher in Atx3 1−259 homozygous compared to wildtype mice in 3 months old ( * p = 0.047) but not in 12 months old animals (Figures 6E,F). Analyzing the mRNA level of antiapoptotic Bcl2 and pro-apoptotic Bax revealed an upregulation of both in 3 months old homozygous mice. However, Bax was highly expressed in brain lysates of 12 months old Atx3 1−259 homozygous mice and, therefore, indicate an activation of apoptotic pathways (Figure 6B, * p < 0.05). Combining these data with the increased susceptibility to endoplasmic reticulum stress and ribosomal dissociation described for phenotypic ataxin-3 Atx3 1−259 homozygous mice in a previous study (Hübener et al., 2011), indicates that autophagy plays a role in the early disease pathogenesis, whereas the chronic stress at late disease stages is triggered by the ER stress response system. To analyze this effect in detail, wildtype and Atx3 1−259 homozygous MEF were co-transfected with pDsRed-ER and pEGFP-LC3 vectors and mild ER stress was induced by tunicamycin treatment (1 µg/ml) for 24 and 48 h. Both wildtype and Atx3 1−259 homozygous MEF revealed comparable numbers of autophagosomes under untreated conditions. After 24 h of tunicamycin treatment, both genotypes produced significantly more autophagosomes ( * * p < 0.01). Additionally, Atx3 1−259 homozygous MEF showed ER swelling and "bubble-like" structures as previously observed (Hübener et al., 2011). Treatment for 48 h resulted in autophagosome numbers, which were comparable to numbers observed in untreated cells, in both genotypes. However, ER swelling was constantly visible in Atx3 1−259 homozygous MEF but was never observed in treated wildtype cells (Figures 7A,B). Cells transfected either with pDsRed-ER or pGFP-LC3 and treated with mild ER stress for 48 h demonstrated similar results as shown before by double transfection experiments ( Figure 7C). ER swelling was also observed in wildtype MEF transfected with polyglutamine-expanded pEGFP-N1 ataxin-3 70Q and 147Q after 48 h of tunicamycin treatment. This was never observed in tunicamycin-treated MEFs transfected with non-expanded ataxin-3 (15Q) or under unstressed conditions (Supplementary Figure S5). In conclusion, inducing mild ER stress showed that wildtype cells react to misfolded/ unfolded proteins within the ER by activating the autophagic machinery and thereby maintain normal cell homeostasis. In contrast, overactivation of the autophagy in Atx3 1−259 homozygous MEF prevented efficient refolding of unfolded/misfolded proteins, which finally resulted in accumulation of unfolded proteins within the ER after 24 h of tunicamycin treatment.
To further support our data on autophagy, lysosomes were analyzed in Atx3 1−259 homozygous MEF and Atx3 1−259 homozygous mice. Counterstaining of MEF with MitoTracker green and LysoTracker red demonstrated significantly fewer lysosomes in Atx3 1−259 homozygous MEF compared to wildtype MEF ( Figure 8A) that were also less dynamic (data not shown). Similar results were found by Western blot analysis of the lysosomal marker protein Lamp1, which revealed no Lamp1 protein expression in Atx3 1−259 homozygous MEF ( Figure 8B) and less in 12 months old phenotypic Atx3 1−259 homozygous mice (Figures 8C-E) compared to respective wildtype controls.
In summary, an N-terminal ataxin-3 fragment (Atx3 1−259 ) similar to a fragment produced by calpain cleavage (Atx3 1−256 ) in human SCA3 patient neurons disrupts mitochondrial function independently from the polyglutamine stretch. Altogether, the above-mentioned results indicate that full-length ataxin-3 has indeed a role in mitochondrial function, biogenesis and homeostasis. Additionally, ataxin-3 fragmentation plays a role in the autophagic lysosomal degradation systems due to misregulation of important autophagic and lysosomal proteins especially at earlier disease stages (Figure 9).
DISCUSSION
Neurons are characterized by particularly high-energy demands to maintain extensive physiological functions. ATP production in neurons mainly relies on functional mitochondria as they cannot switch to glycolysis if oxidative phosphorylation is impaired. Therefore, metabolic defects and loss of body weight, which have been linked to mitochondrial dysfunction, are well described symptoms in neurodegenerative diseases including polyglutamine disorders like HD and SCA3 (reviewed in Weber et al., 2014). Understanding this link between neurodegenerative diseases and mitochondria may pave the way for future therapeutic interventions in neurodegenerative disorders. Both wildtype and polyglutamine-expanded ataxin-3, the disease-causing protein in SCA3, were shown to interact with mitochondria (Pozzi et al., 2008;Kristensen et al., FIGURE 6 | Autophagy is activated in 3 months old but not 12 months old homozygous Atx3 1-259 mice. (A,B) Quantitative real-time PCR revealed differences in expression levels of important autophagic genes and the counter player Bax and Bcl2 in homozygous Atx3 1-259 whole brain lysates. (A) In whole brain lysates of 3 months old homozygous Atx3 1-259 mice, significantly higher Atg5 and Becn1 mRNA levels ( * * p < 0.01) and Map1lc3a mRNA levels ( * p < 0.05) were found. No changes were found for Mtor mRNA level. Analyzing the same mRNAs at the age of 12 months revealed only a slight upregulation of Atg5 and Mtor ( * p < 0.05) and no changes in Becn1 and Map1lc3a expression in whole brain lysates of three homozygous Atx3 1-259 mice compared to wildtype controls. (B) Analyzing the mRNA level of pro-apoptotic Bax and anti-apoptotic Bcl2 revealed a upregulation of both in 3 months old homozygous Atx3 1-259 mice and an overactivation of Bax in 12 months old Atx3 1-259 homozygous brain lysates ( * p = 0.047). (C-F) Protein expression analyses of Map1lc3a (LC3; C,D) and Atg5 (E,F) demonstrated a slightly increased Atg5 protein level ( * p = 0.047) and no differences for the LC3 II protein levels (p = 0.149) in 3 months old homozygous Atx3 1-259 mice. Comparable to mRNA levels, no differences were found for Atg5 and LC3 protein expression at the age of 12 months comparing 3 animals per genotype. β-actin is shown as loading control.
2018). Up to date, it is unclear, if full-length wildtype and/ or polyglutamine-expanded ataxin-3 or N-and C-terminal fragments generated by proteolytic cleavage events during the pathogenesis are responsible for the observed impaired mitochondrial function and homeostasis. In the last years it was demonstrated that truncated forms and cleavage fragments of ataxin-3 resulted in elevated cytotoxicity and higher aggregation propensity, which in the end lead to neurodegeneration (Ikeda et al., 1996;Haacke et al., 2006;Weber et al., 2017). Last year we identified four calpain FIGURE 7 | Mild ER stress leads to early autophagy activation but still results in accumulation of unfolded proteins within the ER in homozygous Atx3 1-259 cells.
(A) Living wildtype and homozygous Atx3 1-259 MEF were transfected with pDsRed-ER and pEGFP-LC3 vectors and treated with 1 µg/ml tunicamycin (Tm) for 24 and 48 h. Wildtype cells reacted with producing more autophagosomes after 24 h of treatment and came back to normal steady-state autophagy levels after 48 h of treatment. The ER was not affected. In comparison, homozygous Atx3 1-259 MEF also started with producing more autophagosomes after 24 h of treatment but at the same time point unfolded proteins started to accumulate within the ER. This accumulation resulted in "bubble-like" DsRed-positive structures found in homozygous Atx3 1-259 MEF. After 48 h of treatment the accumulation of ER proteins was constantly going on but the number of autophagosomes resembled the number of autophagosomes detected under untreated conditions. Bar indicates 20 µm, represented pictures of three independent experiments is shown. (B) Counting the number of autophagosomes in 50 cells from three independent experiments revealed a significantly higher number of autophagosomes in 24 h Tm-treated wildtype and homozygous Atx3 1−259 cells compared to untreated cells and cells, which were treated for 48 h with tunicamycin. Additionally, the number of autophagosomes was significantly higher in homozygous Atx3 1−259 MEF compared to wildtype cells after 24 h of treatment ( * p < 0.05, * * p < 0.01). (C) As a control experiment, cells were transfected with either pDsRed-ER or pEGFP-LC3 and treated with 1 µg/ml tunicamycin for 48 h. Comparable results were seen as demonstrated for double-transfected wildtype and homozygous Atx3 1-259 cells. Scale bar indicates 20 µm. cleavage sites in wildtype and polyglutamine-expanded ataxin-3 at amino acid positions H187, D208, S256, and G259 by mass spectroscopy and confirmed the importance of cleavage in position D208 and S256 for human SCA3 pathology (Weber et al., 2017). Hsu et al. (2017) overexpressed an artificial N-terminally truncated wildtype and polyglutamineexpanded ataxin-3 fragment, spanning from amino acid 163 to the proteins C-terminal end, and analyzed its involvement in mitochondrial function. They showed that the ataxin-3 fragment is directly causing increased mitochondrial fission, a decreased mitochondrial membrane potential, increased reactive oxygen species and finally increased cell death rates (Hsu et al., 2017). We generated and characterized an ataxin-3 mutant mouse model using the genetrap approach. This mouse model endogenously expresses C-terminally truncated murine ataxin-3, which ends at the recently identified calpain cleavage site G259 and, moreover, resembles a prominent cleavage fragment of human SCA3 pathology resulting from calpain cleavage at amino acid position S256 (Hübener et al., 2011;Weber et al., 2017). Although the endogenous murine ataxin-3 misses the polyglutamine repeat, mice expressing the truncated protein develop neurological symptoms with gait FIGURE 8 | Less lysosomes and abolished expression of lysosomal protein Lamp1 in MEF and slightly reduced expression in whole brain lysates of homozygous Atx3 1-259 mice. (A) Living wildtype and homozygous Atx3 1-259 MEF were stained with LysoTracker red and MitoTracker green FM and analyzed by life cell microscopy at 37 • C using ApoTome R optical slides with 0.300-0.350 z-stacks. Immunofluorescence microscopy demonstrated less lysosomes in homozygous Atx3 1-259 MEF compared to wildtype controls (15 cells from 3 independent experiments). Bar indicates 10 µm. (B) Analyzing the expression level of an important lysosomal protein, namely Lamp1, revealed no Lamp1 expression in homozygous Atx3 1-259 MEF compared to wildtype controls. Sec61b and coomassie staining are shown as loading control. (C,D) Western blot analyses of whole brain lysates of 3 mice per genotype demonstrated no Lamp1 expression differences at the age of 3 months and a reduced Lamp1 level in homozygous Atx3 1−259 mice compared to wildtype mice at the age of 12 months, without reaching statistical significance in densitometric quantification (E; p = 0.06).
ataxia, coordination and balance problems, tremor, weight loss, and premature death. Regarding molecular neuropathology, the model is characterized by neuronal cytoplasmic inclusion bodies, neuronal cell death and increased susceptibility to endoplasmic reticulum stress including ribosomal dislocation (Hübener et al., 2011). To study mitochondrial (dys)function and autophagic impairment, brain lysates and MEFs obtained from wildtype and homozygous Atx3 1−259 mutant mice were analyzed. However, there is knowledge that fibroblast cultures are not able to mimic the brain region-specific patterns of morphological alterations in MJD. Due to their nature, these primary cells are unable to form neuronal structures as synapses and as synaptic mitochondria are known to behave differently from non-synaptic mitochondria, respective effects cannot be investigated in this model (Lai et al., 1977;Brown et al., 2006). Nevertheless, MEF isolated from animal models of neurodegenerative disorders are often used as ex vivo model to study the influence of the mutation without the need of an artificial overexpression. Additionally and despite their limitations, fibroblast cultured from human disease patients and the respective animal models are often used along with biomaterials isolated from patients/ animal models as these cells are easy to generate and to handle (Auburger et al., 2012;Lei, 2013).
FIGURE 9 | Interplay of mitochondrial homeostasis and mitophagy as well as apoptosis in the pathogenesis of the C-terminally truncated Atx3 1-259 mouse model. Our results demonstrated that at early disease stages (3 months) the autophagic machinery is activated shown by increased expression of Atg5, Becn1, LC3 and the anti-apoptotic protein Bcl2. After disease onset an impaired mitochondrial homeostasis (increased expression of pro-fission protein Dnm1l, mitochondrial fragmentation, cristae remodeling, reduced mitochondrial membrane integrity) and biogenesis (decreased PGC1α expression) accompanied by an induced apoptosis (increased Bax expression, more cytochrome C release) were found. Red label indicate increased expression, green label demonstrate decreased expression.
Additional to the fact that in vivo expression of the N-terminal ataxin-3 cleavage fragment lacking the polyglutamine stretch led to a phenotype reminiscent of SCA3 pathology, we found in the present study comparable mitochondrial deficits as earlier shown for N-terminally truncated ataxin-3 fragments with expanded polyglutamine tract (Hsu et al., 2017). Both studies demonstrated fragmented mitochondria, changes in fission and fusion, decreased mitochondrial membrane potential and higher cell death rate. Additionally, we found an impaired mitophagy with an activation of autophagy only in early pathogenesis combined with a reduced lysosomal activity. Changes in mitochondrial morphology are already linked to pathogenesis of other polyglutamine diseases, especially shown for HD. Here, HeLa cells overexpressing polyglutamine-expanded huntingtin as well as lymphoblast's of HD patients revealed fragmented mitochondria and disrupted cristae structure (Costa et al., 2010). Costa and colleagues demonstrated that mitochondrial fragmentation is associated with increased activity of the pro-fission protein Dnm1l, which is regulated by the calcium-dependent phosphatase calcineurin (Costa et al., 2010). Additionally, a direct interaction of mutant huntingtin and Dnm1l was shown, which influences the GTPase activity of Dnm1l (Song et al., 2011;Shirendeb et al., 2012). Similar to these results, we found fragmented mitochondria and a disrupted cristae structure in our C-terminally truncated Atx3 1−259 homozygous mice. Additionally, the pro-fission protein Dnm1l was significantly increased at the protein level in Atx 1−259 homozygous mice, whereas the other mitochondria shaping proteins were not differentially regulated. So far, our study showed that ataxin-3 cleavage fragments influence mitochondrial morphology and function independent of its polyglutamine tract.
In order to learn whether ataxin-3 fragmentation also influences mitochondrial biogenesis, the expression of PGC1α, peroxisome proliferator-activated receptor-γ coactivator 1α, a master regulator of energy homeostasis and mitochondrial biogenesis, was investigated. Differential expression of PGC1α is known to influence mitochondrial dysfunction in HD, but previous studies also linked regulation of PGC1α activity to general aging and other neurodegenerative diseases including AD and PD (reviewed in Austin and St-Pierre, 2012). As ataxin-3 can regulate nuclear gene expression (Evert et al., 2006), mRNA as well as protein levels of full-length and N-terminally truncated PGC1α were analyzed in this study. Whether fulllength or N-terminally truncated PGC1α affects mitochondrial biogenesis is still under discussion (Cui et al., 2006;Weydt et al., 2006;Johri et al., 2011). We found significantly reduced mRNA levels of N-terminally truncated PGC1α in Atx3 1−259 homozygous mice, whereas changes in the level of full-length PGC1α did not reach statistical significance due to the high variability within one genotype. This variability may be explained by different metabolic situations, which have been shown to finetune PGC1α expression (Cantó and Auwerx, 2009). At protein level, no expression differences were found for N-terminally truncated PGC1α, whereas the full-length PGC1α is clearly reduced in homozygous Atx3 1−259 C-terminally truncated mice. In addition, Atx3 1−259 homozygous mice demonstrated a drastic reduction of body weight (up to 20-30 g of total weight) and reduced amounts of dopamine in the striatum at 12 months of age, the beginning of their neurological phenotype (Hübener et al., 2011(Hübener et al., , 2012. Reduction of dopamine content, which resulted from selective loss of dopaminergic neurons and increased dopamine levels, has also been reported for HD models steadily overexpressing PGC1α (Ciron et al., 2012).
While the exact correlation between disruption of the mitochondrial network and mitochondrial function and biogenesis is still a matter of debate in the case of HD (reviewed in Costa and Scorrano, 2012), complex-II impairment emerges as key event of mitochondrial dysfunction in SCA3 and has been observed in different SCA3 cell and animal models as well as in peripheral cells of SCA3 patients (Laço et al., 2012). Additionally, mass spectroscopy analyses of polyglutamine-expanded ataxin-3 identified SDHA and SDHB, which are constitute part of the complex-II, as potential interactors (Kristensen et al., 2018). Concordantly, the fragmented mitochondrial network observed in the SCA3 model analyzed in the present study came along with reduced complex-I and complex-II activities and reduced uncoupled respiration upon FCCP treatment. These respiratory deficiencies finally resulted in impaired mitochondrial membrane integrity and an increased rate of mitochondrial-associated apoptosis. Cytochrome C release from the mitochondrial intermembrane space into the cytosol, which is a key event in triggering intrinsic apoptosis, depends on two changes of mitochondrial morphology influenced by Dnm1l, i.e., mitochondrial fragmentation and cristae remodeling (Frank et al., 2001;Scorrano et al., 2002). Indeed, we detected increased rates of cytochrome C release in brain lysates of our Atx3 1−259 homozygous mice and reduced mitochondrial membrane integrity. Moreover, a higher susceptibility to apoptosis was confirmed in homozygous Atx3 1−259 MEFs. Earlier studies on neuronal SK-N-SH cells expressing polyglutamine-expanded ataxin-3 demonstrated increased cytochrome C levels under basal conditions in combination with a significantly decreased expression of the anti-apoptotic protein Bcl2 (Tsai et al., 2004). Bcl2 is localized in the outer membrane of mitochondria, where it plays an important role in promoting cellular survival and inhibiting the actions of pro-apoptotic proteins like Bax. The mRNA levels of Bcl2 in our C-terminally truncated Atx3 1−259 homozygous mice revealed an increased expression of Bcl2 at early disease stages, but no differences at 12 months where we found increased cytochrome C levels as described by Tsai et al. (2004). Additionally, the mRNA levels of the pro-apoptotic protein Bax were increased in Atx3 1−259 homozygous mice at the age of 3 and 12 months comparable to a study from Chou et al. (2011). Chou and coworkers demonstrated that polyglutamine-expanded ataxin-3 led to upregulation of the pro-apoptotic proteins Bax and PUMA and caused apoptotic death via enhancing the phosphorylation and transcriptional activity of p53 (Chou et al., 2006(Chou et al., , 2011. Recently, evaluation of the activation of the intrinsic apoptotic pathway by determining the Bcl2/Bax ratio demonstrated lower values in SCA3 mutation carriers as well as in preataxic subjects compared to controls. In combination with the also analyzed accumulation of mitochondrial mtDNA deletions, it was hypothesized that apoptosis is initiated during the pre-ataxic stage, which might lead to an upregulation of cell death in subsequent disease stages (Raposo et al., 2018).
Mitochondrial deficiencies, as observed in the herein analyzed Atx3 1−259 C-terminally truncated mice, can lead to intrinsic apoptosis as described above but may also account for increased mitophagy, a process which selectively degrades mitochondria by autophagy (Twig and Shirihai, 2011). Induction of mitophagy is tightly linked to mitochondrial quality control. Depending on the dimension of mitochondrial damage, fragmented mitochondria either fuse back to a functional network or undergo mitophagy. Dysregulation of autophagy has been demonstrated in SCA3 patients' post-mortem brain samples. In the cerebellum and oculomotor nucleus of SCA3 patients, autophagic proteins such as autophagy-related gene (Atg) protein ATG-7, -12, -16L2 and microtubule-associated proteins 1A/1B light chain 3A and B (MAP1LC3A/B) were significantly increased (Sittler et al., 2018). In the present study, investigation of Map1lc3a and the Atg12-Atg5 conjugate, two factors essential for autophagosome formation, revealed an increased autophagy at early stages of the disease but not at stages when first neurological symptoms appear in the Atx3 1−259 homozygous mice. Inducing mild ER stress in MEF by tunicamycin treatment resulted in an increased activation of autophagy in Atx3 1−259 MEFs after short treatment periods and in increased ER stress response upon continued treatment. This crosstalk between autophagy and ER stress was already described for neurodegenerative diseases like HD (Vidal and Hetz, 2012;. Upon autophagosome maturation, autophagosomes fuse with lysosomes containing different enzymes, which are required for degradation of the autophagosomal content. Hence, normal lysosomal function and organization are essential for autophagy and lysosomal disturbances have been associated with neurodegeneration (reviewed in Appelqvist et al., 2013). In 2016 a direct link between mitochondrial dysfunction and disruption of structure and function of lysosomes was demonstrated in cells and brains (Demers-Lamarche et al., 2016). Mitochondrial dysfunction is characterized by mitochondrial fragmentation, decreased mitochondrial ATP and increased ROS production, which all can potentially influence lysosomal structure and activity. Demers-Lamarche and colleagues demonstrated that lysosomal impairment is mainly caused by increased ROS production and linked to large Lamp1-positive vacuoles (Demers-Lamarche et al., 2016). The present study also detected lysosomal disturbances in form of a decreased number of lysosomes and impaired lysosomal dynamics in Atx3 1−259 MEF. Lamp1, a marker for late-endosomes, lysosomes and autolysosomes, is known to be involved in lysosome biogenesis and autophagy (Eskelinen, 2006), and differential expression as well as colocalization with polyglutamine-containing aggregates in HD were already reported (King et al., 2008;Zheng et al., 2010). In our study, the reduction in the number of lysosomes was accompanied by a reduced expression of Lamp1 in brain samples of phenotypic Atx3 1−259 homozygous mice.
Summing up, this study shows that an N-terminal S256 ataxin-3 cleavage fragment impairs mitochondrial morphology, function and homeostasis as well as disturbs proper clearance of impaired mitochondria via mitophagy. Interestingly, together with Hsu et al. (2017) we could demonstrate that both N-and C-terminal fragments of ataxin-3 generated by calpain cleavage may lead to a mitochondrial phenotype independent of the expression of the polyglutamine tract. Therefore, further research on the influence of cleavagederived fragments in different areas as well as in vitro and in vivo models that are relevant for SCA3 pathogenesis is demanded to elucidate whether the function of full-length ataxin-3 is directly compromised by the pathologically elongated polyglutamine stretch or by consequently occurring ataxin-3 fragments.
AUTHOR CONTRIBUTIONS
TH, CP, JW, CF, SD, JM handled animal studies, molecular analyses, and analyzed the data. FG performed the mitochondria respiration studies. GK and RK helped with FACS analyses and live cell microscopy. HW performed the electron microscopy. SNH, SH, and LS generated and characterized iPSCs and iCNs from SCA3 patients and controls. OR and JH designed the experiments and oversaw the progression of the study. TH, CP, JW, and JH drafted the paper. All the authors read and approved the final manuscript.
FUNDING
The fortüne program of the University of Tübingen (1987-10) and the German Research Foundation (DFG, HU 1770/3-1) supported this work and JH. The German Center for Neurodegenerative Diseases (DZNE) supported this work by funding TH. We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of University of Tübingen. | v3-fos-license |
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} | pes2o/s2orc | Interactions of platinum metals and their complexes in biological systems.
Platinum-metal oxidation catalysts are to be introduced in exhaust systems of many 1975 model-year automobiles in the U.S. to meet Clean Air Act standards. Small quantities of finely divided catalyst have been found issuing from prototype systems; platinum and palladium compounds may be found also. Although platinum exhibits a remarkable resistance to oxidation and chemical attack, it reacts chemically under some conditions producing coordination complex compounds. Palladium reacts more readily than platinum. Some platinum-metal complexes interact with biological systems as bacteriostatic, bacteriocidal, viricidal, and immunosuppressive agents. Workers chronically exposed to platinum complexes often develop asthma-like respiratory distress and skin reactions called platinosis. Platinum complexes used alone and in combination therapy with other drugs have recently emerged as effective agents in cancer chemotherapy. Understanding toxic and favorable interactions of metal species with living organisms requires basic information on quantities and chemical characteristics of complexes at trace concentrations in biological materials. Some basic chemical kinetic and thermodynamic data are presented to characterize the chemical behavior of the complex cis-[Pt(NH3)2Cl2] used therapeutically. A brief discussion of platinum at manogram levels in biological tissue is discussed.
Introduction
The platinum metals comprise the rare metals in two triads of Group VIII of the periodic classification of elements: ruthenium (Ru), rhodium (Rh), palladium (Pd), and osmium (Os), iridium (Ir), and platinum (Pt). Standards for mobile emission sources (primarily automobiles) have been promulgated; manufacturers have chosen to meet the standards by equipping automobiles with devices using platinum-group catalysts. Losses of the active metals from these devices will expose large numbers of people to a new environmental contaminant whose biological effects may be important. Interactions of platinum metals and their compounds with biological systems have been reported over the last 140 years. The discovery that platinum metal complexes have a potent effect on cell division in bacteria and some animal tumors prompted more intensive study of their effects on cancers of many types. There is evidence that these platinum-metal complexes interact strongly with some amino acids, peptides, proteins, nucleotides, and nucleosides, specifically with deoxyribonucleic acid (DNA).
This paper summarizes information available on behavior of platinum metals and compounds and effects these materials produce in biological systems exposed to them. It describes a method of quantitation of platinum at nanogram levels in biological samples by atomic absorption spectrometry.
Catalytic Converters
Catalytic converters containing an estimated 1-3 g (approximately 0.1 oz troy), of platinum (Pt) and palladium (Pd) metal have been developed to oxidize unburned hydrocarbon (HC) and carbon monoxide (CO) emissions in internal combustion engine (ICE) exhaust to water (H20) and carbon dioxide (CO.,) to meet the standards established in the Clean Air Act (1). Antiknock additives containing lead alkyls and organic halide scavengers will not be used in the fuels for converter-equipped vehicles because they inactivate the catalysts. Sulfur in the fuel is oxidized to sulfur dioxide (SO2) in the engine; this SO2 is further oxidized in the converter to the trioxide, SO3, that combines with water to produce a mist of sulfuric acid (H2SO0) in the exhaust gas stream.
Conversion equipment involving use of a ruthenium-based catalyst is under test to diminish oxides of nitrogen (NO,) in the exhaust by chemical reduction to molecular nitrogen (N2) (2).
Catalyst and Emission Characteristics
The catalysts under consideration may be monolithic or pelletized, with an alumina matrix to support the active metal. Detailed procedures used to manufacture these catalysts are proprietary information that is not generally available.
Industrial vehicles such as ICE-powered fork-lift trucks used in ships' holds, warehouses, and mines have been equipped with catalytic converters for a decade or more. Although vehicle operating conditions and catalyst types may differ, this application seems to offer the best available basis for assessment of these converters in actual use. The U.S. Environmental Protection Agency has made tests on a Ford Motor Company automotive engine prototype using a monolithic catalyst in its exhaust system and an Oldsmobile (General Motors Corp.) test vehicle equipped with a pelletized catalyst.
Data available on operating parameters (reaction rates, temperature profiles, flow rates and configurations) in catalytic converters of each type under various operating conditions have been closely held by the automakers and catalyst manufacturers and have not been disclosed.
In industrial applications, platinum gauze oxidizing catalysts are known to undergo substantial surface rearrangements during catalysis at temperatures far below the melting point of the metal. Dendritic growths appear, grain sizes increase, and attrition occurs (3). In vehicles, there may be significant differences in the form of catalyst emissions from the different types under test, e.g., alumina particles are found in the exhaust from a vehicle with a pelletized catalyst. Catalytic noble metals in the automobile exhaust gas stream are expected to be very finely divided and predominantly water-insoluble. Loss rates of platinum and palladium of the order of micrograms of metal per mile have been estimated from regularly serviced test vehicles under running conditions stipulated for the modified California test cycle. Definitive data are lacking on this point. Industry spokesmen expect recovery of the catalytic metals to be economically feasible. Until the recovery rate is established, we note that the automakers have arranged substantial supply contracts. For the coming decade, GM agreed to buy 120,000 troy oz Pd annually and 300,000 troy oz Pt annually from Impala Platinum Ltd. of South Africa. (4).
In addition to their catalytic activity, platinum metals are well known for their resistance to chemical attack, even at elevated temperature. Indeed, many applications of the group are predicated on this inertness. It is less well known that the platinum group metals do react to a degree Environmental Health Perspectives that depends on their fineness (or compactness), on the presence and character of impurities, metallurgical history, and conditions of dissolution (5).
There may be a tendency to think the nobility and chemical resistance of the bulk metal extends to the finely divided form and conclude that it is nearly unreactive and must therefore be innocuous. According to Beamish (5), given sufficient fineness, the platinum metals may be expected to respond to the corrosive action of even single mineral acids, particularly in the presence of air. Losses of 1-8 g of platinum per metric ton of sulfuric acid, depending on concentration, were reported for some of the early methods of production of the acid in platinum-lined vessels (6). Palladium is subject to attack by hot concentrated mineral acids.
It is not possible to state, definitively, the extent to which sulfates issuing from a catalytic exhaust system may include those of platinum or palladium, without accurate information on the conditions that obtain in the system.
Platinum is subject to net weight loss when heated strongly in air or oxygen. Depending on conditions, it may incur a loss of weight due to volatility of an oxide or a gain resulting from formation of a stable oxide (5). This point has been treated in an extensive discussion of a century's conflicting reports of reactions under many different conditions (7). Formation of intermetallic alloys within the platinum group or with associated base metals is well known; the effect of such alloying on the resistance to dissolution cannot be simply inferred from the behavior of the components (5).
Biological Response to Metals and Metal Complexes
The critical question in biological term is: What interactions obtain in living systems exposed to trace concentrations of these highly active metals, whether soluble or insoluble, and to the interconversion products they may form in the environment? Microbial Viral and Fungal Interactions Zinno and Cutolo (8) reported an iridium chloride solution to be highly potent against Staphylococcus aureus and the causative organisms of typhoid, cholera, and anthrax. A 10-min exposure to a 0.O1N solution rendered spores of the anthrax bacillus nonviable.
Shulman and Dwyer (9) in an extensive review, compared the bacteriostatic behavior of 1,10-phenanthroline and 2,2'-bipyridine bases, their quaternary salts, and their metal chelates containing identical or mixed ligands on gram-positive (Staphylococcus pyogenes, Streptococcus pyogenes, and Clostridium welchii), gram-negative (Escherichia coli and Proteus vulgaris), and acid-fast (Mycobacterium tuberculosis H37Rv) organisms. They found ruthenium complexes moderately active against the gram-positive organisms and showed some activity against the gramnegative microorganisms. They also report that the expected development of drug resistance with Staph. pyogenes and M. tuberculosis did not occur with the highly active metal chelates of ruthenium and iron. They report further on the inhibitory and mutagenic activity of metal chelates on a yeast, Saccharomyces cerevisiae, and fungistatic activity on several pathogenic fungi. Bromfield
Systemic Administration of Transition Metal Compounds in Mice
Collier and Kraus (12) treated tumorous mice with some 64 transition metal chlorides, including those of three platinum metals. They reported a slight activity on mouse sarcoma by two ruthenium compounds and no significant effect by the rhodium and osmium compounds they used. Taylor and Carmichael (13) studied effects of some 37 transition metal chlorides and nitrates on mouse mammary adenocarcinoma and transplantable sarcoma in DBA mice. The results were favorable with rhodium and iridium chlorides, but apparently these agents were not studied further. Rosenberg (14) also found that the square-planar complexes cis-
Metal Implants in Rats and Mice
Nothdurft (16)(17)(18) reported a high frequency of appearance of tumors in rats and mice at the sites of subcutaneously and intraperitoneally implanted discs, spheres, and powderlike forms of the metals platinum, gold, and silver and the nonmetal ivory. He states, categorically, that the appearance of tumors at the site of implantation is not related to the chemical properties of the metals, but unfortunately he does not adduce results with positive and negative controls to support the conclusion.
Human Chronic Exposure to Precious Metals in Industry Workers chronically exposed to precious metal dusts and their complex salts in platinum metal refineries and in catalyst manufacture are subject to platinosis, a condition whose signs are: cold symptoms, tightness in the chest, dermatitis, eczema and skin ulcerations. Fothergill (19) found none of the symptoms of platinosis in workers exposed to 5-70 4g/m3 of platinum dust only or to complexes of precious metals other than platinum. Karasek and Karasek (20) identified eight cases of allergic phenomena occurring in the nasal mucosa, bronchi, and skin of photographic studio personnel. They worked with a sensitized paper containing potassium chloroplatinite. Hunter et al. (21) described the same syndrome in 52 of 91 men exposed to dust or spray of complex salts of platinum in four metal refineries in England. It started with repeated sneezing, followed by profuse running of the nose. Tightness of the chest with shortness of breath, wheezing, cough, and cyanosis ensued. Some 13 men also experienced a scaly erythematous dermatitis and some an urticarial rash. These conditions did not occur in workers exposed to much higher concentrations of metallic platinum in the atmosphere or to men exposed to the complex salts of other precious metals including palladium.
Two case histories reported were characteristic: One person worked in the refinery 18 years and had symptoms after the first year which became slowly but progressively worse until he was forced to give up this work. On leaving, the symptoms disappeared and did not recur. The second person presented symptoms after six years of service, becoming worse until asthma appeared in the 10th year. After transfer to another department, the employee no longer suffered from asthma. Roberts (22) reviewed the previous reports and cited the experience gained in a five-year study of 21 employees of the Bishop & Co. Platinum Works. He described factors predisposing to platinosis: previous history of allergy, strong family history of hives, hay fever, asthma, contact dermatitis.
Individuals who have skin blemishes, moles, acne, sebaceous cysts and other lesions are prone to become sensitive to complex platinum salts. Those with blond hair, blue eyes, and thin or transparent skin that tolerates irritants poorly are prone to have platinosis. More swarthy types, free from skin blemishes with darker, coarse hair and thicker, less sensitive skin do not usually develop the platinum intolerance.
Biological Effects of Platinum Complexes
Since Rosenberg (14) reported the activity of platinum complexes as antitumor agents in test animals, considerable effort has been made to study the effects in detail. The most widely investigated of the compounds is Peyrone's salt (23)(24)(25)(26), cis-dichlorodiamminoplatinum (II) (cis-[Pt (NH,)2C1] ). It has displayed promising results in arresting tumor growth in animals and has been introduced into clinical trials for cancer treatment in humans to establish appropriate dose levels and schedules.
Preclinical Toxicology: Studies of toxicology of cis-[Pt (NH) 2C12] have been conducted on mice and rats (27)(28)(29)(30), and have been summarized by Rosenberg (31). The important histological changes observed in these rodents were: denudation of intestinal epithelium, bone marrow depression, thymic and splenic atrophy, and acute nephrosis.
Trials with dogs and monkeys revealed similar toxic effects with damage to the renal tubules a prominent effect, especially at high dose levels. Other important effects are damage to the bone marrow and gastrointestinal epithelium. The clinical brochure (32) for cis-[Pt (NH)2C12] includes the findings of clinicians studying its behavior in humans. Therapeutic efficacy was seen clinically in Phase I trials in treatment of several types of tumors. Some of the most encouraging results were reported by Higby (33) on testicular tumors. The major limitation on the use of this complex as an antitumor agent is its renal toxicity, usually manifested as tubular epithelial damage. It has been estimated that single IV doses above 2 mg/kg leads to unacceptable toxic effects. Other toxic effects reported include ototoxicity, high-frequency hearing loss, at total doses above 1 mg/kg. Wallace (34) has reported no evidence of cumulative renal toxicity in one patient who received 545 mg/M2 ( -..14 mg/kg) over a 104-day period (schedule unspecified). Another of his patients received a 479 mg cumulative dose over a period of 200+ days; the patient experienced a BUN elevation of 38 and a creatinine of 4.4 at the very terminal stage of her disease with other complicating factors.
Mechanism of Action: Extensive research is under way to establish the mode of antitumor action of this and related complexes. Several interactions with the nucleic acid constituents have been observed and are under study. It has been suggested that tumor destruction results from drug action at sites in the cell resulting in primary lesions on the nuclear DNA (31). The principal mechanism appears to involve inhibition of DNA synthesis and, to a lesser degree, inhibition of RNA and protein synthesis. Harder and Rosenberg (35) reported selective inhibition of DNA synthesis in vitro by cis-[Pt(NH3)2C12 below 5 XM.
Characterization of Platinum Group Metals in Biological Systems
Understanding the detailed interactions of platinum group metals with biological systems requires extensive quantitation and characterization of the chemical species involved. Studies of chemical behavior of complexes in relatively simple systems in vitro suggest the large number of variables that affect ligand substitution reactions and thus the diversity of the reaction products.
The most stable square-planar complexes are those of divalent platinum, Pt(II) with dsp2 hybridization. Divalent palladium, Pd (II), produces complexes of the same geometry; both are low-spin d8 systems.
Most of the cis-trans isomers of squareplanar complexes that have been isolated are the relatively inert complexes of Pt(II). In fact, the concept of a square configuration, rather than a tetrahedral one was introduced by Werner (36) because the tetrahedral structure could not account for the two forms of [Pt(NH3) :.Cl] prepared more than a century ago by Peyrone (23) and by Reiset (37).
Thermodynamic versus Kinetic Properties of Complexes
The terms "stable" and "unstable" are used to refer to the thermodynamic properties of the complex species considered. The term "inert" is used in the kinetic sense, to describe complexes which engage in ligand replacement reactions slowly; those which undergo such reactions rapidly are described as "labile", as suggested by Taube (38).
While it is often true that stable substances are slow to react and unstable compounds react rapidly, there is no absolute requirement that this be the case. Synthesis of various Pt (II) compounds rests on utilization of competing thermodynamic and kinetic factors. The behavior of the complexes with biological materials will depend on the same considerations; the distinction between thermodynamic and kinetic factors may be important in understanding the interactions. Some metal complexes are so stable they do not react with the biological system. Thus, the stability (stability constant, K-044) and inertness (kinetics extremely slow) of the complex [Fe (CN)G] 2are so great, even in acid solution, that it yields no significant HCN; this cyanide complex could be ingested with no ill effects.
Kinetics of the ligand substitution reaction are important, since the half-times of the reactions may range from less than a second to months or years. The latter may appear to be "stable" if only short studies are made. The rates are functions of the geometry of the complex, the metal and its oxidation state, and the polarizability of the ligands. Photochemical effects and pH may also affect reaction rates.
Effect of Metal Oxidation State
Stability of oxidation states of metal complexes in the biological medium must be considered. Low-spin complexes generally undergo rapid one-electron oxidation or reduction reactions. Since biological systems operate at low redox potential, approximately -0.5 to 0 V, reduced low oxidation states usually obtain. Platinum group complexes would be reduced to the metallic state under these conditions but for their inert reduction kinetics. Although Pt(IV) amines would be expected to penetrate biological systems more rapidly than Pt(II) amines, the latter produce the biological effect.
Common Chemical Features of Antitumor Complexes
Of the transition-metal complexes tested for antitumor activity, only a small number have shown it; some active compounds are highly toxic. The effective compounds have features in common that may aid us in understanding their interactions with biological systems.
Some of these features are outlined here. The complexes exchange some ligands rapidly; other ligands are exchanged slowly or not at all. This behavior is usually found in low-spin complexes having electrons paired in the t29 level d-oribitals. Strongly bound ligands are transported with the metal through membranes like cell walls. The geometric configurations of transition metal cations are: square-planar (usually low-spin, d8), pentagonal square-pyramidal (usually low-spin, d7), octahedral (all other low-spin electronic configurations).
Active antitumor agents usually have two exchangeable ligands in cis positions. The ligand donor atoms most probable for the low-oxidation state cations in a biological system are N, 0, S, Cl, Br; these donor atoms may be incorporated into a large variety of ligands. Some ligands are monodentate; others may be mono-or multidentate. Chelation may occur with the latter. Halide ligands can form bridges between metals.
The activity of cis-[Pt(NH,),2Cl2] against tumors is associated with reactions replacing the halides. These complexes are bifunctional reagents that may undergo nucleophilic substitution at two cis positions.
Antitumor behavior is apparently not related to oxidation-reduction reactions of the metal.
Detailed Reaction Scheme
Square-planar complexes undergo predominantly bimolecular nucleophilic displacement reactions in contrast to the generally dissociative reactions exhibited by octahedral complexes. Cis-[Pt (NH3)2C12], for example, is injected as a neutral molecule in physiologic saline solution. This neutral species may undergo limited hydrolysis in the extracellular fluid (,..O.lM Cl-). Within the cell, a lower chloride ion concentration obtains (0.004M Cl-) ; thus more extensive hydrolysis may occur as indicated in the stepwise reaction scheme (1), which assumes monomeric species and preservation of the cis configuration of the ammine ligands despite the greater favorability of the trans configuration thermodynamically. The stability constants shown are cited by Balzini (41). The rate constants for the successive aquation reactions (41) shown for cis-[Pt(NH3 )2 C12] are kl,H2o = 2.5 X 10-5 sec-, k2,H2o= 3.3 X 10-5 sec-1; at 200C; thus these reactions have half-times of the order of 6-8 hr (42). Note that both ligand substitution rate and partial equilibria for this type of reaction may be functions of the pH and pC1 of the surrounding medium. In a fluid low in Cland, near neutral pH (e.g. cellular fluid), one could expect to find approximately half of the Pt as [Pt(NH3)2(H2O)OH]+1 and half Electronic absorption spectra yield information on interactions between the metal and bound groups, stability constants and rate constants for complexation. Magnetic and natural circular dichroism (CD) measurements also yield useful information on geometries and metal-ligand interactions. Electron spin resonance (ESR) and nuclear magnetic resonance (NMR) are useful in determining symmetry relations, binding constants and rate data. Vibrational spectra may also be used for characterization of the molecular structures.
A few studies reporting various biological effects reflect known total quantities used in the experiments, but few data are available from analyses for the metal distributed in the biological system. The available data have come principally from tracer experiments with radioactive platinum isotopes. Renshaw Unfortunately, the platinum radionuclides present experimental difficulties because of the short half-life of the radioactive isotopes and relatively lengthy steps in synthesis and purification of complexes. Studies of long duration require low-level counting facilities if tracer levels introduced initially are to be kept at levels that do not require heroic safety precautions.
Techniques for Quantitation
Quantitation of platinum can be performed at relatively low concentrations by several techniques including mass and emission spectrography, arc, spark, and M6ssbauer spectroscopy, x-ray fluorescence, neutron activation, and electronic and atomic absorption.
Standard Reference Materials
The biological Standard Reference Materials (SRM) available from the National Bureau of Standards (NBS) (bovine liver SRM 1577 and mixed orchard leaves SRM 1571) do not contain certified quantities of Pt metals. Laboratories analyzing for these metals have no biological standard to measure their analytical techniques against.
The NBS does offer high purity platinum wire, SRM 680, with a certified analysis from which a hexachloroplatinic acid standard can be prepared. Our determinations by use of atomic absorption spectrophotometry have been based on standards preparedfrom highly purified Pt metal and a commercially available hexachloroplatinic acid standard.
We plan independent analyses of the platinum in biological materials to confirm results obtained by atomic absorption spectrometry. One method to be used is neutron-activation analysis using the reaction (2) (2) 199Au subsequently decays to 199Hg with a 3.15 day half-life.
Quantitation of Platinum in Biological Samples
Analytical techniques have been evolved to overcome difficulties specific to the types of samples analyzed. Biological samples present special considerations and difficulties; they usually contain a substantial proportion of water, and ionic metal species may bind so strongly to amino acids and proteins that the Environmental Health Perspectives latter must be destroyed to free the metal for analysis.
Experience with several procedures for quantitation of platinum in biological samples has led to development of a method that yields reproducible results with tissue samples of approximately 1 g wet weight (or 1 ml samples of fluids like blood plasma or urine) that contain 0.25 ,g Pt or more. Measurements typically use a 20-ul portion of digest taken up in lM HCI. Under favorable conditions, this method yields reliable results with samples containing 2 ng Pt or more.
If analytical results are to be reported on the dry-weight basis, solid tissue is lyophilized. Otherwise, the tissue is weighed wet.
The weighed tissue is wet-ashed in 10 ml of a solution made from 1 volume HNO3 (60 %0) + 1.5 volume HC104 (70 %0). The digestion step need not be carried out under reflux. The temperature of the mixed acid solution rises progressively as the HNO3 is concentrated and volatilized with subsequent concentration of the HCIO, and progressive development of its full oxidation potential (-2 V) at 203°C, the boiling point of the HCIO4-water azeotrope (acid strength 72.5 %0). Experience has shown that some tissues (e.g., liver, fat) may require repeated treatment to yield a clear digest. Heating is continued to incipient dryness of the digest, whereupon 5 ml lM HCl are added and evaporated again to incipient dryness. Finally, the contents of the beaker are taken up into 5 ml iM HCI.
Portions of this solution (typically, 20 zd) may be introduced directly into the heated graphite analyzer (HGA-2000) coupled to a Perkin-Elmer 303 atomic absorption spectrophotometer to give consistent results at the 5 ng level or higher by using the 265.9 nm resonance line for Pt. An additional step using solvent extraction increases the sensitivity of the method. The solvent, methyl isobutyl ketone (MIBK) is used to extract the platinum species from the aqueous phase. This solvent affords a distribution coefficient virtually independent of HCI concentration in the range 1-12M. The atomic absorption measurement is then made on a 20 pl portion of the solvent. Although this step improves results, we have experienced some unexplained variations in yield with it and consider it premature to recommend.
The solvent extraction technique and variations of it using the aliphatic secondary amines LA-1 or LA-2 liquid ion-exchangers in xylene as extractants are being developed further. We have conducted some preliminary separations using LA-1 since it offers higher distribution coefficients than MIBK over the 1-9M HCI acidity range. Davidson and Jameson (44,45), showed that the extraction depends not only on the HCl molarity in the aqueous phase, but also on the concentration of the background electrolyte anions in the aqueous phase that compete for exchange sites. Work is currently under way to increase the sensitivity of the analysis and improve the reliability of the extraction step.
Summary
We have cited information on known interactions of platinum-metal complexes with bacteria, viruses and mammals. Effects in humans have been noted for chronic industrial exposure to platinum complex compounds; clinical therapeutic experience with cis-[Pt (NHW) 2CX] has been reported. Knowledge of biological and chemical behavior of platinum metals is limited. Data available on the quantities and chemical nature of the materials issuing from converter equipped test vehicles are insufficient to permit definitive assessment of the risk posed by introduction of these systems on a large scale. | v3-fos-license |
2018-04-03T02:59:05.836Z | 2012-12-03T00:00:00.000 | 8607569 | {
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} | pes2o/s2orc | Characterizing protein domain associations by Small-molecule ligand binding
Background: Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to smallmolecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods: We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results: We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions: A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing.
Background
Protein domains are evolutionarily conserved units in protein sequence, structure and function, which can be recombined in different arrangements to create new proteins in biological organisms [1][2][3][4][5][6]. The interactions between protein domains and other molecules play a fundamental role in molecular recognition in living organisms. Small molecule binding domains are of particular interest, as many of them represent targets for biologically important ligands including drugs [7,8]. Studies on small molecule-protein domain interactions have received increasing attention for their potential to advance chemogenomics research and drug development [9][10][11].
Many studies have investigated the interactions between small molecules and protein domains. For example, Yamanishi et al., [12] used the canonical correspondence analysis method to investigate the rules governing the recognition of chemical substructures and protein domains. Bender et al., [13] built a statistical model on chemical structures and protein domains to triage the affinity chromatography data. Wang et al., [14] used protein domains and therapeutic information to predict drug targets. Besides, Kruger and Overington [15] incorporated protein domain information to analyze small-molecule bindings of homologous proteins in human and rat. Collectively, the underlying assumption of these studies is that small molecule-protein recognitions are accomplished through small molecule-protein domain interactions. However, due to the lack of accurate binding site information, these interactions are usually assumed according to the presence of protein domain(s) within a protein, yet a specific connection between a domain and its ligand is not guaranteed. This strategy may work well for single-domain proteins, while it may fail for multidomain proteins that are usually observed in human genome. To address such issues, Kruger and Overington [15] proposed to derive small molecule-domain interactions based on the observed frequency in single-domain proteins. However, the results based on such empirical assignment are nonetheless compromised. Meanwhile, proteins are conventionally grouped into individual families based on sequence or structure similarity [1][2][3][4][5][6]16]. The interrelationship across such families, especially for smallmolecule binding, is seldom studied, though they are important for understanding the regulatory roles of small molecules in biological systems.
In the present study, we attempted to address such issues by first collecting the physical interactions between small molecules and protein domains derived from the experimentally determined structures in Protein Data Bank (PDB) [17] and then characterizing the protein domain inter-relationship with respect to small-molecule binding on a large scale. As PDB contains protein 3D structures and accurate structural information of protein-ligand interactions, several secondary databases have been developed to include small molecule-protein domain links as recently reviewed by Bashton and Thornton [18]. For example, the PDBLIG [19] database associates small molecules contained in PDB to the CATH domains [1]. Likewise, PROCOGNATE [20] links small molecules in PDB to three distinct domain databases including CATH, SCOP [2] and Pfam [3,4], with a special highlight on cognate molecules that are endogenous in living organisms for enzymes [21]. In addition, the Inferred Biomolecular Interactions Server (IBIS) [22] contains detailed description and classification of binding sites between small molecules and proteins. The interactions compiled in IBIS are integrated with the Conserved Domain Database (CDD) [5,6] and PubChem database [23,24], a protein domain annotation database and a chemical structure database, respectively. The above three databases, i.e. IBIS, CDD and PubChem, were used in this work to derive pairwise associations between small molecules and protein domains.
By analyzing these small molecule-protein domain interaction data, we identified promiscuous small-molecule ligands that bound to two or more protein domains, which subsequently led to the generation of an inter-connected protein domain network. By analyzing this network, we found that many protein domains, despite belonging to various families, can bind common or similar ligands. Moreover, tightly connected domains were observed to form modules in the network, which often share similar biochemical mechanisms, or are involved in related biological pathways. This study provides a global view of the complex role of small molecules in biological systems and reveals a novel relationship among protein domains, complementary to the traditional classifications derived solely from protein sequences or structures. Meanwhile, the success of identifying potential targets for marketed drugs based on this network may shed light on network pharmacology study and systematic identification of novel targets for drug repurposing.
Physical interaction data for small molecules and protein domains
Three databases were used to derive the physical interactions between small molecules and protein domains, including IBIS [22] (updated Oct 25, 2011), CDD [5,6] (version 3.01) and PubChem [23,24]. IBIS contains binding site information of small molecules and proteins in PDB; the CDD database consists of both manually curated protein domain models and those imported from other resources, such as Pfam [3,4], SMART [25] and COG [26,27]; PubChem comprises standardized and validated chemical structures of smallmolecule ligands, in which a Compound Identifier (CID) represents a unique chemical structure.
Identification of small molecule-protein domain interactions
For each small molecule-protein interaction, we mapped the binding sites obtained from IBIS to the domain footprint annotations provided by CDD. A flowchart of our approach is shown in Figure 1.
Firstly, we retrieved a total of 88,774 small moleculeprotein interactions derived from IBIS, corresponding to 13,851 unique small molecule structures and 67,619 distinct protein sequences. Here, we used the IBIS criteria to define a small-protein interaction that five or more amino acid residues of a protein are within 4Å from its small-molecule ligand (heavy atom). We excluded the 'non-biological' interactions marked by IBIS, as most of them were resulted by auxiliary molecules, such as buffers, salts, detergents, solvents and ions, used for crystallization or purification. Moreover, we confined our study to the small molecules with two properties: (1) molecular weight between 100 and 800; (2) containing only organic elements (H, C, N, O, F, P, S, Cl, Br and I) in one covalent unit (i.e. non-mixtures). As a result, we obtained a dataset containing 11,582 unique small molecules and 51,594 protein sequences with accurate binding site information.
Secondly, we annotated each protein sequence obtained in the previous step with domain footprints, i.e. domain positions, by searching against CDD with default parameters. In the retrieved results, we selected the manually curated domain models (CDD accession starting with 'cd') ranked on the top of the hit list (if available); otherwise, we used the Pfam models (CDD accession starting with 'pfam'). At last, we obtained 3,012 distinct protein domains in total. Additionally, we retrieved the superfamily information (CDD accession starting with 'cl') for these protein domains from CDD as well.
Thirdly, we mapped each small-molecule binding site obtained in the first step onto a specific protein domain (if possible), according to domain footprint annotations. A small molecule-protein domain interaction was determined if more than 75% of the contact residues were within the domain region. This process produced 13,822 small molecule-protein domain pairs, corresponding to 9,529 unique small-molecule structures and 2,125 distinct protein domains.
Drug and cognate molecules
Some of the small molecules obtained in the previous step are marketed drugs according to the DrugBank [28,29] annotations. These can be easily accessed through the PubChem CIDs, as DrugBank has deposited drug data into PubChem.
A cognate ligand is an endogenous small molecule in biological organisms. To identify such cognate molecules, we used a similar strategy reported by Bashton et al., [20]: a small molecule was 'cognate' if it has a similar compound (with the Tanimoto coefficient above 0.9 by using the PubChem fingerprint [23,31]) in the KEGG Reaction database [30], which consists of detailed annotations of the biological reactions in organisms.
Protein domain network
To study the relationship of the small-molecule binding domains, we constructed a domain network (Figure 1), in which a node represents a protein domain, and an edge links two protein domains if they bind common or similar ligand(s). Here, we considered two ligands similar if their Tanimoto similarity is above 0.9, as calculated by using the PubChem fingerprint [23,31]. To characterize network properties, the following metrics were used: Node degree (k i ) measures the number of edges connecting to node i.
Shortest path (L i,j ) is defined as the shortest distance or minimum number of steps between any two given nodes (i and j) over the domain network. The average shortest path (<L>) is a mean of the shortest paths of all possible node pairs.
Clustering Coefficient (C i ) is defined as C i =2n/k i (k i -1), where n denotes the number of edges connecting the nearest neighbors (k) of node i [32]. The value of C i is equal to 1 for a node at the center of a fully interconnected cluster, while the value of 0 indicates a node in a loosely connected group. The average clustering coefficient (<C>) over all nodes of a network is a measure of the network's potential modularity.
The network was drawn by using Cytoscape [33,34] (version 2.81) and the network properties were calculated with the igraph library (version 0.5.4, http://igraph.sourceforge.
Results
In this work, we compiled 13,822 small molecule-protein domain interactions (See Method), corresponding to 9,529 unique small molecules and 2,125 distinct protein domains. Originally, we identified 3,012 protein domains in total from these small-molecule binding proteins. Some proteins contained multiple domains and the domains (30%) that had no bound small-molecule ligand (see Method) were excluded in the following study.
Small molecule-protein domain interactions: a many to many relationship
We observed that the number of small-molecule ligands varied by each domain, with a ligand count of five on average. The overall distribution is shown in Figure 2A. The majority of the protein domains bound few small-molecule ligands; however, some domains interacted with hundreds of distinct small molecules, such as the trypsin-like serine protease domain (CDD accession: cd00190), carbonic anhydrase alpha I-II-III-XIII domain (CDD accession: cd03119) and HIV retropepsin domain (CDD accession: cd05482) ( Table 1). In addition, we found that, although the smallmolecule ligands of many protein domains spread over a wide range in chemical space, they have preferential zones in terms of physicochemical properties as indicated by the molecular weight and octanol-water partition coefficient (Supplement figure S1-A). For example, the HIV retropepsin like domain (CDD accession: cd05482) tended to bind larger molecules (Supplement figure S1-B); while the trypsin-like serine protease domain was prone to bind relatively diverse ligands (Supplement figure S1-C).
On the other hand, we found that 1,168 out of the 9,529 small molecules, including drugs, were promiscuous because they bound to two or more protein domains. For an example, dexibuprofen (PubChem CID: 39912), a nonsteroidal anti-inflammatory drug (NSAID), bound to both of the phospholipase A2 domain (PLA2c, CDD accession: cd00125) and albumin domain (CDD accession: cd00015). The overall distribution of the number of protein domains targeted by small molecules is shown in Figure 2B. It is worth noting that 73% (852) of the promiscuous small molecules were observed to bind multiple domains from different domain superfamilies. For instance, nicotinamide adenine dinucleotide phosphate (NADP, PubChem CID: 5886) bound to 103 distinct protein domains from over 20 domain superfamilies; and adenosine diphosphate (ADP, PubChem CID: 6022) interacted with as many as 191 protein domains, belonging to 57 superfamilies that are widely distributed in a biological system. Especially, 72% (842) of the total 1,168 promiscuous molecules were cognate (endogenous) molecules. These results demonstrate the versatility of small molecules, including cognate molecules and drugs, in regulating biological processes. Therefore, our analysis unveiled a many-to-many relationship between small molecules and protein domains, which led us to further investigate the relationship among protein domains as resulted from interacting with small-molecule ligands.
Pairwise protein domain associations
Based on the observation in the previous section, we noted that about 89% (1,883) of the 2,125 domains were associated with at least one other domain through binding common ligands, producing 79,160 domain pair associations. The rest 11% (242 domains) bound with "selective" ligands that interacted with only one single domain target observed in the current dataset, hence these domains did not demonstrate domain associations regarding to share common ligands. Surprisingly, among the domain pair associations, we found that 86% (67,976) of them were from different superfamilies. This clearly indicates that distinct protein domains may associated with each other in terms of small-molecule binding, despite of the differences in protein sequences or structures. Furthermore, we investigated the strength of these domain associations. Intuitively, the more ligands sharing between two domains, the stronger the association is. In this study, we not only considered the number of common ligands, but also took similar ligands into account, as we noticed that certain ligands shared significant similarity in structure, such as ADP and adenosine triphosphate (ATP, PubChem CID: 5957). We set a similarity (Tanimoto coefficient) threshold of 0.90 to ensure high-quality domain associations identified. By incorporating ligand similarity, we observed a 6% increase in the number of domain associations identified.
For any two domains, the ligand structures of them were compared in pairwise. The number of similar ligand pairs, named NSLP score, was calculated to represent the strength of a domain association. By systematically evaluating the NSLP score for each domain pair, we found a great variation among the domain association strength (Figure 3). Some domain pairs from the same superfamily tended to have high NSLP scores. For example, the bacterial photosynthetic reaction center complex M domain (CDD accession: cd09291) and bacterial photosynthetic reaction center complex L domain (CDD accession: cd09290) had an NSLP score of 926, both of which belong to the photosynthetic reaction center superfamily (CDD accession: cl08220). Particularly, we observed that certain domain pairs from different superfamilies also had high NSLP scores, indicating considerable similarities among their ligands. For instance, the nucleoside diphosphate kinase group I domain (CDD accession: cd04413) and canonical ribonuclease A domain (CDD accession: cd06265), despite that they belong to the nucleoside diphosphate kinase superfamily (CDD accession: cl00335) and ribonuclease A superfamily (CDD accession: cl00128), respectively, had an NSLP score of 151, with many being nucleotide derivative ligands. More examples of protein domain associations with high NSLP scores are listed in Table 2.
In fact, we found that the majority of the domain associations identified in the present study were across different superfamilies. Hence, we further investigated domain superfamily associations and their strength in the same way as that for the domain association study. As a result, a number of closely related superfamilies were identified, such as the P-loop NTPase superfamily (CDD accession: cl09099) and Rossmann-fold NAD(P)(+)-binding protein superfamily (CDD accession: cl09931) were associated with a NSLP score of 625. Additional examples of superfamilies with significantly strong associations regarding to small molecule binding are listed in Supplement table S1. This analysis demonstrates, to some extent, the deficiency of the conventional classifications based protein sequences or structures, because they cannot well represent such relationship resulted by small-molecule binding. Therefore, it indicates that our work on identifying protein domain associations based on small-molecule binding may complement the conventional approaches in protein family studies.
Protein domain network
In the previous analysis of pairwise domain associations, we not only identified closely related domains with regard to small-molecule binding, but also found some popular domains that were associating with many other domains through binding common or similar ligands. suggest that the small-molecule binding domains are comprehensively associated with each other through binding small-molecule ligands. Among the entire domain network, we observed a power-law like distribution of the node degrees (Figure 4), which indicates that the nodes with higher degree ("hub" nodes) had a lower frequency in general. For example, the canonical ribonuclease A domain (CDD accession: cd06265) and nucleoside diphosphate kinase group I domain (CDD accession: cd04413), connected to as many as 690 and 676 other domains (Supplement table S2 and S3), respectively. Moreover, the shortest path between any two nodes (domains) in the network was 2.9 on average, i.e. any two randomly selected domains were separated by less than three steps, which suggests a small-world property of the network [32,35].
Furthermore, we calculated the clustering coefficient [32] of each node and obtained an average value of 0.5 over the network, which implies potential modularity existing in the domain network. A domain module represents a group of domain nodes that are densely inter-connected within a group, but loosely connected to nodes outside the group. When looking into these domain modules, it is not surprising to observe that domains in such modules often shared a similar biochemical mechanism in vivo or belonged to the same superfamily. For example, the alpha carbonic anhydrase (CA) domains, including types I-II-III-X-III (CDD accession: cd03119), V (CDD accession: cd03118), IX (CDD accession: cd03150), XII-XIV (CDD accession: cd03126) and VII (CDD accession: cd03149) that catalyze CO 2 hydration to bicarbonate and protons in living organisms, formed doi: 10.7243/2050-2273-1-6 a fully inter-connected module in the network (the red module in Figure 5, referred as the CA module in this work) through binding acetazolamide, the first non-mercurial diuretic drug [36].
In addition, we also found that some domains within a module were involved in relevant biological processes. One such example was the blue module in Figure 5, which consisted of six protein domains including the PLA2c domain (CDD accession: cd00125), prostaglandin endoperoxide synthase domain (PES, CDD accession: cd09816), lipocalin domain (CDD accession: pfam00061), albumin domain (CDD accession: cd00015), the ligand binding domain of peroxisome proliferator-activated receptors (NR-LBD-PPAR, CDD accession: cd06932) and the ligand binding domain of hepatocyte nuclear factor 4 (NR-LBD-HNF4-like, CDD accession: cd06931). These domains were closely inter-connected in the network as they bound various fatty acids or derivatives. Especially, the PLA2c domain, PES domain, lipocalin domain and albumin domain had relatively stronger associations (higher NSLP scores) to each other, in which the first two domains were closely related to prostaglandin biosynthesis in arachidonic acid metabolism pathway and considered as main targets for NSAIDs; while, the latter two were responsible for transporting lipids, fatty acids and their metabolites in vivo [37,38]. More interestingly, the NR-LBD-HNF4-like domain was also identified in this module, which was recently 'deorphanized' because it could be regulated by fatty acids [39]. This result suggests that domains involved in relevant biological processes/pathways can be identified through the domain network analysis.
On the other hand, some domains involved in different pathways and superfamilies were also observed to form modules through binding common cognate molecules. For instance, the ligand binding domain of thyroid hormone receptors (NR-LBD-TR, CDD accession: cd06935), TLP-Transthyretin domain (CDD accession: cd05821) and the ligand binding domain of androgen receptors (NR-LBD-AR, CDD accession: cd07073) formed a three-node domain module (the green module in Figure 5), because they bound thyroid hormones, thyroxine (PubChem CID: 5819), triiodothyronine (PubChem CID: 5920) and a derivative, triac (PubChem CID: 5803). Despite of belonging to different superfamilies, the first two domains are known to participate in the thyroid hormone transportation and signaling process; while the NR-LBD-AR domain was recently reported to bind thyroid hormones [40]. In fact, some modules consisting of hundreds of domains, such as the NADP or ATP binding domains, were also observed. Thus, proteins containing these highly associated domains can be effectively regulated by few common molecules in vivo.
Notably, domain modules were often inter-connected to some extent, e.g. the three modules shown in Figure 5. Even within the fatty acids related module (colored in blue), we can clearly identify a sub-module consisting of the PLA2c domain, PES domain, albumin domain and lipocalin domain, which inter-connected to each other with strong associations. Indeed, these four domains were also observed in larger modules including the ATP related module and NADP related module. To characterize how the domains or domain modules were organized over the entire network, we investigated the distribution of clustering coefficient and node degree. For a node, the higher the clustering coefficient is, the more likely its neighbors are inter-connected. We found that the clustering coefficients were inversely proportional to the node degrees in general (Supplement figure S2), suggesting that the nodes within a module tend to have higher clustering coefficients, and the nodes with relatively lower clustering coefficients but higher degrees are responsible for integrating domain modules. Similar phenomenon was also observed in other networks that were in hierarchical organization [41][42][43].
In summary, these results indicate that small-molecule binding domains, sharing the same biochemical mechanism (or within one superfamily), being involved in relevant biological pathways, or binding common cofactors, can be identified in the network as domain modules. The results reveal new relationships of protein domains, which may be hardly detected through conventional protein sequence or structure based approaches.
Protein domain associations for drug target identification
It is widely accepted that many marketed drugs are derived from natural products or known drugs [44][45][46]. Thus, it is of great interest to study whether the domain associations identified in this work can be used to infer potential drug targets for drug repurposing. Among the small moleculedomain interaction dataset, we found a total of 252 drugdomain pairs, corresponding to 147 marketed drugs and 135 protein domains (Supplement table S4). A domain network showing interactions between drugs and their protein domain targets was built, and a sub-network including the three domain modules discovered in the previous section is shown in Figure 6.
Based on this network, we successfully identified potential targets for some known drugs, which were retrospectively verified by literature search (shown in Figure 6). For example, in the fatty acids related module (colored in blue), we observed that three NSAIDs, i.e. dexibuprofen, indomethacin (PubChem CID: 3715) and diclofenac (PubChem CID: 3033), respectively interacted with several domains (solid lines in grey in Figure 6), including the PLA2c domain and PES domain. Considering the strong associations among domains in this module, one may be interested in repositioning these drugs to other domain members. Some of the predicted drug-domain associations were confirmed by literature mining (dashed line in green in Figure 6). For instance, diclofenac was reported to bind to NR-LBD-PPAR [47], albumin [48] and lipocalin [49]; and doi: 10.7243/2050-2273- [1][2][3][4][5][6] indomethacin was found binding to albumin as well [50]. Especially, it has been reported that the NR-LBD-PPAR domain contained proteins, such as peroxisome proliferatoractivated receptor gamma, can be activated by many NSAIDs, including ibuprofen (PubChem CID: 3672) and flufenamic acid (PubChem CID: 3371) that produce adipogenesis and peroxisome activity in vivo [51]. Thus, we may anticipate more hidden interactions with NSAIDs to be discovered by conducting a systemic assay against all protein domains in this module. Likewise, ethoxzolamide (PubChem CID: 3295) could be successfully repositioned as a ligand for other member domains in the CA module (dashed green line in Figure 6), though it only bound to two domains according to the current dataset (solid grey line in Figure 6). In fact, this CA inhibitor can inhibit almost all CA isoforms in many tissues and organs, producing various inhibitory profiles and clinical applications [36].
Moreover, we could infer potential domain targets from neighboring modules. For instance, the TLP-Transthyretin domain (colored in green in Figure 6), which is responsible for transporting thyroid hormones and retinol in vertebrates, connected to several domains in the fatty acid module (colored in blue in Figure 6), though the associations were relatively weak compared to the ones within the modules. Several drugs, including levothyroxine and diflunisal, were found binding to both the fatty acid module (colored in blue in Figure 6) and the thyroid hormone related module (colored in green in Figure 6) based on the current network, hence it would be interesting to explore whether other drugs can bind to the domains across these two modules as well. From literatures, we found that flufenamic acid, a ligand of the PES domain from the fatty acid module [52], was able to bind to the NR-LBD-PPAR domain of the same module [51], as well as the other two domains, the NR-LBD-AR domain and TLP-Transthyretin domain, in the thyroid hormone related module (dashed line in green in Figure 6). In addition, a plant-derived naphthoquinone, shikonin (PubChem CID: 479503), which did not show interaction with either the fatty acid module or the thyroid hormone related module based on the current dataset, was reported to bind to both NR-LBD-TR domain contained receptors (PubChem AID: 1479) and PES domain contained receptors, including cyclooxygenase-1 and -2 (COX1 and COX2) [53] (dashed line in green in Figure 6). Furthermore, it has also been reported that NSAIDs indeed compete with thyroid hormone binding in vivo [54,55]. Similarly, based on the observed connection between the two neighboring modules (CA module and fatty acid module) due to celecoxib (PubChem CID: 2662), a selective COX2 inhibitor with nanomolar activity against the carbonic anhydrase [56], we successfully verified a hidden interaction of alpha-CA-I-II-III-XIII domain with indomethacin, a ligand of the PES domain [57,58].
Our analysis indicates that additional drug targets may be suggested based on the modules from the domain interaction network. Thus, it demonstrates again that the constructed domain network can be used in drug target identification for drug repurposing.
Discussion
In the present study, we systematically investigated the protein domain associations from the small-molecule binding point of view on a large scale, based on the physical interactions extracted from the PDB structures. To the best of our knowledge, this is the first large-scale study on protein domain associations in with respect to smallmolecule binding. Conventionally, proteins and protein domains are classified into families or superfamilies according to the similarity in protein sequence, structure or biochemical reaction. Thus, proteins from the same family or superfamily are believed to have similar or relevant functions in vivo. But, the inter-relationship among families or superfamilies, especially regarding to their interactions with small molecules, has rarely been investigated. In this work, we identified a novel relationship that most small-molecule binding protein domains, despite distributing over different superfamilies, biological pathways, tissues or organs, were comprehensively associated through binding the same or similar small molecules. On the other hand, the development of systems biology provides great opportunities to interfere biological organisms on the system level, for example, modulating multiple targets for disease treatment. The approach in the present study can be used, not only to identify protein targets that are potentially modulatable by small molecules within a pathway, but also to detect the associations among these targets with respect to small-molecule interactions for the multiple-point control of a biological pathway. Notably, this strategy can also identify the inter-connections across biological pathways by using protein domain associations obtained in this study. Through interacting with such protein targets, the involved biological pathways may be affected or regulated by few small molecules including drugs, to generate various biological effects or pharmacological efficacies in vivo. Therefore, this study may provide a complementary insight into the complex biological systems from the small-molecule binding point of view, compared to the traditional approaches.
Moreover, the identification of comprehensive associations among small-molecule binding domains coincides with the fact that an increasing number of drugs are found to bind to multiple protein targets [59][60][61]. The concept of "one drug, one target, on disease" has dominated the field of drug discovery for years, and there has been a long-standing controversy over the count of drug targets in human genome [28,[62][63][64][65][66][67][68]. Until recently, substantial evidences [69,70] have shown that many successfully marketed drugs, especially those for polygenic diseases (eg. cancer, cardiovascular diseases [60] and depression [71]) turn out to interact with multiple targets, though they were originally developed against a single or specific target [60,72]. The mechanisms of action of these drugs for curing polygenic diseases suggest that the count of drug targets may no longer be a substantial question, and the challenge is how to identify potential targets including anti-targets for known drugs, and how to combine multiple drug targets to produce a desirable therapeutic effect. The domain network constructed in this study, though based on an arguably limited dataset of the PDB structures, has demonstrated its capability of inferring potential targets for marketed drugs. The present study may shed a light on systematic identification of drug targets for drug repurposing and network pharmacology.
In addition, the other side of the coin is that a considerable number of adverse drug reactions are due to drug interaction with unintended targets [73]. Similar to drug repurposing, the strategy reported in this study may be used to predict potential off-target interactions for drugs based on the domain binding profile, and to suggest a group of off-target candidates for drug safety evaluation in preclinical research. In the future studies, we will aim to build an interactive web service and a tool for researchers to explorer protein domain network with additional links to biological pathways, disease information and bioactive molecules including drugs available in public domains.
Conclusions
In this work, we studied the protein domain associations with respect to small-molecule binding on a large scale. Based on the physical interactions of small molecules and protein domains derived from the PDB structures, we characterized the pairwise domain associations, as well as the global relationship from a network point of view. The results indicate that protein domains are widely inter-connected through binding the same or similar small molecules, which can hardly be found via traditional protein sequence or structure based approaches. Most closely related domains further constituted domain modules in the network, through sharing similar mechanism, being involved in relevant biological processes/pathways, or binding common cofactors,. Moreover, using the domain associations identified in this study as guidance, we successfully inferred potential targets for marketed drugs and verified them by literature mining. Collectively, the results reported in the present study, not only provide an insight into the complex role of small molecules involved in biological systems, but also demonstrate a global view of protein domain inter-relationship for small-molecule bindings. The strategy used in this work may shed a light on network pharmacology study and target identification for drug repurposing, as well as chemogenomic research. | v3-fos-license |
2018-10-17T02:12:55.945Z | 2012-05-23T00:00:00.000 | 11119665 | {
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} | pes2o/s2orc | Proteins Involved in Otoconia Formation and Maintenance
The vestibule of the inner ear senses head motion for spatial orientation and bodily balance. In vertebrates, the vestibular system consists of three fluid filled semicircular canals, which detect rotational acceleration, and two gravity receptor organs, the utricle and saccule, which respond to linear acceleration and gravity (Figure 1). The utricle and saccule are also referred to as the otolithic organs because they contain bio-crystals called otoconia (otolith in fish). These crystals are partially embedded in a honeycomb layer atop a fibrous meshwork, which are the otoconial complex altogether. This complex rests on the stereociliary bundles of hair cells in the utricular and saccular sensory epithelium (aka macula). When there is head motion, the otoconial complex is displaced against the macula, leading to deflection of the hair bundles. This mechanical stimulus is converted into electrical signals by the macular hair cells and transmitted into the central nervous system (CNS) through the afferent vestibular nerve. In the CNS, these electrical signals, combined with other proprioceptive inputs, are interpreted as position and motion data, which then initiate a series of corresponding neuronal responses to maintain the balance of the body. Electrophysiological and behavioral studies show that the size and density of these tiny biominerals determine the amount of stimulus input to the CNS (Anniko et al. 1988; Jones et al. 1999; Jones et al. 2004; Kozel et al. 1998; Simmler et al. 2000a; Trune and Lim 1983; Zhao et al. 2008b).
Introduction
The vestibule of the inner ear senses head motion for spatial orientation and bodily balance.In vertebrates, the vestibular system consists of three fluid filled semicircular canals, which detect rotational acceleration, and two gravity receptor organs, the utricle and saccule, which respond to linear acceleration and gravity (Figure 1).The utricle and saccule are also referred to as the otolithic organs because they contain bio-crystals called otoconia (otolith in fish).These crystals are partially embedded in a honeycomb layer atop a fibrous meshwork, which are the otoconial complex altogether.This complex rests on the stereociliary bundles of hair cells in the utricular and saccular sensory epithelium (aka macula).When there is head motion, the otoconial complex is displaced against the macula, leading to deflection of the hair bundles.This mechanical stimulus is converted into electrical signals by the macular hair cells and transmitted into the central nervous system (CNS) through the afferent vestibular nerve.In the CNS, these electrical signals, combined with other proprioceptive inputs, are interpreted as position and motion data, which then initiate a series of corresponding neuronal responses to maintain the balance of the body.Electrophysiological and behavioral studies show that the size and density of these tiny biominerals determine the amount of stimulus input to the CNS (Anniko et al. 1988;Jones et al. 1999;Jones et al. 2004;Kozel et al. 1998;Simmler et al. 2000a;Trune and Lim 1983;Zhao et al. 2008b).
Otoconia dislocation, malformation and degeneration can result from congenital and environmental factors, including genetic mutation, aging, head trauma and ototoxic drugs, and can lead to various types of vestibular dysfunction such as dizziness/vertigo and imbalance.In humans, BPPV (benign paroxysmal positional vertigo), the most common cause of dizziness/vertigo, is believed to be caused by dislocation of otoconia from the utricle to the ampulla and further in the semicircular canals (Salvinelli et al. 2004;Schuknecht 1962;Schuknecht 1969;Squires et al. 2004).In animals, otoconial deficiency has been found to produce head tilting, swimming difficulty, and reduction or failure of the airrighting reflexes (Everett et al. 2001;Hurle et al. 2003;Nakano et al. 2008;Paffenholz et al. 2004;Simmler et al. 2000a;Zhao et al. 2008b).
Despite the importance of these biominerals, otoconial research is lagging far behind that of other biomineralized structures, such as bone and teeth, partly due to anatomical and methodological constraints.The mechanisms underlying otoconia formation and maintenance are not yet fully understood.In this review, we will summarize the current state of knowledge about otoconia, focusing on the identified compositions and regulatory proteins and their roles in bio-crystal formation and maintenance.Homologs and analogs of these proteins are also found in fish with similar functions but varied relative abundances, but the review will focus on studies using mice as the latter have similar otoconia and inner ear properties as humans.
The roles of otoconial component proteins in crystal formation
Otoconia from higher vertebrates have a barrel-shaped body with triplanar facets at each end (Figure 1C).The core is predominantly organic with a low Ca 2+ level, and is surrounded by a largely inorganic shell of minute crystallites outlined by the organic matrix (Lim 1984;Lins et al. 2000;Mann et al. 1983;Steyger and Wiederhold 1995;Zhao et al., 2007).Most primitive fishes have apatite otoliths, more advanced fishes have aragonite otoliths, whereas higher levels of vertebrates have calcite otoconia (Carlstrom D 1963;Ross and Pote 1984).Otoliths in lower vertebrates display a daily growth pattern, whereas otoconia in mammals are formed during late embryonic stages, become mature shortly after birth and may undergo maintenance thereafter (Salamat et al. 1980;Thalmann et al. 2001) (Lundberg, unpublished data).Because otoconia/otoliths from animals of different evolutionary levels all have the common CaCO 3 component but have various morphologies and crystalline structures and different protein compositions, otoconins (a collective term for otoconial component proteins) must be important for otoconia formation.More importantly, as the mammalian endolymph has an extremely low Ca 2+ concentration, otoconins may be essential for CaCO 3 crystal seeding.
Otoconin-90 (Oc90) is the essential organizer of the otoconial matrix
Oc90 is the first identified otoconin, and accounts for nearly 90% of the total protein content of otoconia (Pote and Ross 1991;Verpy et al. 1999;Wang et al. 1998).Subsequent studies have revealed that Oc90 is the essential organizer of the otoconial organic matrix by specifically recruiting other matrix components and Ca 2+ (Yang et al. 2011;Zhao et al. 2007).
Oc90 is structurally similar to secretory phospholipase A2 (sPLA2).Although it likely does not have the catalytic activity of the enzyme due to the substitutions of a few essential residues in the active site (Pote and Ross 1991;Wang et al. 1998), Oc90 possesses the other features of sPLA2.It is a cysteine-rich secretory protein, and has several glycosylation sites and calcium binding capability.The enriched cysteine residues are likely involved in the formation of higher-order protein structures via intra-and inter-molecular disulfide bonds.The intra-molecular disulfide bonds play an important role in protein folding and the stabilization of the tertiary structure, while the disulfide bonds formed between subunits allow dimerization and oligomerization of the protein.
Type
Protein name Otoconia phenotype of mutant mice Reference
------Table 1. Identified and validated murine otoconial proteins and their importance in otoconia formation by genetic mutation studies.Shaded ones have no measurable impact on bio-crystal formation.---, no mutant mice available or unknown otoconia/otolith phenotype.
The Ca 2+ concentrations of the mammalian endolymph are extremely low at ~20 µM (Ferrary et al. 1988;Salt et al. 1989), with a few reporting much higher in the vestibule (Marcus and Wangemann 2009;Salt et al. 1989).This is much lower than what is necessary for the spontaneous formation of calcite crystals, therefore, otoconial proteins are speculated to sequester Ca 2+ .Indeed, most of the otoconial proteins have structural features for Ca 2+ binding.Oc90 has 28 (~6%) Glu and 39 (~8%) Asp out of the total 485 amino acids, endowing the molecule with a calculated acidic isoelectric point (pI = 4.5).The measured pI of mature Oc90 is even lower (2.9) due to post-translational modifications such as N-linked glycosylation (Lu et al. 2010).This extreme acidic feature may help Oc90 recruit Ca 2+ and/or interact with the surface of calcium carbonate crystals to modulate crystal growth.Deletion of Oc90 causes dramatic reduction of matrix-bound Ca 2+ in the macula of the utricle and saccule (Yang et al. 2011).In the absence of Oc90, the efficiency of crystal formation is reduced by at least 50%, and www.intechopen.com Proteins Involved in Otoconia Formation and Maintenance 7 the organic matrix is greatly reduced, leading to formation of a few giant otoconia with abnormal morphology caused by unordered aggregation of inorganic crystallites (Zhao et al. 2007).A subsequent in vitro experiment has also demonstrated that Oc90 can facilitate nucleation, determine the crystal size and morphology in a concentration-dependent manner (Lu et al. 2010).Recent evidence suggests that the formation of otoconia at all in Oc90 null mice may be partially attributed to the compensatory deposition of Sc1 (Xu et al. 2010).
The expression of Oc90 temporally coincides that of otoconia development and growth, also providing evidence for the critical requirement of Oc90 in this unique biomineralization process.Oc90 expression is the earliest among all otolith/otoconia proteins in fish and mice (before embryonic day E9.5 in mice) (Petko et al. 2008;Verpy et al. 1999;Wang et al. 1998), much earlier than the onset of any activities of ion channels/pumps, or the onset of otoconia seeding at around E14.5.Oc90 then recruits other components at the time of their expression to form the organic matrix for calcification (Zhao et al. 2007).When otoconia growth stops at around P7 (postnatal day 7), the expression level of Oc90 significantly decreases in the utricle and saccule (Xu and Lundberg 2012).Although Oc90 has a relatively low abundance in zebrafish otoliths (known as zOtoc1) (Petko et al. 2008), Oc90 morphant fish show more severe phenotypes than morphants for the main otolith matrix protein OMP1 (Murayama et al. 2005;Petko et al. 2008), suggesting that zOc90 (zOtoc1) is essential for the early stages of otolith development (i.e.crystal seeding) whereas OMP regulates crystal growth.Thus, the structure and function of Oc90 is conserved from bony fish to mice (two model systems whose otoconia/otolith are the most studied) regardless of the abundance of the protein in each species.
Sc1 can partially compensate the function of Oc90
Sc1 was first isolated from a rat brain expression library (Johnston et al. 1990).It is widely expressed in the brain and can be detected from various types of neurons (Lively et al. 2007;McKinnon and Margolskee 1996;Mendis and Brown 1994).As a result, studies of Sc1 have focused on the nervous system.Recently, Thalmann et al. identified Sc1 from mouse otoconia by mass spectrometry (Thalmann et al. 2006).However, Xu et al. (Xu et al. 2010) found that Sc1 was hardly detectable in the wild-type otoconia.Instead, the deposition of Sc1 was drastically increased in otoconia crystals when Oc90 is absent, suggesting a possible role for Sc1 as an alternative process of biomineralization (Xu et al. 2010).Sc1 knockout mice did not show any obvious phenotypic abnormalities, including vestibular functions (McKinnon et al. 2000)(S. Funk andH. Sage, communication through Thalmann et al. 2006).
Although Sc1 and Oc90 have no significant sequence similarity, the two proteins share analogous structural features.Murine Sc1 is a secreted, acidic and Cys-rich glycoprotein, and belongs to the Sparc family.Its Sparc-like domain consists of a follistatin-like domain followed by an -helical domain (EC) containing the collagen-binding domain and 2 calcium-binding EF-hands (Maurer et al. 1995).All of these features likely render Sc1 an ideal alternative candidate for otoconia formation in the absence of Oc90.The high abundance of Glu/Asp residues (52 Glu and 87 Asp out of 634 aa) makes the protein highly acidic (pI = 4.2), which, together with the EF-hand motif, provides Sc1 a high affinity for calcium and calcium salts (e.g.calcium carbonate and phosphate).The collagen-binding site in the EC domain can recognize the specific motif of the triple-helical collagen peptide and form a deep 'Phe pocket' upon collagen binding (Hohenester et al. 2008;Sasaki et al. 1998).
The follistatin domain was reported to modulate the process of collagen-binding even though it does not interact with collagen directly (Kaufmann et al. 2004).In addition, the enriched cysteines in the polypeptide backbone of Sc1 may enable the formation of numerous intra-and inter-molecular disulfide bridges, as well as dimerization or even oligomerization of the protein, all of which enable the protein to serve as a rigid and stable framework for inorganic crystal deposition and growth (Chun et al. 2006;Xu et al. 2010).
Otolin may function similarly to collagen X
Otolin is a secreted glycoprotein present in both otoconial crystals and membranes.The expression level of otolin mRNA in the utricle and saccule is much higher than that in the epithelia of non-otolithic inner ear organs (Yang et al. 2011), implicating a potentially critical role of this molecule in otoconia development.In fish, knockdown of otolin led to formation of fused and unstable otoliths (Murayama et al. 2005).
Otolin contains three collagen-like domains in the N-terminal region and a highly conserved globular C1q (gC1q) domain in the C-terminal region, and belongs to the collagen X family and C1q super-family (Deans et al. 2010;Kishore and Reid 1999;Yang et al. 2011).Like collagen X, the N-terminal collagen domains of otolin contain tens of characteristic Gly-X-Y repeats, which can facilitate the formation of collagen triple helix and higher-order structures.Such structural features in otolin may render the protein extremely stable.The Cterminal gC1q domain is more like a target recognition site which may mediate the interaction between otolin and other extracellular proteins.Co-immunoprecipitation experiments demonstrated that Oc90 can interact with both the collagen-like and C1q domains of otolin to form the otoconial matrix framework and to sequester Ca 2+ for efficient otoconia calcification.Co-expression of Oc90 and otolin in cultured cells leads to significantly increased extracelluar matrix calcification compared with the empty vector, or Oc90 or otolin single transfectants (Yang et al. 2011).Analogously, otolith matrix protein-1 (OMP-1), the main protein in fish otoliths, is required for normal otolith growth and deposition of otolin-1 in the otolith (Murayama et al. 2004;Murayama et al. 2005).
Keratin sulfate proteoglycan (KSPG) may be critical for otoconia calcification
Proteoglycans are widely distributed at the cell surface and in the extracellular matrix, and are critical for various processes such as cell adhesion, growth, wound healing and fibrosis (Iozzo 1998).A proteoglycan consists of a 'core protein' with covalently attached glycosaminoglycan (GAG) chains.They can interact with other proteoglycans and fibrous matrix proteins, such as collagen, to form a large complex.In addition, proteoglycans have strong negative charges due to the presence of sulfate and uronic acid groups, and can attract positively charged ions, such as Na + , K + and Ca 2+ .All those features make proteoglycans important players in the extracellular calcification processes.Indeed, both heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) are critical for bone and teeth formation.Deletion of those proteins results in various calcification deficiencies (Hassell et al. 2002;Viviano et al. 2005;Xu et al. 1998;Young et al. 2002).
In the inner ear, however, KSPG appears to be the predominant proteoglycan (Xu et al. 2010).KSPG has been detected in chicken and chinchilla otoconia, and shows strong staining in murine otoconia as well (Fermin et al. 1990;Swartz and Santi 1997;Xu et al. 2010).The role of KSPG in otoconia development has not been elucidated yet.It may participate in sequestering and retaining Ca 2+ for crystal formation because of its strong negative charges.In vitro immunoprecipitation results demonstrated that it may interact with Oc90 and otolin to form the matrix framework for the deposition of calcite crystals (Yang et al. 2011).
Some low abundance otoconins may be dispensable for otoconia formation
Most of the low abundance otoconial proteins play critical roles in bone and/or tooth formation.In contrast, studies by us and other investigators using existing mutant mice have demonstrated that a few of these proteins are dispensable or functionally redundant for otoconia development.
For example, osteopontin, a multifunctional protein initially identified in osteoblasts, is a prominent non-collagen component of the mineralized extracellular matrices of bone and teeth.Osteopontin belongs to the small integrin-binding N-linked glycoprotein (SIBLING) family.As a SIBLING member, osteopontin has an arginine-glycine-aspartate (RGD) motif, which plays an essential role in bone resorption by promoting osteoclast attachment to the bone matrix through cell surface integrins (Oldberg et al. 1986;Rodan and Rodan 1997).
Similar to the role of Oc90 in otoconia development, osteopontin acts as an important organizer in bone mineralization.It modulates the bone crystal sizes by inhibiting the hydroxyapatite formation and growth (Boskey et al. 1993;Hunter et al. 1994;Shapses et al. 2003).Osteopontin null mice have altered organization of bone matrix and weakened bone strength, leading to reduced bone fracture toughness (Duvall et al. 2007;Thurner et al. 2010).However, despite its presence in otoconia and vestibular sensory epithelia, osteopontin is dispensable for otoconia formation, and osteopontin knockout mice show normal vestibular morphology and balance function (Zhao et al. 2008a).
Dentin matrix acidic phosphoprotein 1 (DMP1) is another protein that belongs to the SIBLING family.DMP1 was first cloned from dentin and then found in bone.It plays a critical role in apatite crystal seeding and growth in bone and teeth (George et al. 1993;Hirst et al. 1997;MacDougall et al. 1998).DMP1 null mice show severe defects in bone structure.Lv et al. (Lv et al. 2010) recently found that DMP1 null mice developed circling and head shaking behavior resembling vestibular disorders.They attributed these phenotypes to bone defects in the inner ear.However, it should not be excluded that DMP1 deficiency may affect otoconia as the protein is also present in mouse otoconia at a low level (Xu et al. 2010).
Sparc, aka BM-40 or osteonectin, is generally present in tissues undergoing remodeling such as skeletal remodeling and injury repair (Bolander et al. 1988;Hohenester et al. 1997;Sage and Vernon 1994).The protein is a normal component of osteiod, the newly formed bone matrix critical for the initiation of mineralization during bone development (Bianco et al. 1985;Termine et al. 1981).Sparc has a high affinity for both Ca 2+ and several types of collagen (Bolander et al. 1988;Hohenester et al. 2008;Maurer et al. 1995).These features likely account for the importance of Sparc in bone formation, and possibly in otoconia formation.Indeed, Sparc is also required for otolith formation in fish (Kang et al. 2008).In the wild-type murine otoconia, however, Sparc is present at an extremely low level (Xu et al. 2010) that it may not play a significant role in crystal formation.Instead, the longer form Sc1 is the preferred scaffold protein when Oc90 is absent (Xu et al. 2010).
Fetuin-A, also known as 2-HS-glycoprotein or countertrypin, is a hepatic secreted protein that promotes bone mineralization.It is among the most abundant non-collagen proteins found in bone (Quelch et al. 1984).Several recent studies demonstrated that fetuin-A can bind calcium and phosphate to form a calciprotein particle and prevent the precipitation of these minerals from serum (Heiss et al. 2003;Price et al. 2002), which may explain the role of fetuin-A in bone calcification and its potent inhibition of ectopic mineralization in soft tissues (Schafer et al. 2003;Westenfeld et al. 2007;Westenfeld et al. 2009).However, fetuin-A null mice have normal bone under regular dietary conditions (Jahnen-Dechent et al. 1997).
Fetuin-A is present in otoconia crystals (Zhao et al. 2007), but null mice for the protein do not show balance deficits (Jahnen-Dechent, communication in Thalmann et al., 2006), therefore, it is unlikely that the protein has a major impact on otoconia genesis.
Taken together, findings on these low abundance otoconins indicate similarities and differences between bone and otoconia biomineralization.
The roles of regulatory proteins in otoconia formation
Otoconia formation depends on both organic and inorganic components that are secreted into the vestibular endolymph.Non-component regulatory proteins affect otoconia development and maintenance likely by several ways: (1) by influencing the secretion (Sollner et al. 2004), structural and functional modification of the component and anchoring proteins (Lundberg, unpublished data), and (2) by spatially and temporally increasing chemical gradients of Ca 2+ , HCO 3 -, H + and possibly other ions/anions to establish an appropriate micro-environmental condition for crystal seeding and growth.
NADPH oxidase 3 (Nox3) and associated proteins are essential for otoconia formation
The Noxs are a family of enzymes whose primary function is to produce ROS (reactive oxygen species).These proteins participate in a wide range of pathological and physiological processes.To date, seven Nox family members, Nox1-Nox5, Duox1 and Duox2, have been identified in mammals (Bedard and Krause 2007).Noxs serve as the core catalytic components, and their activities are regulated by cytosolic partners such as p22 phox , Nox organizers (Noxo1, p47 phox and p40 phox ), and Nox activators (Noxa1 and p67 phox ).
Among the identified Nox family members, Nox3 is primarily expressed in the inner ear and is essential for otoconia development (Banfi et al. 2004;Cheng et al. 2001;Paffenholz et al. 2004).It interacts with p22 phox and Noxo1 to form a functional NADPH oxidase complex, and all three components are required for otoconia development and normal balance in mice (Kiss et al. 2006;Nakano et al. 2007;Nakano et al. 2008;Paffenholz et al. 2004).However, the mechanisms underlying the requirement of Nox-related proteins for otoconia formation are poorly understood.One possible role of Nox3 is to oxidize otoconial proteins, including Oc90, which then undergo conformational changes to trigger crystal nucleation.Indeed, our recent unpublished data show that Nox3 modifies the structures of a few otoconia proteins (Xu et al. 2012).
A novel mechanism proposed by Nakano et al. (Nakano et al. 2008) states that while the Nox3complex passes electrons from intracellular NADPH to extracellular oxygen, the plasma membrane becomes depolarized.Such depolarization of the apical membrane would elevate endolymphatic Ca 2+ concentration by preventing cellular Ca 2+ uptake from endolymph, and by increasing paracellular ion permeability to allow Ca 2+ influx from perilymph to endolymph.In addition, Nox3-derived superoxide may react with endolymphatic protons and thereby elevate the pH so that CaCO 3 can form and be maintained.
Otopetrin 1 may mobilize Ca 2+ for CaCO 3 formation
Otopetrin (Otop1), a protein with multiple transmembrane domains, is essential for the formation of otoconia/otolith in the inner ear (Hughes et al. 2004;Hurle et al. 2003;Sollner et al., 2004).The protein is conserved in all vertebrates, and its biochemical function was first revealed by studying the phenotypes of two mutants, the tilted (tlt) and mergulhador (mlh) mice, which carry single-point mutations in the predicted transmembrane (TM) domains (tlt, Ala 151 Glu in TM3; mlh, Leu 408 Gln in TM9) of the Otop1 gene.Both tlt and mlh homozygous mutant mice show non-syndromic vestibular disorders caused by the absence of otoconia crystals in the utricle and saccule (Hurle et al. 2003;Zhao et al. 2008b).
Those mutations in Otop1 do not appear to affect other inner ear organs, making tlt and mlh excellent tools to investigate how Otop1 participates in the development of otoconia and in what aspects the absence of otoconia impacts balance functions.
In fish, expression of Otop1 is in both hair cells and supporting cells before otolith seeding, but is restricted in hair cells during otolith growth (Hurle et al. 2003;Sollner et al. 2004).In mice, Otop1 exhibits complementary mRNA expression pattern with Oc90 in the developing otocyst, and high Otop1 protein level is visible in the gelatinous membrane overlying the sensory epithelium, suggesting that it may be integral to the membrane vesicles released into the gelatinous layer (Hurle et al. 2003).However, a more recent study by Kim and colleagues using a different antibody (Kim et al. 2010) demonstrated that Otop1 is expressed in the extrastriolar epithelia of the utricle and saccule, and is specifically localized in the apical end of the supporting cells and a subset of transitional cells.They also found that the tlt and mlh mutations of Otop1 change the subcelluar localization of the mutant protein, and may underlie its function in otoconia development (Kim et al. 2011).
Both in vitro and ex vivo studies demonstrated that one of the functions of Otop1 is to modulate intra-and extracellular Ca 2+ concentrations by specifically inhibiting purinergic receptor P2Y, depleting of endoplasmic reticulum Ca 2+ stores and mediating influx of extracellular Ca 2+ (Hughes et al. 2007;Kim et al. 2010).Under normal conditions, the concentration of Ca 2+ in the mammalian endolymph is much lower than that in the perilymph and other extracellular fluids, and is insufficient to support normal growth of otoconia.Hence, Otop1 may serve as the indispensible Ca 2+ source that supports otoconia mineralization.
Moreover, Otop1 may also regulate the secretion of components required for otoconia formation.In zebrafish, Otop1 was shown to affect the secretion of starmaker, a protein essential for otolith formation, in the sensory epithelia (Sollner et al. 2004).
PMCA2 is a critical source of Ca 2+ for CaCO 3 formation
Calmodulin-sensitive plasma membrane Ca 2+ -ATPases (PMCAs) are vital regulators of otoconia formation by extruding Ca 2+ from hair cells and thereby maintaining the appropriate Ca 2+ concentration near the plasma membrane.There are four isoforms of mammalian PMCA (PMCA1-4) encoded by four distinct genes and each of them undergoes alternative exon splicing in two regions (Keeton et al. 1993).All four PMCAs are expressed in the mammalian cochlea and extrude Ca 2+ from hair cell stereocilia, whereas PMCA2a, a protein encoded by Atp2b2 gene, is the only PMCA isoform present in vestibular hair bundles (Crouch and Schulte 1996;Dumont et al. 2001;Furuta et al. 1998;Yamoah et al. 1998).Null mutation in Atp2b2 results in the absence of otoconia and subsequent balance deficits (Kozel et al. 1998), underpinning the importance of PMCA2 in otoconial genesis.
Pendrin regulates endolymph pH, composition and volume
Pendrin, encoded by Slc26a4, is an anion transporter which mediates the exchange of Cl -, I -, OH -, HCO 3 -, or formate, across a variety of epithelia (Scott et al. 1999;Scott and Karniski 2000).In the inner ear, pendrin is primarily expressed in the endolymphatic duct and sac, the transitional epithelia adjacent to the macula of the utricle and saccule, and the external sulcus of the cochlea (Everett et al. 1999).Pendrin is critical for maintaining the appropriate anionic and ionic composition and volume of the endolymphatic fluid, presumably due to HCO 3 -secretion.Mutations in human SLC26A4 are responsible for Pendred syndrome, a genetic disorder which causes early hearing loss in children (Dai et al. 2009;Luxon et al. 2003).Studies using an Slc26a4 knockout mouse model have revealed that pendrin dysfunction can cause an enlargement and acidification of inner ear membrane labyrinth and thyroid at embryonic stages, leading to deafness, balance disorders and goiter similar to the symptoms of human Pendred syndrome (Everett et al. 2001;Kim and Wangemann 2010;Kim and Wangemann 2011).The mice have much lower endolymphatic pH, resulting in the formation of giant crystals with reduced numbers in both the utricle and saccule (Everett et al. 2001;Nakaya et al. 2007).Recently, Dror et al. have also demonstrated that a recessive missense mutation within the highly conserved region of slc26a4 results in a mutant pendrin protein with impaired transport activity.This mutant mouse has severely abnormal mineral composition, size and shape of otoconia, i.e., giant CaCO 3 crystals in the utricle at all ages, giant CaOx crystals in the saccule of older adults, and ectopic giant stones in the crista (Dror et al. 2010).Therefore, pendrin participates in otoconia formation through providing HCO 3 -, which is essential for forming CaCO 3 crystals and for buffering the endolymphatic pH.Pendrin can also buffer pH through other anions such as formate.
3.5 Carbonic anhydrase (CA) provides HCO 3 -and maintains appropriate pH for otoconia formation and maintenance CA catalyzes the hydration of CO 2 to yield HCO 3 -and related species, and is thus thought to be important for otoconia formation by producing HCO 3 -and keeping appropriate endolymph pH.CA is widely present in the sensory and non-sensory epithelia of the inner ear (Lim et al. 1983;Pedrozo et al. 1997), especially the developing endolymphatic sac of mammalian embryos contain high levels of CA.Administration of acetazolamide, a CA inhibitor, in the latter tissue can decrease the luminal pH and HCO 3 -concentration (Kido et al. 1991;Tsujikawa et al. 1993).Injection of acetazolamide into the yolk sac of developing chick embryos alters and inhibits normal otoconial morphogenesis (Kido et al. 1991).Activation/deactivation of macular CA under different gravity is associated with changes in otolith sizes in fish (Anken et al. 2004).Immunohistochemstry shows that CAII is coexpressed with pendrin in the same cells in the endolymphatic sac, suggesting that those two proteins may cooperate in maintaining the normal function of the endolymphatic sac (Dou et al. 2004), which is an important tissue for endolymph production.
In addition to CA, HCO 3 --ATPase and Cl -/HCO 3 --exchangers are involved in the transepithelial transport of bicarbonate ions to the endolymph, and affect carbon incorporation into otoliths (Tohse and Mugiya 2001).
Transient receptor potential vanilloids (TRPVs) may also regulate endolymph homeostasis
Studies suggest that TRPVs may also play an important part in fluid homeostasis of the inner ear.All TRPVs (TRPV1-6) are expressed in vestibular and cochlear sensory epithelia (Ishibashi et al. 2008;Takumida et al. 2009).In addition, TRPV4 is also present in the endolymphatic sac and presumably acts as an osmoreceptor in cell and fluid volume regulation (Kumagami et al. 2009).Both TRPV5 and TRPV6 are found in vestibular semicircular canal ducts (Yamauchi et al. 2010).In pendrin-deficient mice, the acidic vestibular endolymphatic pH is thought to inhibit the acid-sensitive TRPV5/6 calcium channels and lead to a significantly higher Ca 2+ concentration in the endolymph, which may be another factor causing the formation of abnormal otoconia crystals (Nakaya et al. 2007).However, direct evidence has yet to be presented on whether TRPV-deficiency will lead to otoconia abnormalities.
The roles of anchoring proteins in the pathogenesis of otoconia-related imbalance and dizziness/vertigo
The inner ear acellular membranes, namely the otoconial membranes in the utricule and saccule, the cupula in the ampulla, and the tectorial membrane in the cochlea, cover their corresponding sensory epithelia, have contact with the stereocilia of hair cells and thus play crutial role in mechanotransduction.In the utricle and saccule, otoconia crystals are attached to and partially embedded in a honeycomb layer above a fibrous meshwork, which are collectively called otoconial membranes, and are responsible for the site-specific anchoring of otoconia.Disruption of the otoconial membrane structure may cause the detachment and dislocation of otoconia and thus vestibular disorders.
The acellular structures of the inner ear consist of collagenous and non-collagenous glycoproteins and proteoglycans.Several types of collagen, including type II, IV, V and IX, have been identified in the mammalian tectorial membrane (Richardson et al. 1987;Slepecky et al. 1992).In the otoconial membranes, however, otolin is likely the main collagenous component.As to the noncollagenous constituents, three glycoproteins, otogelin, -tectorin and -tectorin, have been identified in the inner ear acellular membranes in mice to date (Cohen-Salmon et al. 1997;Legan et al. 1997).The proteoglycan in mouse otoconia is keratin sulfate proteoglycan (KSPG) (Xu et al. 2010).
Otogelin is a glycoprotein that is present and restricted to all acellular membranes of the inner ear (Cohen-Salmon et al. 1997).At early embryonic stages, otogelin is produced by the supporting cells of the sensory epithelia of the developing vestibule and cochlea, and presents a complementary distribution pattern with Myosin VIIA, a marker of hair cells and precursors (El-Amraoui et al. 2001).At adult stages, otogelin is still expressed in the vestibular supporting cells, but become undetectable in the cochlear cells.Otogelin may be required for the attachment of the otoconial membranes and consequently site-specific anchoring of otoconia crystals.Dysfunction of otogelin in either the Otog knockout mice or the twister mutant mice leads to severe vestibular deficits, which is postulated to be caused by displaced otoconial membranes in the utricle and saccule (Simmler et al. 2000a;Simmler et al. 2000b).
-tectorin and -tectorin, named with reference to their localization, are major noncollagenous glycoproteins of the mammalian tectorial membrane (Legan et al. 1997).In addition, these two proteins are abundant constituents of the otoconial membranes, but are not present in the cupula (Goodyear and Richardson 2002;Xu et al. 2010).In the mouse vestibule, -tectorin is mainly expressed between E12.5 and P15 in the transitional zone, as well as in a region that is producing the accessory membranes of the utricle and saccule, but absent in the ampullae of semicircular canals (Rau et al. 1999).Mice with targeted deletion of -tectorin display reduced otoconial membranes and a few scattered giant otoconia (Legan et al. 2000).
-tectorin has a spatial and temporal expression pattern distinct from that of -tectorin in the vestibule.It is expressed in the striolar region of the utricule and saccule from E14.5 until at least P150 (Legan et al. 1997;Rau et al. 1999), suggesting that the striolar and extrastriolar region of the otoconial membranes may have different composition.Tectb null mice show structural disruption of the tectorial membrane and hearing loss at low frequencies (Russell et al. 2007).However, no vestibular defects have been reported.
Interestingly, both otogelin and -tectorin possess several von Willebrand factor type D (VWFD) domains containing the multimerization consensus site CGLC (Mayadas and Wagner 1992).This structural feature is probably essential for the multimer assembly of those proteins to form filament and higher order structures.
Otoancorin is a glycosylphosphatidylinositol (GPI)-anchored protein specific to the interface between the sensory epithelia and their overlying acellular membranes of the inner ear (Zwaenepoel et al. 2002).In the vestibule, otoancorin is expressed on the apical surface of the supporting cells in the utricle, saccule and crista.Although the function of otoancorin has not been elucidated, the C-terminal GPI anchor motif of this protein likely facilitates the otoancorin-cell surface adhesion.It is proposed that otoancorin may interact with the other components of the otoconial membranes, such as otogelin and tectorins, and with the epithelial surface, thus mediating the attachment of otoconial membranes to the underlying sensory epithelia (Zwaenepoel et al. 2002).
Summary and future direction
Like other biominerals such as bone and teeth, otoconia primarily differ from their nonbiological counterparts by their protein-mediated nucleation, growth and maintenance processes.With only CaCO 3 crystallites and less than a dozen glycoprotein/proteoglycan components, otoconia are seemingly simple biological structures compared to other tissues.Yet, the processes governing otoconia formation are multiple and involve many more molecules and much complicated cellular and extracellular events including matrix assembly, endolymph homeostasis and proper function of ion channels/pumps.Expression of the involved genes is well orchestrated temporally and spatially, and the functions of their proteins are finely coordinated for optimal crystal formation.Some of these proteins also play vital roles in normal cellular activities (e.g.hair cell stimulation) and other vestibular function.Some other proteins (e.g.otolin, tectorins and otoancorin) still need to be further investigated of their functions.Animal models with targeted disruption of otolin and otoancorin are not yet available, and animal models with double mutant genes (e.g.Oc90 and Sc1) have not been studied but can yield more information on the precise role of the organic matrix in CaCO 3 nucleation and growth.Additional studies are needed to further uncover the mechanisms underlying the spatial specific formation of otoconia.The high prevalence and debilitating nature of otoconia-related dizziness/vertigo and balance disorders necessitate these types of studies as they are the foundation required to uncover the molecular etiology. | v3-fos-license |
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} | pes2o/s2orc | Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T
The transition of intrinsically disordered, monomeric α-synuclein into β-sheet–rich oligomers and fibrils is associated with multiple neurodegenerative diseases. Fibrillar aggregates possessing distinct structures that differ in toxicity have been observed in different pathological phenotypes. Understanding the mechanism of the formation of various fibril polymorphs with differing cytotoxic effects is essential for determining how the aggregation reaction could be modulated to favor nontoxic fibrils over toxic fibrils. In this study, two morphologically different α-synuclein fibrils, one helical and the other ribbon-like, are shown to form together. Surprisingly, a widely used small molecule for probing aggregation reactions, thioflavin T (ThT), was found to tune the structural heterogeneity found in the fibrils. The ribbon-like fibrils formed in the presence of ThT were found to have a longer structural core than the helical fibrils formed in the absence of ThT. The ribbon-like fibrils are also more toxic to cells. By facilitating the formation of ribbon-like fibrils over helical fibrils, ThT reduced the extent of fibril polymorphism. This study highlights the role of a small molecule such as ThT in selectively favoring the formation of a specific type of fibril by binding to aggregates formed early on one of multiple pathways, thereby altering the structural core and external morphology of the fibrils formed.
The conversion of soluble, functionally active proteins into insoluble, -sheet-rich, aggregated structures is associated with a variety of neurodegenerative diseases such as Parkinson's disease (PD) 2 and Alzheimer's disease (AD) (1)(2)(3)(4). Detailed structural analyses of the characteristics of cross-sheet architecture (5-7) present in these aggregates reveal their polymorphism (8). It is now known that fibrils formed by a single protein can show multiple distinct conformations under different growth conditions (9 -11) as well as under identical growth conditions (12)(13)(14)(15). The importance of studying fibril-lar heterogeneity originated from the prion strain phenomenon, in which a single prion protein is known to cause multiple different pathologies by adopting amyloid-like conformations that differ mainly in their external morphologies and molecular structures (16 -18). A prion strain propagates a specific pathology faithfully by presenting a specific amyloid template for existing monomer to add on to. Proteins other than the prion protein can also acquire different fibrillar morphologies, showing different levels of toxicity, and propagate faithfully (5,10). Importantly, fibrils of different morphologies, which differ in their cytotoxicity levels, have been shown to exist in the brains of different AD patients (8,19). The differences in the toxicity potentials suggest that various fibrils may have different levels of stability, packing, and hydrophobicity (20 -22). It is now clear that fibril polymorphism is responsible for different pathological phenotypes (23). It is therefore important to understand the origin of fibril polymorphism.
In PD, the central molecular species is the protein ␣-synuclein, which is an intrinsically disordered protein of 140 residues. It is expressed mainly in the neurons of the central nervous system (CNS). It acquires helical and -hairpin structure upon binding to membranes and -wrap proteins, respectively (27,28). The function of this protein has not yet been ascertained conclusively, although some studies suggest that it is involved in the process of vesicle release and trafficking (29). ␣-Synuclein is known to aggregate and form Lewy bodies in dopaminergic neurons of the brain. Lewy bodies are made of cross--sheet-rich structured aggregates called amyloid fibrils. The process of ␣-synuclein fibrillation is associated with a variety of neurodegenerative diseases besides PD, including dementia with Lewy bodies (1), AD, and multiple system atrophy (2,3). ␣-Synuclein has been shown to form differently structured amyloid aggregates (10,12,13,15), but there is little understanding of how this happens.
Structural heterogeneity in ␣-synuclein fibrils originates mainly during the multi-step process of fibrillation in which monomers self-assemble into various on-and off-pathway oligomers that vary in size, shape, structure, stability, and packing (30,31). Structurally distinct oligomers appear to grow into different types of fibrillar aggregates with structural cores that resemble the oligomers (31). The structural core of ␣-synuclein fibrils have been characterized by solid-state NMR (10,12,(32)(33)(34), hydrogen-deuterium exchange mass spectrometry (HDX-MS) (31,35), HDX-NMR (13,33), electron paramag-netic resonance (EPR) spectroscopy (36,37), and proteinase K treatment (38). Most of the studies indicate that residues 31-109 form the structural core of fibrils (10,12,13,15,(31)(32)(33)(34)(35)(36)(37)(38). However, fibrils with a very different structural core, comprising residues 1-43 and 58 -97, have also been characterized (10). A recent structural model for ␣-synuclein fibrils based on solid-state NMR, electron microscopy, and X-ray fiber diffraction data suggests a Greek-key topology (32). The atomic structures of fibrils made from peptides corresponding to the NACore and preNAC segments (residues 68 -78 and 47-56, respectively) of the protein have been characterized in a microelectron diffraction study (39). Both of these sequence segments are present in the structural core of fibrils formed by full-length ␣-synuclein (39). ␣-Synuclein monomers were found to form antiparallel -sheet structures that stack on top of each other to form a protofilament (39). The protofilaments twist together to form fibrils (39). Cryo-electron microscopy and scanning transmission electron microscopy studies have also been used to characterize the structure of ␣-synuclein protofibrils. Two protofilaments, each made of three -strands from the same subunit, form the protofibrils in which sequence segment 8 -94 forms the structural core (15). Two protofibrils were found to associate with each other asymmetrically to form fibrils (15). It was hypothesized that the number of protofibrils and their nature of association in the fibrils may determine the final morphology of ␣-synuclein fibrils and, hence, their structural heterogeneity (15).
The process of ␣-synuclein fibrillation has been described by the nucleation-dependent polymerization model, which involves a lag phase followed by an exponential phase (40). During the lag phase, monomers undergo structural rearrangements to form transient nuclei, which further grow by the addition of monomers to form fibrils. The molecular structure of the nucleus is likely to determine the molecular structure of the fibrils (41), and modulation of the nucleation and elongation rates by varying the growth conditions is expected to modulate the amount of various fibrillar polymorphs (42).
Under different solution conditions, ␣-synuclein has been shown to form two types of fibrils, which differ in their morphology, structural core, toxicity, and infectivity (10). Even under the same aggregation conditions, ␣-synuclein can form morphologically and structurally different fibrils (12,13,15). Understanding the molecular mechanism by which different types of aggregates arise and how fibril formation can be modulated is crucial for the development of therapeutics for PD and other protein aggregation diseases.
Small molecules have been used extensively to modulate and inhibit the process of aggregation for several disease-linked proteins (43)(44)(45) including ␣-synuclein (46,47). The binding of small molecules to fibrils can reduce the amount of toxic oligomers by blocking fibril dissociation (48), by modifying the fibril surface, which acts as an efficient catalyst to generate toxic oligomers (49), or by driving the equilibrium toward fibril formation (45). The small molecule thioflavin T (ThT) is used widely to monitor the aggregation process for many proteins because of its ability to bind to cross--sheet structures found in amyloid fibrils, which modulates the fluorescence properties of ThT (50). ThT binds to different types of fibrils with different affinities (51), and it can accelerate protein aggregation by binding to monomer or fibrils (52,53). It is, however, not known whether small molecules such as ThT that bind fibrils can modulate a fibril formation reaction, such that one type of fibrils is preferred over another, or whether the presence of such small molecules during fibril formation affect the internal structure as well as the external morphology of the fibrils.
In this study, the process of ␣-synuclein fibrillation in the absence and presence of ThT was studied. In the absence of ThT, two types of coexisting fibrils were observed. 70% of the fibrils were helical in external morphology and had a shorter structural core, whereas the remaining 30% of the fibrils had a flat, ribbon-like morphology and an extended structural core. The addition of ThT during aggregation enhances the rate constant of ␣-synuclein fibril formation and reduces structural heterogeneity with only ribbon-like fibrils being formed.
Effects of ThT on the fibrillation of ␣-synuclein
To study the effects of ThT on the fibrillation of ␣-synuclein, 100 M protein was incubated at pH 7.0 and 37°C in the absence and presence of 1 mM ThT. The fibrillation process was monitored by measuring the ThT fluorescence emission at 482 nm ( Fig. 1). In the absence of ThT, fibril formation by ␣synuclein followed a nucleation-dependent polymerization mechanism with a lag phase of ϳ30 h duration. In contrast, the presence of ThT accelerated the fibrillation of the protein by reducing the lag phase and accelerating the elongation phase. The ThT fluorescence emission signal obtained at saturation for the reaction in the presence of ThT was 2-fold higher than for the reaction in the absence of ThT, indicating that either the amount of fibrils was greater or the fibrils differed in their binding ability to ThT. For fibrils formed in the absence and presence of 1 mM ThT, similar amounts of monomer were found to have converted into fibrils at saturation (supplemental Fig. S1). Hence, it was likely that the fibrils formed in the absence and presence of 1 mM ThT differed in their ability to bind ThT.
Effect of ThT on the size and morphology of the fibrils
Atomic force microscopy (AFM) images were obtained for fibrils formed by 100 M protein both in the absence and presence of ThT (Fig. 2, a and b). Two types of fibrils were observed to have formed in the absence of ThT (Fig. 2c). One type was helical, with a periodicity of 74 Ϯ 6 nm (Fig. 2e), and the other was flat and ribbon-like with no periodicity. The mean heights of the helical and ribbon-like fibrils were 5.7 Ϯ 0.7 and 6.3 Ϯ 1.5 nm, respectively. Interestingly, only one type of fibril was obtained in the presence of ThT; these were flat and ribbon-like with a mean height of 7.4 Ϯ 0.8 nm (Fig. 2, d and f). Comparable results were obtained when the fibrils were formed from 50 M protein (data not shown). The flat, ribbon-like fibrils formed in the presence of ThT had a larger diameter and less heterogene-ity than the flat, ribbon-like fibrils formed in the absence of ThT (Fig. 2, c and d); the standard deviation of the fibril height distribution was 0.8 nm in the former case and 1.5 nm in the latter case. The difference in the heights of the ribbon-like fibrils formed in the absence and presence of ThT could conceivably result from intercalation of the ThT molecules in the fibrils formed in the presence of ThT.
To check whether the two types of fibrils behaved like prion strains, seeds were formed by sonicating the fibrils formed in the absence and presence of ThT, and seeding assays were carried out both in the absence and presence of ThT. It was found that both types of seeds (at 3% concentration) abolished the lag phase regardless of whether ThT was present during the aggregation of 100 M ␣-synuclein (supplemental Fig. S2). Further- Thioflavin T modulates fibrillar heterogeneity more, the nature of the fibrils formed depended not on the nature of the seed but on whether ThT was present or not during aggregation (data not shown).
Binding of ThT to fibrils and its effect on fibril secondary structure
To determine whether the fibrils made in the presence of ThT had ThT incorporated into their structure, 100 M fibrils made in the presence and absence of ThT were incubated with the same concentration (1 mM) of ThT. Free and loosely bound ThT was then removed by washing the fibril pellet with buffer following centrifugation. ThT fluorescence was measured for equal concentrations of the fibrils to compare the extent of ThT bound to fibrils made in the absence and presence of ThT (Fig. 3a). The ThT fluorescence of fibrils made in the presence of ThT was about 5-fold higher than that of the fibrils made in the absence of ThT but to which ThT was subsequently added. Comparable results were obtained when the concentration of associated ThT was determined by measuring the absorbance at 412 nm (supplemental Fig. S3a) after first dissolving the fibrils in 8 M GdnHCl. Hence, ThT remained tightly bound to the fibrils made in its presence, presumably by intercalation inside the fibrils. These results suggested that the fibrils made in the absence and presence of 1 mM ThT differed in their structures.
To determine whether the fibrils made in the presence and absence of ThT differed in their secondary structures, infrared spectra were acquired ( Fig. 3b and supplemental Fig. S3, b and c). The presence of the peak near 1630 cm Ϫ1 for both types of fibrils suggested that the fibrils had typical parallel -sheet structures. This peak near 1630 cm Ϫ1 appeared at a lower wave number for the fibrils made in the presence of ThT compared with the fibrils made in the absence of ThT, which suggested an increase in the number of -strands in the fibrils or a change in the twist angle of the -sheet in fibrils made in the presence of ThT (54,55).
Structural characterization of fibrils by hydrogen-deuterium exchange mass spectrometry
HDX-MS was used to further characterize the difference in the internal structures of the fibrils made in the absence and presence of 1 mM ThT. In HDX-MS studies, the amide hydrogen sites that are protected against HDX can be localized to specific segments of the protein sequence by proteolytic fragmentation at low pH after the HDX reaction is complete. A peptide map of ␣-synuclein, covering 100% of the sequence, was first generated by controlled proteolysis using pepsin at low pH (supplemental Fig. S4). The measured mass of each peptide was found to be the same as its calculated mass (supplemental Table S1), except for peptide 95-109 with a mass 18 daltons less than its calculated mass, probably due to water loss. A 5-min labeling pulse was given by incubating the fibrils as well as monomeric protein in deuterated buffer at pH 7.0, 25°C. Control samples having no deuteration (0% D) and complete deuteration (95% D) were also run to calculate the amount of deuterium incorporation in different samples (supplemental Fig. S5). As expected for an intrinsically disordered protein, the monomeric protein showed complete labeling in all the sequence segments after 5 min of HDX (supplemental Fig. S6). Regions of the protein that are highly flexible or that remain unfolded in the fibrils would get labeled to the same extent as they do in the monomer. Regions that are part of the structural core of the fibrils would remain protected and not be labeled. In all fibrils obtained under the two differing conditions, the sequence segments spanning residues 39 to 94 were found to remain highly protected and unlabeled, whereas sequence segments spanning residues 95 to 140 were fully labeled ( Fig. 4 and supplemental Fig. S5).
Interestingly, sequence segments spanning residues 1 to 38 showed bimodal mass distributions for the fibrils formed in the absence of ThT but highly protected unimodal mass distributions for the fibrils formed in the presence of 1 mM ThT (Fig. 4). The bimodal mass distributions for the sequence segments spanning residues 1 to 38 indicated the existence of two different conformations differing in structure in this region. One of the conformations showed protection against HDX and hence was structured, whereas the other conformation showed no protection against HDX and hence was not structured in the sequence segment 1-38. Taken together, the data presented in Fig. 4 show that fibrils formed in the absence of ThT existed in at least two conformations. One of the conformations had a structured core from residues 1 to 94, whereas the other conformation had a structured core only from residues 39 to 94 (Fig. 5, a and b). On the other hand, fibrils formed in the presence of 1 mM ThT had only a single conformation with a structured core from residues 1 to 94 (Fig. 5c).
Differences in molecular structures of fibrils with different morphologies
To correlate the structural core with external morphology for fibrils formed in the absence of ThT, the relative amounts of the two conformations were quantified by fitting the bimodal mass distributions obtained for sequence segments 1-17 and 18 -38 to the sum of two Gaussian distributions (Fig. 6a). In addition, the fractions of helical and flat fibrils were quantified by counting the numbers of the two types of fibrils from AFM images (Fig. 6a). About 70% of the fibrils were found to be helical, and the remaining ϳ30% fibrils had a flat morphology. Interestingly, about 70% of the fibrillar protein molecules had a
Thioflavin T modulates fibrillar heterogeneity
structural core region extending from residues 39 to 94, whereas the remaining ϳ30% of the fibrillar protein molecules had a fibril core that extended from residues 1 to 94. These observations suggested that the helical fibrils are likely to have a core region extending from residues 39 to 94, whereas the flat, ribbon-like fibrils are likely to have a core region extending from residues 1 to 94. To further establish whether the flat, ribbon-like and helical fibrils had core regions extending from residues 1 to 94 and residues 39 to 94, respectively, the structural cores and morphologies of the fibrils were studied at 0.3 mM ThT instead of in the absence of ThT. Importantly, the AFM and HDX-MS studies showed that about 30% of the fibrils were helical in morphology and had a core region extending from residues 39 to 94, and about 70% of them were flat fibrils and had a core region extending from residues 1 to 94 (Fig. 6b). Hence, these data suggested that the helical fibrils had a core region extending from residues 39 to 94 and the flat fibrils had a core region extending from residues 1 to 94. These results also suggested that the average helical fibril and the average flat, ribbon-like fibril contained similar numbers of protein molecules.
The structure of the N-terminal region was found to differ in the two types of ␣-synuclein fibrils. In the ribbon-like fibrils, but not in the helical fibrils, the N-terminal region was found to be part of the structural core. This suggested that the N-terminal region plays an important role in determining the morphology of the fibrils. Helical fibrils formed in the absence of ThT did not convert into ribbon-like fibrils after incubating the fibrils in 1 mM ThT for 24 h at 25°C (data not shown), suggesting that the fibrils have a stable structure and do not interconvert.
The presence of ThT modulated the fibrillar conformation, and it was important to determine how this occurred. To this end, ␣-synuclein aggregation reactions were carried out at different concentrations of ThT ranging from 0 to 1 mM, and AFM imaging was used to determine the relative fraction of helical fibrils formed at each concentration of ThT (Fig. 6c). We found that the relative fraction of helical fibrils decreased monotonically with an increase in the concentration of ThT present and that no helical fibrils had formed at 1 mM ThT concentration. The structure of the final aggregates was also determined using HDX-MS (supplemental Fig. S7). The relative amounts of the two fibrillar conformations were quantified by fitting the bimodal mass distributions obtained for the sequence segment 18 -38 to the sum of multiple Gaussian distributions (supplemental Fig. S7). Interestingly, the relative amount of fibrillar
Thioflavin T modulates fibrillar heterogeneity
protein with a core extending from residues 39 to 94 decreased with increasing concentration of ThT ( Fig. 6c and supplemental Fig. S7). Thus, the relative amount of helical fibrils decreased from 70% when the fibrillation was carried out in the absence of ThT to 0% when fibrillation was carried out in the presence of 1 mM ThT. These results showed that ThT reduced structural heterogeneity in the fibrils formed by ␣-synuclein.
Monitoring the formation of ␣-synuclein oligomers during fibrillation
To determine whether oligomer formation occurred during the fibril formation reaction carried out in either the absence or presence of ThT, ␣-synuclein was incubated at two different concentrations (100 and 690 M (10 mg/ml)) under the fibrillation conditions (pH 7.0 and 37°C) for 5 h. Size-exclusion chromatography was used to detect whether oligomers had formed. Oligomers were not observed to have formed in either the absence or presence of ThT (supplemental Fig. S8, a and b). Oligomers could only be observed (supplemental Fig. S8c) when aggregation was carried out at high a protein concentration (10 mg/ml) in a different (PBS) buffer at pH 7.4 and 37°C for 5 h, as described previously (31).
Characterization of the toxicity levels of the fibrils
To determine whether the fibrils with different structures were differentially toxic to cells, the toxicities of the fibrils formed in the absence and presence of 1 mM ThT were measured using HEK-293T cells (human embryonic kidney cells). Equal amounts of fibrils (2.5 M) were incubated with HEK-293T cells for 24 h, and toxicity was measured using the Wst-1 assay (Fig. 7). To eliminate the effect of any free ThT, the fibrils were first washed multiple times with Milli-Q water to remove any free and loosely bound ThT. For toxicity assays, 1 M ThT was used as a buffer control, and cells without the addition of any buffer served as the control. It was found that about 113 Ϯ 22% of the cells were viable after incubating them with fibrils formed in the absence of ThT, whereas about 76 Ϯ 8% of the cells were viable in the case of fibrils formed in the presence of ThT. It could be concluded that the flat, ribbon-like fibrils formed in the presence of ThT were significantly more toxic to the cells than the fibrils formed in the absence of ThT. It would therefore appear that fibrils with a flat, ribbon-like morphology are more toxic than fibrils with a helical morphology. Control experiments were carried out to ensure that the observed toxicity was due to fibrils and not to any free (unbound) ThT. To this end, toxicity assays were carried out at various free ThT concentrations (supplemental Fig. S9). Free ThT was found to be toxic to cells at concentrations greater than 1 M (supplemental Fig. S9) but not at lower concentrations.
Discussion
The current study was focused on understanding the structural and mechanistic basis for heterogeneity in ␣-synuclein fibrils and its modulation by the small molecule ThT. In the case of several neurodegenerative diseases, the aggregating protein concerned has been found to adopt distinct fibrillar conformations (5,8,15,19). Thus, understanding the structural and physical basis for fibril heterogeneity may shed light on the different pathological behaviors of distinct fibrillar conformations.
Structural heterogeneity in ␣-synuclein fibrils and their toxicity levels
In this study, both helical and flat, ribbon-like fibrils were found to form under the same aggregation conditions (Fig. 2, a and c). In previous studies as well, ␣-synuclein was found to form two types of fibrils, under particular aggregation conditions, which differed in their secondary structures, morphologies, folds, and the extent and distribution of -sheets but had the same structural core encompassing residues 38 -95 (12,13). In contrast, the two types of fibrils observed to form together in the current study had different structural cores (Fig. 5, a and b). The structural core of the helical fibrils comprises residues 39 -94, and that of the flat, ribbon-like fibrils comprises residues 1-94 (Fig. 5, a and b). Multiple studies using solid-state NMR (10, 12, 32-34), HDX-MS (31,35), HDX-NMR (13,33), EPR spectroscopy (36,37), and proteinase K treatment (38) have previously identified the structural core of the ␣-synuclein fibrils. Most of the studies have determined that residues 31-109 form the structural core of the ␣-synuclein fibrils (10, 12,13,15,[31][32][33][34][35][36][37][38]. In a previous study, ribbon-like fibrils were found to form under one aggregation condition and cylindrical fibrils under another aggregation condition, with the two types also differing in their morphology, structural core, toxicity, and infectivity (10). The cylindrical fibrils observed in that study resembled the helical fibrils observed in the current study in having a structural core composed of residues 39 -94; and the ribbon-like fibrils observed in that study were similar to the ribbon-like fibrils observed in the current study in having a structural core formed by residues 1-94 (10). Although the structurally distinct fibrils were shown to differ in their toxicity potentials (10), the properties of the fibrils that determine their toxicity potentials are not well-known. The factors that are likely to affect the toxicity of fibrils include their stability, their ability to interact with membranes, and their surface hydrophobicity (20 -22).
Thioflavin T modulates fibrillar heterogeneity
The flat, ribbon-like fibrils, which possess the extended structural core, are somewhat more toxic than the helical fibrils (Fig. 7). Interestingly, in a previous study, fibrils formed by peptides of two different lengths, which were derived from the ␣-synuclein NACore (segment 68 -78) and subNAC (segment 69 -77) regions, which differed in their structural core, were also found to differ in their cytotoxicity (39). Fibrils made of the longer peptide (NACore), which had a longer structural core, were more cytotoxic than those made by the shorter peptide (39). Further studies are required to delineate the biological importance of the length of the amyloid core.
It is interesting to note that although the fibrils formed in the absence of ThT include flat ribbon-like fibrils similar to those formed in the presence of ThT, which are toxic, little if any toxicity was observed for them. It seems that this might be due to the relatively low proportion of ribbon-like fibrils formed in the absence of ThT.
It should be noted that in this study, toxicity was measured after the addition of the fibrils to cells for 24 h. It is possible that during this incubation with the cells, the fibrils break down into smaller aggregates and that it is these smaller aggregates that are toxic. In this context, it should also be noted that oligomers and prefibrillar aggregates formed by ␣-synuclein are reported to be toxic (56 -60) as are fibrils (10, 61, 62). After incubating the cells for 24 h, Wst-1 reagent was added to the wells. After incubating them for 2.5 h, the OD at 477 nm was measured. Absorbance data were normalized to that of the untreated cells (Control) having 100% cell viability (ns ϭ non-significant; ****, p Ͻ 0.0001 using an unpaired t test). Buffer represents cells treated with only 1 M ThT as described under "Experimental procedures." The error bars represent S.E. from five independent experiments, each with three replicates.
Thioflavin T modulates fibrillar heterogeneity Different internal structures lead to different external fibril morphologies
Different fibril morphologies can originate from distinct molecular structures or from different arrangements of the same molecular structure (6). In the current study, ␣-synuclein fibrils possessing different morphologies were found to comprise internal structural cores of different lengths. It is likely that the fibrils differing in the length of the structural core also differ in extent and organization of -sheets. The observation that the flat, ribbon-like fibrils with an extended structural core had a larger diameter/height than the helical fibrils (Fig. 2, c and d) suggests that they are likely to have a higher fold symmetry than the helical fibrils, as observed previously for A fibrils (6). In the case of the small protein barstar as well, fibrils of very different morphology and diameter, which were, however, formed under different solution conditions, were found to have inner structural cores of different lengths (63,64). At present, the link between a short structural core and helical fibrillar morphology, and between a longer structural core and flat, ribbon-like fibrillar morphology is not understood in the case of ␣-synuclein.
Origin of ␣-synuclein fibril heterogeneity and its reduction by ThT
Structural heterogeneity in ␣-synuclein fibrils could arise from heterogeneity at the monomer level, which would lead to the formation of distinct nuclei. Because of its intrinsically disordered nature, ␣-synuclein can adopt various conformations in a given solution condition as characterized by electrospray ionization mass spectrometry (65), single-molecule AFM (66), and NMR (67). Modifications in the amino acid sequence and solution conditions have been shown to modulate the conformational composition of the ensemble of ␣-synuclein molecules (66). The conformational diversity could determine the relative proportion of different aggregates that can be formed, as suggested previously for other proteins (23). It can then be expected that the stabilization of a specific conformational state upon binding to a small molecule might lead to the formation of specific aggregates.
Structural heterogeneity in ␣-synuclein fibrils could also arise from the utilization of multiple pathways for fibril formation. For several other proteins, including barstar (64,68) and the mouse prion protein (70), structurally distinct amyloid fibrils have been shown to form on different aggregation pathways under different aggregation conditions. In the case of ␣-synuclein, morphologically and structurally different aggregates have been shown to form from structurally distinct oligomers (31). One oligomer was found to be on pathway to fibril formation and had a structural core similar to that of fibrils, and the other oligomer with a different structural core grew into amorphous aggregates (31). In the current study, the inability to detect the formation of oligomers during fibril formation under the aggregation conditions used, either in the absence or presence of ThT (see "Results" and supplemental Fig. S8), did not permit us to determine whether the ribbon-like and helical fibrils arose from structurally distinct oligomers formed on different pathways of fibril formation.
The formation of distinct ␣-synuclein fibrils suggests that multiple nucleation events can take place under the same aggregation condition. Changes in the aggregation condition may affect the nucleation rate and elongation rate differently for distinct nuclei, which would play an important role in modulating structural heterogeneity. In the current study, the observation that the presence of ThT accelerates nucleation and elongation (Fig. 1) suggests that ThT binds to early species formed during the fibrillation of ␣-synuclein. Binding of ThT to early aggregates on one pathway, when multiple pathways are operative, will stabilize those aggregates and result in a reduction in fibril heterogeneity.
Role of ThT in modulating the fibril formation reaction of ␣-synuclein
ThT is known to bind to cross--sheet structures found in amyloid fibrils and has been used widely to monitor the amyloid fibril formation reaction of many proteins (50). The binding of ThT to fibrils can occur in different ways. ThT can bind perpendicular to the long fibril axis in the cavities formed by the side chains of aromatic/hydrophobic residues across consecutive -strands on the surface of the -sheet in the fibrils (51,(71)(72)(73). ThT is also known to bind in a parallel orientation to the peptide strand (74). It also appears that ThT can bind to the peptide backbone viainteractions (75). Hence, it is not surprising that ThT can bind diverse types of amyloid fibrils with different affinities (51). If fibrillar structures (nuclei), which form very early, differ in their binding affinity for ThT, then the early structure (nucleus) that binds most tightly to ThT will be stabilized and, hence, populated the most. The relative amounts of different fibrils formed will reflect the relative amounts of the initial aggregates (nuclei). Hence, the relative amounts of different fibrils that form will depend on the concentration and binding affinity of the ThT present. Thus, ThT must bind strongly to the early fibrillar structures (nuclei) that lead to the formation of the flat, ribbon-like fibrils. In fact, ThT is seen to remain bound to the final, mature, flat, ribbonlike fibrils (Fig. 3a).
The observation that ThT accelerates the fibrillation of ␣-synuclein by increasing the nucleation rate (as indicated in the reduction of the lag phase) (Fig. 1) suggests that ThT does indeed bind to early structures (nuclei) formed during the fibrillation process, resulting in an increase in the nucleus concentration and the elongation rate. In the case of A, a previous study shows that the nucleation rate is affected more than the elongation rate in the presence of ThT, which also suggests that ThT binds to the early structures on the fibrillation pathway (52,76).
It is also possible that ThT accelerates the amyloid fibril formation reaction by binding to monomer. In the case of ␣-synuclein, the electrostatic interaction of ThT with monomeric ␣-synuclein might favor nucleation or the conformation conversion of monomer to aggregation-prone structures that ultimately form the nucleus. Indeed, ThT is known to bind to the negatively charged C terminus of monomeric ␣-synuclein (53). Nevertheless, fibrils made from ␣-synuclein truncated at its C terminus can still bind ThT (77), indicating that ThT binds differently to monomer and to fibril.
Thioflavin T modulates fibrillar heterogeneity
The C terminus is known to be the most solvent-exposed region in both oligomers (31,78,79) and fibrils (10,12,13,15,(31)(32)(33)(34)(35)(36)(37)(38). The C terminus is also known to stabilize the disordered conformation of the protein by interacting with the N terminus or NAC domain (80 -83). It is likely that the interaction of the C terminus with the N-terminal region suppresses the participation of the latter in forming the structural core of amyloid fibrils (53). Not surprisingly, then, C-terminal truncation significantly accelerates the fibrillation of ␣-synuclein (77,84). Binding of the C-terminal domain to the NAC region protects the NAC region from participating in aggregation. Binding of the C terminus to other proteins (85), polyamines (86,87), and metal ions (9,88) increases the rate of fibril formation, suggesting that neutralization of the negatively charged C terminus prevents its binding to the NAC region and the N terminus of the protein. Thus, the interaction of ThT with the C terminus of ␣-synuclein may play an important role in modulating the fibrillation reaction.
It should be noted that, in this study, a range of ThT concentrations was used to investigate the effect of ThT in modulating structural heterogeneity in ␣-synuclein fibrils (Fig. 6). It was found that fibril formation by ␣-synuclein was not modulated at ThT concentrations below 30 M, a concentration known to be the critical micelle concentration for ThT (89). It seems therefore that structural modulation by ThT during fibril formation, as seen in this study, is effected by micellar ThT. ThT is used widely to monitor fibril formation reactions, and the present study suggests that it is safe to do so at concentrations below its critical micelle concentration.
In summary, the small molecule ThT has been shown to modulate the fibril formation reaction of ␣-synuclein and to thereby modulate the structural heterogeneity of the fibrils that form. The presence of ThT during the fibril formation favors the formation of flat, ribbon-like fibrils over the formation of helical fibrils. The flat, ribbon-like fibrils have inner structural core extending from residues 1 to 94, whereas the helical fibrils have an inner structural core extending from residues 39 to 94. The current study highlights the potential use of small molecules in modulating the fibril formation reaction of proteins so that the less toxic aggregates are favored over the more toxic aggregates.
Protein expression and purification
The plasmid pRK172 containing the human ␣-synuclein gene was a kind gift from Prof. A. L. Fink. The protein was expressed and purified as described previously (90) with some modification to the procedure. Escherichia coli BL21(DE3) codon plus (Stratagene) cells transformed with pRK172 were grown overnight at 37°C in LB medium containing 100 g/ml ampicillin and then subcultured into 1000 ml of LB containing 100 g/ml ampicillin. At an OD 600 of 0.8 -1.0, the cells were induced by adding IPTG at a final concentration of 10 g/ml and pelleted down after 5 h. They were resuspended in osmotic shock buffer (30 mM Tris-HCl, 40% w/v sucrose, 2 mM EDTA, pH 7.2), incubated at 25°C for 15 min, and centrifuged to remove supernatant. The pellet was resuspended in Milli-Q water containing about 1 mM MgCl 2 , and the supernatant was obtained after centrifugation. This supernatant was dialyzed twice against 20 mM Tris-HCl buffer, pH 8.0, at 4°C. The protein was denatured by adding 8 M urea solution and loaded onto a DEAE FF ion-exchange column (5 ml, HiTrap, GE Healthcare). The loaded protein was washed and then eluted out using a gradient of 100 -300 mM NaCl. The eluted protein was concentrated by ultrafiltration (Millipore), flash-frozen in liquid N 2 , and stored at Ϫ80°C.
Chemicals, buffers, and aggregation condition
All of the reagents used were of the highest purity grade available from Sigma-Aldrich unless specified otherwise. GdnHCl was procured from USB Corp. The stored protein was concentrated by filtration using a YM3 filter (Millipore) and denatured in 6 M GdnHCl before injecting it into a size-exclusion column (Superdex 200 10/300 GL). The protein was eluted out in 20 mM sodium phosphate, 0.01% sodium azide, and 0.1 mM EDTA, pH 7.0 (aggregation buffer) and passed through a YM100 filter to remove any aggregated protein. For aggregation reactions, 500 l of 100 M protein was agitated at 750 rpm and 37°C (Eppendorf ThermoMixer) in a 1.5-ml centrifuge tube. For aggregation reactions in the presence of ThT, 500 l of 100 M protein solution containing 0.01 to 1 mM ThT was agitated at 750 rpm and 37°C.
Aggregation studies
Aliquots were withdrawn from the aggregation reaction at various time points, and the amount of fibrils was monitored by measuring the ThT fluorescence at pH 8.0 in 20 mM Tris-HCl buffer. In the assay, 10 M ThT and 1 M protein were used. For aggregation reactions carried out in the absence of ThT, 5-l aliquots were withdrawn and mixed with 495 l of 20 mM Tris-HCl buffer containing 10 M ThT. For aggregation reactions carried out in the presence of 1 mM ThT, 5-l aliquots were withdrawn and mixed with 495 l of 20 mM Tris-HCl buffer. Then fluorescence was monitored using a Fluoromax-4 spectrofluorometer with the excitation and emission wavelengths set at 440 and 482 nm, respectively.
Quantification of ThT bound to ␣-synuclein fibrils
To 45 l of 100 M fibrils prepared in the absence of ThT, 4.5 l of 10 mM ThT was added. To 45 l of 100 M fibrils prepared in the presence of 1 mM ThT, 4.5 l of Milli-Q water was added. In both cases, the fibrils were then incubated at 25°C for 90 min and then spun down at 20,000 ϫ g for 10 min at 9°C. The pellet was resuspended in 50 l of aggregation buffer (without ThT). This was repeated four times to remove the free/loosely bound ThT. Fibril concentration was determined using the BCA assay kit (Thermo Scientific), and equal amounts of fibrils formed in the absence and presence of ThT were used to monitor the ThT fluorescence and compare the amount of ThT retained in each fibril sample. The amount of ThT bound to the fibrils was also measured by dissolving the fibrils in 8 M GdnHCl and monitoring the absorbance at 412 nm.
Atomic force microscopy
50 l of fibrils applied onto freshly cleaved mica were incubated for 3 min. The mica surface was rinsed twice with filtered
Thioflavin T modulates fibrillar heterogeneity
Milli-Q water and dried under vacuum for 45-60 min. The AFM images were acquired using a FastScan Bio (Bruker) instrument and analyzed using WSxM software. The height distribution of the fibrils was obtained by measuring the heights of about 200 fibrils from 10 images of fibrils formed in the presence of ThT and about 1000 fibrils from 36 images of fibrils formed in the absence of ThT. The nature of the periodicity seen in the helical fibrils was determined by measuring 32 fibrils from nine images.
Fourier transform infrared (FTIR) spectroscopy
For FTIR measurements, samples were spun down at 20,000 ϫ g for 10 min at 9°C, and fibrils were washed three times with aggregation buffer (without EDTA) made in D 2 O and then resuspended in the same buffer. A thin film was prepared on the diamond crystal by drying 3 l of sample using N 2 gas. FTIR spectra were acquired using a Thermo Nicolet 6700 FT-IR spectrometer. The FTIR spectra of the fibrils were analyzed after background subtraction of the spectrum of the appropriate aggregation buffer.
Peptide mapping
To generate a peptide map of ␣-synuclein, the protein, dissolved in water, was subjected to online pepsin digestion in 0.05% formic acid using an immobilized pepsin cartridge (Applied Biosystems) at a flow rate of 50 l/min on a nano-Acquity UPLC (Waters). The eluted peptides were collected using a peptide trap column (C18 reversed-phase chromatography column), and eluted into an analytical C18 reversedphase chromatography column using a gradient of 3-40% acetonitrile (0.1% formic acid) at a flow rate of 45 l/min. The peptides were directed to the coupled Synapt G2 HD mass spectrometer (Waters). The peptides were sequenced using the MS/tandem MS (MS E ) method followed by analysis with ProteinLynx Global Server software (Waters) and manual inspection.
HDX-MS measurements
Fibrils were prepared as described above. 80 l of 100 M fibrils were spun down at 20,000 ϫ g for 20 min at 9°C. The pellet was resuspended in 20 l of aggregation buffer. To initiate deuterium labeling, 10 l of the above sample was diluted into 190 l of aggregation buffer (without EDTA) made in D 2 O and incubated at 25°C for 5 min. After a 5-min pulse, 200 l of the above sample was mixed with 400 l of ice-cold quench buffer (0.1 M glycine-HCl, 8.4 M GdnHCl, pH 2.5) and incubated for 1 min on ice to dissolve the fibrils. The samples were desalted using a Sephadex G-25 HiTrap desalting column equilibrated with water at pH 2.5 and Akta Basic HPLC. The desalted samples were injected into the HDX module coupled with a nanoACQUITY UPLC system (Waters) for online pepsin digestion using an immobilized pepsin cartridge (Applied Biosystems). Further processing of the sample for mass determination using a Waters Synapt G2 mass spectrometer was carried out as described previously (91).
Peptide masses were calculated from the centroid of the isotopic envelope using MassLynx software, and the shift in the mass of labeled peptide relative to the unlabeled peptide was used to determine the extent of deuterium incorporation. As the sample was in 95% D 2 O during labeling and was exposed to H 2 O after dissolution in GdnHCl, control experiments were carried out to correct for back-exchange and forward-exchange. To this end, monomeric ␣-synuclein was completely deuterated by incubating it in 20 mM sodium phosphate buffer, pH 7.0 (95% D 2 O) at 25°C for 5 min. The fully deuterated ␣-synuclein sample was then processed in exactly the same way as the aggregates. The extent of deuterium incorporation in each peptide, % D, was determined using the following equation, where m(t) is the measured centroid mass of the peptide at time point t, m(0%) is the measured mass of an undeuterated reference sample, and m(95%) is the measured mass of a fully deuterated reference sample (in 95% D 2 O) (69).
The percent deuterium incorporation for peptides showing a bimodal distribution was calculated as described previously (14,91). The centroid mass for each peak was obtained by fitting the bimodal mass distributions to the sum of two Gaussian distributions using OriginPro 8. The % D for each peak was determined using Equation 1.
Cytotoxicity assay
Equal concentrations (1 mM) of ThT were added to the fibrils made under the two different conditions, and the fibrils were incubated at 25°C for 15 min. The fibrils were spun down at 20,000 ϫ g for 20 min at 9°C, the supernatant was removed, and the fibrils were washed with an equal volume of autoclaved Milli-Q water. The washing process was repeated three times, and then the fibrils were resuspended in autoclaved Milli-Q water. The concentration of the fibrils was determined using the BCA assay after dissolving the fibrils in 4 M GdnHCl. Equal concentrations (2.5 M) of fibrils made under the two different conditions were used for the cytotoxicity experiments. Autoclaved Milli-Q water containing 1 M ThT was used as a buffer control in the cytotoxicity assay. For the assay, HEK-293T cells (ATCC) were cultured in DMEM (Gibco) supplemented with 10% FBS at 37°C in a 5% CO 2 -humidified environment. Cells were plated in a 96-well plate at a density of 5000 cells/well to a final volume of 100 l. After incubation for 25 h, 10 l of fibrils (25 M) or ThT only (0.1-5 M) was added to each well, and the cells were further incubated for 24 h at 37°C in 5% CO 2 . The Wst-1 assay kit (Roche) was used to measure the viability of the cells. 9 l of Wst-1 reagent was added into each well, and the cells were incubated for 2.5 h at 37°C in 5% CO 2 . The optical density at 477 nm was measured with a microplate reader (SpectraMax M5). Data were normalized with respect to the data obtained with untreated (control) cells. | v3-fos-license |
2019-04-10T13:11:56.409Z | 2018-09-10T00:00:00.000 | 105382862 | {
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} | pes2o/s2orc | A Hybrid Reactor System Comprised of Non-Thermal Plasma and Mn / Natural Zeolite for the Removal of Acetaldehyde from Food Waste
The degradation of low concentrations of acetaldehyde while using a non-thermal plasma (NTP)/catalyst hybrid reactor system was investigated while using humidified air at ambient temperature. A series of highly active manganese-impregnated natural zeolite (Mn/NZ) catalysts were synthesized by the incipient wetness method using sonication. The Mn/NZ catalysts were analyzed by Brunauer-Emmett-Teller surface area measurements and X-ray photoelectron spectroscopy. The Mn/NZ catalyst located at the downstream of a dc corona was used for the decomposition of ozone and acetaldehyde. The decomposition efficiency of ozone and acetaldehyde was increased significantly using the Mn/NZ catalyst with NTP. Among the various types of Mn/NZ catalysts with different Mn contents, the 10 wt.% Mn/NZ catalyst under the NTP resulted the highest ozone and acetaldehyde removal efficiency, almost 100% within 5 min. Moreover, this high efficiency was maintained for 15 h. The main reason for the high catalytic activity and stability was attributed to the high dispersion of Mn on the NZ made by the appropriate impregnation method using sonication. This system is expected to be efficient to decompose a wide range of volatile organic compounds with low concentrations.
Introduction
The increase in volatile organic compounds (VOCs) has become an important environmental issue worldwide, because VOCs are typically odorous substances that can cause discomfort and have an adverse impact on human health [1,2].In addition, VOCs that are emitted from food waste are considered to be a severe problem in Korea, and the government has been trying to reduce the levels of VOCs to improve health and quality of life.Especially, acetaldehyde is one of the main odorous components generated during food waste decomposition, and it is generally difficult to degrade than other components.Various methods, including adsorption [3][4][5][6][7], thermal and catalytic oxidation [8], and photo-catalysis [9,10], have been applied for the removal of high concentrations of VOCs in air.
On the other hand, conventional removal methods are unsuitable for removing low concentrations of VOCs because of their low efficiency and high cost [11].Therefore, alternative technologies need to be developed to overcome the limitations of conventional methods, which cannot be applied effectively for the removal of low concentrations of VOCs.
Non-thermal plasma (NTP) techniques offer an innovative approach for the removal of VOCs [1,12,13].The oxygen and other oxidative species generated by plasma are able to oxidize VOCs.On the other hand, the formation of highly toxic and reactive by-products, such as NO x and ozone [14,15], during the removal of VOCs while using the NTP technology is considered to be a problem with this system.Since ozone is a powerful oxidant and residual ozone can damage facility or hurt life, ozone is the most undesirable by-products in the NTP discharge zone and is thermally stable above 250 • C [16].The additional use of a catalyst for the removal of VOCs using NTP technology might remove VOCs and reduce the undesirable by-products of NTP technology.The removal efficiency of ozone depends on the position of the catalyst.A comparison of in-plasma catalysis (IPC) and post-plasma catalysis (PPC) processes has shown that PPC technology has higher performance for the removal of ozone [16].The temperature and humidity of the PPC process are also very important for the removal of VOCs emitted from food waste.Stable activity of PPC under ambient temperature and humidity that is higher than 50% is necessary for the successful application to the removal of ozone and VOCs from food waste.Despite this, most of the experiments were performed at high temperatures, up to 200~700 • C [17], without considering humidity.
Many types of catalyst have been applied widely for the removal of VOCs while using the PPC process [17].Several catalysts, such as γ-Al 2 O 3 , SiO 2 , TiO 2 , and zeolites, have been used for the complete oxidation of VOCs, and various types of metal, such as Ag, Cu, Pd, Pt, Ni, Co, Fe, and Mn, have been used as coating or impregnation materials on these catalysts to provide additional catalytic activity [18].Among them, Mn has been widely used as an impregnated metal for the removal of VOCs [11,19,20].On the other hand, its actual application is limited to vehicles and indoor air control because high temperatures are required to provide the high and rapid response for odor removal.The negative impact of humidity in air and the high cost of the catalyst limit the applicability of the catalytic process to the removal of VOCs from food wastes.In this aspect, the PPC technology has a potential application for the removal of VOCs.A successful approach for the removal of VOCs and ozone while using the cost-efficient PPC process needs to be tested under high humidity and ambient temperature, so that this process can be applied to food waste.
In this study, a post-NTP/catalyst hybrid system was developed for the removal of VOCs from food waste.To enhance economic feasibility, natural zeolite (NZ) was applied as a catalyst support instead of synthesized catalysts.Also, low cost NZ was applied to develop the economic removal process of VOCs from food waste.Because NZ has showed good catalytic activity for toluene oxidation activity [21,22], high catalytic activity of NZ for acetaldehyde oxidation could be expected.However, NZ has never been applied to the plasma/catalyst hybrid system for decomposition of acetaldehyde.Mn/NZ, which was prepared by the incipient wetness method with sonication, was used as a catalyst at the downstream of the NTP reactor, and acetaldehyde was used as a model food waste VOCs pollutant.A higher relative humidity with 60% and ambient temperature was applied to form a plasma at the discharge zone and the effect of humidity on the removal of VOCs and the formation of ozone in the NTP/catalyst hybrid system was evaluated by comparing the removal efficiency of acetaldehyde and ozone in the system.
Characterization of the Catalyst
The Mn/NZ by the incipient wetness method with sonication were characterized by Brunauer-Emmett-Teller (BET) surface area measurements and X-ray photoelectron spectroscopy (XPS).Table 1 lists the specific surface area and the surface atomic concentrations of the NZ or the Mn/NZ.The Mn loading on the NZ had a significant effect on the specific surface area and surface atomic concentration.The specific surface areas of the raw NZ, 1 wt.%Mn/NZ, 3 wt.%Mn/NZ, 5 wt.%Mn/NZ, 10 wt.% Mn/NZ catalyst, and 10 wt.% Mn/NZ catalyst without sonication were 134.8, 29.3, 25.2, 21.4,20.9, and 19.1 m 2 g −1 , respectively.The specific surface area of the Mn/NZ catalyst was much lower than that of raw NZ.The Mn atomic concentration increased with increasing Mn loading on the NZ.The 10 wt.% Mn/NZ showed a higher surface atomic concentration of Mn when compared to the 10 wt.% Mn/NZ calcined at 550 • C or the 10 wt.% Mn/NZ without sonication, despite their equal Mn loading.The surface atomic concentration of impregnated Mn on NZ is related to the dispersion of Mn [23].Thus, this suggests that the introduction of Mn was well dispersed on the surface of the NZ when the Mn/NZ was treated by the incipient wetness method with sonication.The surface elemental compositions, such as the Mn oxidation states and adsorbed oxygen species, were investigated by XPS. Figure 1 shows the XPS spectra in the O 1s and Mn 2p binding energy regions at different Mn loadings.Figure 1A shows the Mn 2p XPS spectra of the samples in the 633~660 eV range.The binding energies at the 640.0~650.0eV and 650.0~660.0eV regions were attributed to Mn 2p 3/2 and Mn 2p 1/2, respectively.The Mn loading of the Mn/NZ catalysts has a significant impact on the distribution of surface Mn.The O 1s spectra in Figure 1B [24].The XPS data show that the MnO x of the 1 and 3 wt.%Mn/NZ had only adsorbed oxygen species, whereas the MnO x of 5 and 10 wt.% Mn/NZ had both lattice oxygen species and adsorbed oxygen species.In the 10 wt.% Mn/NZ, the binding energy of the lattice oxygen species was stronger than that of adsorbed oxygen species.In particular, more oxygen lattices were observed in sonicated 10 wt.% Mn/NZ than 10 wt.% Mn/NZ without sonication.Figure 2 shows the Mn 2p and O 1s XPS spectra of the 10 wt.% Mn/NZ catalysts that were obtained at different calcination temperatures.The Mn 2p spectra of the catalyst can be assigned to three components corresponding to Mn 2+ , Mn 3+ , and Mn 4+ species on the surface of the catalysts [25].As shown in Figure 2A, the catalyst at different calcination temperatures showed peaks for Mn 2+ , Mn 3+ , and Mn 4+ species, but no significant differences were observed in the surface Mn n+ content.The O 1s spectra in Figure 2B show that asymmetrical O 1s can be fitted with three components, such as the adsorbed lattice oxygen species, adsorbed oxygen species, and water.In the 10 wt.% Mn/NZ calcined at 350 • C or 550 • C, the MnO x has lattice oxygen (O 2 − ) and absorbed oxygen species.The 10 wt.% Mn/NZ calcined at 350 • C shows that there is a higher content of lattice oxygen species than absorbed oxygen species.Previous studies reported that the samples that were possessing larger amounts of surface lattice oxygen or adsorbed surface oxygen have higher catalytic activity for VOC oxidation [24,25].Figure S1 shows the X-ray diffraction patterns of the sonication treated Mn/NZ and untreated Mn/NZ by the incipient wetness method.Because low or absent peak intensity of MnOx implies high dispersion of MnOx [26], it can be confirmed again that the sonicated Mn/NZ has a higher dispersion Figure S1 shows the X-ray diffraction patterns of the sonication treated Mn/NZ and untreated Mn/NZ by the incipient wetness method.Because low or absent peak intensity of MnOx implies high dispersion of MnOx [26], it can be confirmed again that the sonicated Mn/NZ has a higher dispersion Figure S1 shows the X-ray diffraction patterns of the sonication treated Mn/NZ and untreated Mn/NZ by the incipient wetness method.Because low or absent peak intensity of MnO x implies high dispersion of MnO x [26], it can be confirmed again that the sonicated Mn/NZ has a higher dispersion of Mn than the Mn/NZ without sonication.This is consistent with data that a higher Mn surface atomic concentration of sonicated Mn/NZ is related to the high dispersion of Mn.
Acetaldehyde Removal Activity
Figure 3 shows the concentration of acetaldehyde removed after 5 min of the NTP/catalyst hybrid reaction while using the NTP and 1 g of the Mn/NZ catalyst at 60% relative humidity.First, acetaldehyde adsorption efficiency of the catalyst was evaluated, but there was no adsorptive removal of acetaldehyde.Although additional adsorption was performed for 1 h, the adsorption of acetaldehyde in high relative humidity atmosphere was not proceeded at all over all the catalysts (data not shown).
Catalysts 2018, 8, x FOR PEER REVIEW 5 of 11 of Mn than the Mn/NZ without sonication.This is consistent with data that a higher Mn surface atomic concentration of sonicated Mn/NZ is related to the high dispersion of Mn.
Acetaldehyde Removal Activity
Figure 3 shows the concentration of acetaldehyde removed after 5 min of the NTP/catalyst hybrid reaction while using the NTP and 1 g of the Mn/NZ catalyst at 60% relative humidity.First, acetaldehyde adsorption efficiency of the catalyst was evaluated, but there was no adsorptive removal of acetaldehyde.Although additional adsorption was performed for 1 h, the adsorption of acetaldehyde in high relative humidity atmosphere was not proceeded at all over all the catalysts (data not shown).The NTP/catalyst hybrid system using the raw NZ catalyst did not decompose acetaldehyde, whereas the removal efficiency of acetaldehyde was increased by applying a catalyst with Mn.The NTP/catalyst hybrid system using the 1, 3, and 5 wt.%Mn/NZ catalysts decomposed 60% of acetaldehyde present.The acetaldehyde removal efficiency was increased further to 70% using the 10 wt.% Mn/NZ catalyst.In addition, 100% ozone decomposition was also achieved after 5 min of using the 10 wt.% Mn/NZ together with the NTP.This indicates that the Mn content in the Mn/NZ catalyst is very important for the decomposition of acetaldehyde and ozone.In addition, the removal of acetaldehyde was strongly related with the decomposition efficiency of ozone.When the decomposition efficiency of ozone was increased using the 10 wt.% Mn/NZ, that of acetaldehyde was also increased.This suggests that the newly generated oxygen species from ozone on the catalyst surface can allow for the additional decomposition of acetaldehyde [16].Although the catalytic activity of the Mn-impregnated catalyst was reported to decrease at high Mn contents [27], in this study, the highest activity for the removal of acetaldehyde was achieved when the 10 wt.% Mn/NZ was applied.
To determine the effect of the catalyst amount on the removal of acetaldehyde, the catalytic decomposition of acetaldehyde using the NTP or the NTP/catalyst hybrid system was also performed, and the results are shown in Figure 4.The removal efficiency of acetaldehyde was extremely low, with almost no decomposition, when the acetaldehyde removal reaction was performed while using the NTP system without the Mn/NZ catalyst at ambient temperature under a humidified atmosphere.In contrast, the NTP/catalyst hybrid system showed much higher efficiency on the removal of acetaldehyde than a single reaction system using plasma only.The removal efficiencies of acetaldehyde were also increased gradually while using a larger amount of catalyst The NTP/catalyst hybrid system using the raw NZ catalyst did not decompose acetaldehyde, whereas the removal efficiency of acetaldehyde was increased by applying a catalyst with Mn.The NTP/catalyst hybrid system using the 1, 3, and 5 wt.%Mn/NZ catalysts decomposed 60% of acetaldehyde present.The acetaldehyde removal efficiency was increased further to 70% using the 10 wt.% Mn/NZ catalyst.In addition, 100% ozone decomposition was also achieved after 5 min of using the 10 wt.% Mn/NZ together with the NTP.This indicates that the Mn content in the Mn/NZ catalyst is very important for the decomposition of acetaldehyde and ozone.In addition, the removal of acetaldehyde was strongly related with the decomposition efficiency of ozone.When the decomposition efficiency of ozone was increased using the 10 wt.% Mn/NZ, that of acetaldehyde was also increased.This suggests that the newly generated oxygen species from ozone on the catalyst surface can allow for the additional decomposition of acetaldehyde [16].Although the catalytic activity of the Mn-impregnated catalyst was reported to decrease at high Mn contents [27], in this study, the highest activity for the removal of acetaldehyde was achieved when the 10 wt.% Mn/NZ was applied.
To determine the effect of the catalyst amount on the removal of acetaldehyde, the catalytic decomposition of acetaldehyde using the NTP or the NTP/catalyst hybrid system was also performed, and the results are shown in Figure 4.The removal efficiency of acetaldehyde was extremely low, with almost no decomposition, when the acetaldehyde removal reaction was performed while using the NTP system without the Mn/NZ catalyst at ambient temperature under a humidified atmosphere.In contrast, the NTP/catalyst hybrid system showed much higher efficiency on the removal of acetaldehyde than a single reaction system using plasma only.The removal efficiencies of acetaldehyde were also increased gradually while using a larger amount of catalyst during the reaction using the NTP/catalyst hybrid system and approached 100% removal when 5 g of the catalyst was used.
These results suggest that the NTP process is essential for the removal of acetaldehyde and the additional use of the 10 wt.% Mn/NZ catalyst as a post plasma reaction can increase the overall efficiency for the removal of acetaldehyde and ozone.
Catalysts 2018, 8, x FOR PEER REVIEW 6 of 11 during the reaction using the NTP/catalyst hybrid system and approached 100% removal when 5 g of the catalyst was used.These results suggest that the NTP process is essential for the removal of acetaldehyde and the additional use of the 10 wt.% Mn/NZ catalyst as a post plasma reaction can increase the overall efficiency for the removal of acetaldehyde and ozone.Also, the removal activity of acetaldehyde over a sonicated Mn catalyst was compared with that over Mn impregnated on the NZ without sonication.The catalytic activity of the sonicated 10 wt.% Mn/NZ was much higher than that of the 10 wt.% Mn/NZ without sonication (Figure 5).This can be explained by the high dispersion efficiency of Mn on the NZ catalyst, which was enhanced by the highly dispersed impregnation or surface atomic concentration of Mn via sonication during the incipient wetness method (Table 1) [28,29], and this leads to high catalytic activation for acetaldehyde removal.The effect of the calcination temperature of the Mn/NZ catalyst on the removal of acetaldehyde was also evaluated.For this, 5 g of the 10 wt.% Mn/NZ calcined at 350 °C and 550 °C were applied for the removal of acetaldehyde while using the NTP/catalyst hybrid system under a high humidity atmosphere.As shown in Figure 6, the removal efficiency of acetaldehyde using the Mn/NZ calcined Also, the removal activity of acetaldehyde over a sonicated Mn catalyst was compared with that over Mn impregnated on the NZ without sonication.The catalytic activity of the sonicated 10 wt.% Mn/NZ was much higher than that of the 10 wt.% Mn/NZ without sonication (Figure 5).This can be explained by the high dispersion efficiency of Mn on the NZ catalyst, which was enhanced by the highly dispersed impregnation or surface atomic concentration of Mn via sonication during the incipient wetness method (Table 1) [28,29], and this leads to high catalytic activation for acetaldehyde removal.during the reaction using the NTP/catalyst hybrid system and approached 100% removal when 5 g of the catalyst was used.These results suggest that the NTP process is essential for the removal of acetaldehyde and the additional use of the 10 wt.% Mn/NZ catalyst as a post plasma reaction can increase the overall efficiency for the removal of acetaldehyde and ozone.Also, the removal activity of acetaldehyde over a sonicated Mn catalyst was compared with that over Mn impregnated on the NZ without sonication.The catalytic activity of the sonicated 10 wt.% Mn/NZ was much higher than that of the 10 wt.% Mn/NZ without sonication (Figure 5).This can be explained by the high dispersion efficiency of Mn on the NZ catalyst, which was enhanced by the highly dispersed impregnation or surface atomic concentration of Mn via sonication during the incipient wetness method (Table 1) [28,29], and this leads to high catalytic activation for acetaldehyde removal.The effect of the calcination temperature of the Mn/NZ catalyst on the removal of acetaldehyde was also evaluated.For this, 5 g of the 10 wt.% Mn/NZ calcined at 350 °C and 550 °C were applied for the removal of acetaldehyde while using the NTP/catalyst hybrid system under a high humidity atmosphere.As shown in Figure 6, the removal efficiency of acetaldehyde using the Mn/NZ calcined effect of the calcination temperature of the Mn/NZ catalyst on the removal of acetaldehyde was also evaluated.For this, 5 g of the 10 wt.% Mn/NZ calcined at 350 • C and 550 • C were applied for the removal of acetaldehyde while using the NTP/catalyst hybrid system under a high humidity atmosphere.As shown in Figure 6, the removal efficiency of acetaldehyde using the Mn/NZ calcined at 550 • C was approximately 60%.On the other hand, that of the Mn/NZ catalyst calcined at 350 • C was 100%.This suggests that 350 • C is a more appropriate calcination temperature of the Mn/NZ than 550 • C for the removal of acetaldehyde via the NTP/catalyst hybrid system.The effects of the Mn calcination temperature on the catalytic activity have also been reported elsewhere [30].In addition, it was reported that the partial oxidation of MnCO 3 could be achieved at calcination temperatures between 300 and 400 • C, which allows for the co-existence of MnCO 3 and MnO x that can increase the performance of the catalyst [24,25].Furthermore, the best reducibility and abundant active lattice oxygen in the catalyst were associated with the excellent performance of VOC oxidation [25].Tian et al. also reported that the lattice oxygen contributed to the catalytic activity [31].Jin et al. also reported that the high activity of the mesoporous α-Mn 2 O 3 calcined at 300 • C might be due to its high oxygen mobility or oxygen vacancies and lattice oxygen.In addition, the low activity of commercial Mn 2 O 3 seemed to be contributed to its lower oxygen mobility and surface area [32].This is consistent with the data showing that more lattice oxygen species (XPS Figure 2B) and surface atomic concentration of Mn (Table 1) after calcination at 350 • C resulted in the best performance for the acetaldehyde decomposition.
Catalysts 2018, 8, x FOR PEER REVIEW 7 of 11 at 550 °C was approximately 60%.On the other hand, that of the Mn/NZ catalyst calcined at 350 °C was 100%.This suggests that 350 °C is a more appropriate calcination temperature of the Mn/NZ than 550 °C for the removal of acetaldehyde via the NTP/catalyst hybrid system.The effects of the Mn calcination temperature on the catalytic activity have also been reported elsewhere [30].In addition, it was reported that the partial oxidation of MnCO3 could be achieved at calcination temperatures between 300 and 400 °C, which allows for the co-existence of MnCO3 and MnOx that can increase the performance of the catalyst [24,25].Furthermore, the best reducibility and abundant active lattice oxygen in the catalyst were associated with the excellent performance of VOC oxidation [25].Tian et al. also reported that the lattice oxygen contributed to the catalytic activity [31].Jin et al. also reported that the high activity of the mesoporous α-Mn2O3 calcined at 300 °C might be due to its high oxygen mobility or oxygen vacancies and lattice oxygen.In addition, the low activity of commercial Mn2O3 seemed to be contributed to its lower oxygen mobility and surface area [32].This is consistent with the data showing that more lattice oxygen species (XPS Figure 2B) and surface atomic concentration of Mn (Table 1) after calcination at 350 °C resulted in the best performance for the acetaldehyde decomposition.Relative Humidity: 60%, The amount of catalysts: 5 g.
To check the removal efficiencies of acetaldehyde and ozone under a high humidity atmosphere, the humidity of the system was also evaluated by the NTP/catalyst hybrid system using 5 g of the 10 wt.% Mn/NZ catalyst (Figure S2).Acetaldehyde and ozone were removed completely under both dry and humidified atmospheres.Even under the highly humidified atmosphere (RH = 80%), 100% of acetaldehyde was removed after 5 min reaction while using the NTP/catalyst hybrid system.This suggests that high humidity does not influence the removal efficiency of acetaldehyde when the NTP/catalyst hybrid system using the Mn/NZ catalyst is applied.This result is superior to previous results that were obtained with a Pd/Al2O3 catalyst for VOCs decomposition using post-plasma catalytic technology [33].They indicate that VOC sorption to the catalyst determined by Van der Waals and H-donor interactions is the most critical parameter of VOC removal efficiency, and humidity affects those interactions.Hydrogen donor interactions are more independent to humidity than Van der Waals force.In the case of polar compounds, such as acetaldehyde tested in this study, both the Van der Waals and hydrogen donor interactions are an important sorption mechanism, which reduces the dependency of humidity on the removal of acetaldehyde.However, further research will be necessary to investigate this phenomena in depth.To check the removal efficiencies of acetaldehyde and ozone under a high humidity atmosphere, the humidity of the system was also evaluated by the NTP/catalyst hybrid system using 5 g of the 10 wt.% Mn/NZ catalyst (Figure S2).Acetaldehyde and ozone were removed completely under both dry and humidified atmospheres.Even under the highly humidified atmosphere (RH = 80%), 100% of acetaldehyde was removed after 5 min reaction while using the NTP/catalyst hybrid system.This suggests that high humidity does not influence the removal efficiency of acetaldehyde when the NTP/catalyst hybrid system using the Mn/NZ catalyst is applied.This result is superior to previous results that were obtained with a Pd/Al 2 O 3 catalyst for VOCs decomposition using post-plasma catalytic technology [33].They indicate that VOC sorption to the catalyst determined by Van der Waals and H-donor interactions is the most critical parameter of VOC removal efficiency, and humidity affects those interactions.Hydrogen donor interactions are more independent to humidity than Van der Waals force.In the case of polar compounds, such as acetaldehyde tested in this study, both the Van der Waals and hydrogen donor interactions are an important sorption mechanism, which reduces the dependency of humidity on the removal of acetaldehyde.However, further research will be necessary to investigate this phenomena in depth.
Long Term Reaction Stability Test
To examine the decrease in acetaldehyde removal due to catalyst poisoning, a long term reaction test using the NTP/catalyst hybrid system was also performed under a humidified atmosphere.The VOCs removal efficiency on the catalytic reaction can be decreased when a large amount metal is impregnated, because high metal contents can make an extremely low dispersion between the active metal phase and supporting catalyst [34,35].Also, it has been reported that the VOCs conversion gradually decreased after 5 h under dry air [29].On the other hand, the removal efficiency of acetaldehyde in this experiment was up to 100% for 15 h, even under a high humidity atmosphere, as shown in Figure 7.This can be explained by the high dispersion of Mn on the NZ surface by the appropriate impregnation method while using sonication and highlights the high stability of the NTP/catalyst system applied in this research for the removal of acetaldehyde.
Long Term Reaction Stability Test
To examine the decrease in acetaldehyde removal due to catalyst poisoning, a long term reaction test using the NTP/catalyst hybrid system was also performed under a humidified atmosphere.The VOCs removal efficiency on the catalytic reaction can be decreased when a large amount metal is impregnated, because high metal contents can make an extremely low dispersion between the active metal phase and supporting catalyst [34,35].Also, it has been reported that the VOCs conversion gradually decreased after 5 h under dry air [29].On the other hand, the removal efficiency of acetaldehyde in this experiment was up to 100% for 15 h, even under a high humidity atmosphere, as shown in Figure 7.This can be explained by the high dispersion of Mn on the NZ surface by the appropriate impregnation method while using sonication and highlights the high stability of the NTP/catalyst system applied in this research for the removal of acetaldehyde.
Catalyst Preparation
NZ, which consisted of SiO2, Al2O3, CaO, Na2O, and K2O, was purchased (Handoo Co. Ltd., Seoul, Korea) and was used as a supporting material for Mn impregnation.The NZ was sieved to achieve a uniform particle size between 2 and 3 mm.Mn was impregnated on the NZ by the incipient wetness method.For this, manganese (II) acetate tetrahydrate (Mn(CH3COOH)2•4H2O, 99.99%), which was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in distilled water and was added to a beaker containing the NZ.As the total pore volume of NZ is 0.1603 cm 3 /g, 0.8015 mL of water was used per 5 g of NZ.After the impregnation, Mn/NZ was sonicated for 2 h.After sonication, the synthesized Mn/NZ was dried overnight at 100 °C and finally calcined at 350 °C or 550 °C for 2 h in air.The weight content of Mn in the Mn/NZ catalyst was controlled to 1, 3, 5, and 10 wt.% to determine the optimal metal content in the catalyst for the removal of VOCs and ozone.The specific amounts of NZ and Mn(CH3COOH)2•4H2O used for the synthesis is listed on Table 2.
Catalyst Preparation
NZ, which consisted of SiO 2 , Al 2 O 3 , CaO, Na 2 O, and K 2 O, was purchased (Handoo Co. Ltd., Seoul, Korea) and was used as a supporting material for Mn impregnation.The NZ was sieved to achieve a uniform particle size between 2 and 3 mm.Mn was impregnated on the NZ by the incipient wetness method.For this, manganese (II) acetate tetrahydrate (Mn(CH 3 COOH) 2 •4H 2 O, 99.99%), which was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in distilled water and was added to a beaker containing the NZ.As the total pore volume of NZ is 0.1603 cm 3 /g, 0.8015 mL of water was used per 5 g of NZ.After the impregnation, Mn/NZ was sonicated for 2 h.After sonication, the synthesized Mn/NZ was dried overnight at 100 • C and finally calcined at 350 • C or 550 • C for 2 h in air.The weight content of Mn in the Mn/NZ catalyst was controlled to 1, 3, 5, and 10 wt.% to determine the optimal metal content in the catalyst for the removal of VOCs and ozone.The specific amounts of NZ and Mn(CH 3 COOH) 2 •4H 2 O used for the synthesis is listed on Table 2.
Catalyst Characterization
All of the samples were degassed at 200 • C prior to the Brunauer-Emmett-Teller (BET) measurements.The BET surface areas (BET) were determined while using a Micromeritics ASAP 2010 (Norcross, GA, USA) nitrogen adsorption apparatus.X-ray photoelectron spectroscopy (XPS) was performed on a PHI 5000 Versa Probe (Ulvac-PHI, Kanagawa, Japan), using Al Kα (1486.6 eV) radiation.The data were charge referenced to the C 1 s peak at 284.6 eV.The crystalline phase of the catalysts was obtained using an X-ray diffractometer (XRD, RINT/PMAX 2500, Rigaku, Tokyo, Japan) within the scanning angle range of 30-70 (2 theta), with CuKα radiations (λ = 0.1541 nm).
NTP/Catalyst System
Figure 8 presents a schematic diagram of the NTP/catalyst hybrid system that was used for the degradation of VOCs.The NTP/catalyst hybrid system consisted of a VOCs generator, a plasma reactor, and a catalyst bed.The Mn/NZ was loaded onto the catalyst bed (height = 24.5 cm, i.d.= 1.27 cm) located at the downstream of the plasma reactor.The concentration of reactant gas, acetaldehyde in a mixture of nitrogen, and air gas, was controlled by adjusting the flow rates of the standard gas (100 ppm of acetaldehyde in nitrogen gas) and air (dilution gas).The gas stream was injected to the reactor through an acrylic box (inner volume: 100 L).After a sufficient stabilization time (120 min), the concentration of reactant was controlled to 10 ppm at the exit line of the acrylic box by fine tuning the flow rates of acetaldehyde standard and dilution gas.The gas hourly space velocity (GHSV) of the reactant gas in the plasma zone, and the catalyst bed were set to 120/h and 222/h at ambient temperature and pressure, respectively.A dc corona reactor was used to produce plasma.The length and gap space of the discharge tube were 15 mm and 5 mm, respectively.Plasma power of 10 W was driven while using a high voltage AC power supply (12 kV, SIE = 120 J/L, 60 Hz, sine wave).During the oxidation reaction, the concentrations of acetaldehyde were measured using detection tubes (GASTEC, 92L, Measurement Range: 1-20 ppm) and ozone concentration was carried out using an ozone analyzer (Aeroqual 200) after gas sampling at the outlet.The effects of humidity in the system on the removal of acetaldehyde was also evaluated by controlling the relative humidity of the reactor to 20%, 60%, and 80%.The experiment was conducted by adjusting the initial humidity and maintaining the humidity continuously.About 25 ppm ozone was produced from NTP reactor.
Catalyst Characterization
All of the samples were degassed at 200 °C prior to the Brunauer-Emmett-Teller (BET) measurements.The BET surface areas (BET) were determined while using a Micromeritics ASAP 2010 (Norcross, GA, USA) nitrogen adsorption apparatus.X-ray photoelectron spectroscopy (XPS) was performed on a PHI 5000 Versa Probe (Ulvac-PHI, Kanagawa, Japan), using Al Kα (1486.6 eV) radiation.The data were charge referenced to the C 1 s peak at 284.6 eV.The crystalline phase of the catalysts was obtained using an X-ray diffractometer (XRD, RINT/PMAX 2500, Rigaku, Tokyo, Japan) within the scanning angle range of 30-70 (2 theta), with CuKα radiations (λ = 0.1541 nm).
NTP/Catalyst System
Figure 8 presents a schematic diagram of the NTP/catalyst hybrid system that was used for the degradation of VOCs.The NTP/catalyst hybrid system consisted of a VOCs generator, a plasma reactor, and a catalyst bed.The Mn/NZ was loaded onto the catalyst bed (height = 24.5 cm, i.d.= 1.27 cm) located at the downstream of the plasma reactor.The concentration of reactant gas, acetaldehyde in a mixture of nitrogen, and air gas, was controlled by adjusting the flow rates of the standard gas (100 ppm of acetaldehyde in nitrogen gas) and air (dilution gas).The gas stream was injected to the reactor through an acrylic box (inner volume: 100 L).After a sufficient stabilization time (120 min), the concentration of reactant was controlled to 10 ppm at the exit line of the acrylic box by fine tuning the flow rates of acetaldehyde standard and dilution gas.The gas hourly space velocity (GHSV) of the reactant gas in the plasma zone, and the catalyst bed were set to 120/h and 222/h at ambient temperature and pressure, respectively.A dc corona reactor was used to produce plasma.The length and gap space of the discharge tube were 15 mm and 5 mm, respectively.Plasma power of 10 W was driven while using a high voltage AC power supply (12 kV, SIE = 120 J/L, 60 Hz, sine wave).During the oxidation reaction, the concentrations of acetaldehyde were measured using detection tubes (GASTEC, 92L, Measurement Range: 1-20 ppm) and ozone concentration was carried out using an ozone analyzer (Aeroqual 200) after gas sampling at the outlet.The effects of humidity in the system on the removal of acetaldehyde was also evaluated by controlling the relative humidity of the reactor to 20%, 60%, and 80%.The experiment was conducted by adjusting the initial humidity and maintaining the humidity continuously.About 25 ppm ozone was produced from NTP reactor.
Conclusions
In this study, the post-NTP/catalyst hybrid system was developed for the removal of VOCs that are emitted from food waste.The Mn-impregnated natural zeolite (Mn/NZ) was prepared, and the
Conclusions
In this study, the post-NTP/catalyst hybrid system was developed for the removal of VOCs that are emitted from food waste.The Mn-impregnated natural zeolite (Mn/NZ) was prepared, and the removal efficiencies of ozone and acetaldehyde were evaluated under a humidified atmosphere.In the presence of plasma, the Mn/NZ enhanced the removal efficiencies of ozone and acetaldehyde.The best efficiency of acetaldehyde removal was achieved when the 10 wt.% Mn/NZ was treated with sonication and calcined at 350 • C for 2 h.As a result, ozone and acetaldehyde were completely removed on 5 g of the 10 wt.% Mn/NZ catalyst with the post plasma treatment.In particular, the removal efficiencies of ozone and acetaldehyde were high, even in the presence of humid air (RH = 80%).Furthermore,
Figure 4 .
Figure 4.The effect of amount of catalysts (10 wt.% Mn/NZ) on the acetaldehyde oxidation in relative humidity 60%.
Figure 5 .
Figure 5.The effect of sonication on the catalytic activity of the 10 wt.% Mn/NZ for the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 4 .
Figure 4.The effect of amount of catalysts (10 wt.% Mn/NZ) on the acetaldehyde oxidation in relative humidity 60%.
Figure 4 .
Figure 4.The effect of amount of catalysts (10 wt.% Mn/NZ) on the acetaldehyde oxidation in relative humidity 60%.
Figure 5 .
Figure 5.The effect of sonication on the catalytic activity of the 10 wt.% Mn/NZ for the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 5 .
Figure 5.The effect of sonication on the catalytic activity of the 10 wt.% Mn/NZ for the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 6 .
Figure 6.The effect of calcination temperature for the 10 wt.% Mn/NZ on the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 6 .
Figure 6.The effect of calcination temperature for the 10 wt.% Mn/NZ on the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 7 .
Figure 7.The long-term catalyst stability test for the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 7 .
Figure 7.The long-term catalyst stability test for the acetaldehyde oxidation.Relative Humidity: 60%, The amount of catalysts: 5 g.
Figure 8 .
Figure 8.A schematic illustration of a non-thermal plasma (NTP)/catalyst hybrid reactor system for the VOCs decomposition from food waste.
Figure 8 .
Figure 8.A schematic illustration of a non-thermal plasma (NTP)/catalyst hybrid reactor system for the VOCs decomposition from food waste.
Table 1 .
Characteristics of the Mn-impregnated natural zeolite catalysts.
Table 2 .
Amounts of NZ and Mn(CH 3 COOH) 2 •4H 2 O used for the synthesis. | v3-fos-license |
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} | pes2o/s2orc | Discrimination of Bacillus cereus Group Members by MALDI-TOF Mass Spectrometry
Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) technology is currently increasingly used in diagnostic laboratories as a cost effective, rapid and reliable routine technique for the identification and typing of microorganisms. In this study, we used MALDI-TOF MS to analyze a collection of 160 strains belonging to the Bacillus cereus group (57 B. anthracis, 49 B. cereus, 1 B. mycoides, 18 B. wiedmannii, 27 B. thuringiensis, 7 B. toyonensis and 1 B. weihenstephanensis) and to detect specific biomarkers which would allow an unequivocal identification. The Main Spectra Profiles (MSPs) were added to an in-house reference library, expanding the current commercial library which does not include B. toyonensis and B. wiedmannii mass spectra. The obtained mass spectra were statistically compared by Principal Component Analysis (PCA) that revealed seven different clusters. Moreover, for the identification purpose, were generated dedicate algorithms for a rapid and automatic detection of characteristic ion peaks after the mass spectra acquisition. The presence of specific biomarkers can be used to differentiate strains within the B. cereus group and to make a reliable identification of Bacillus anthracis, etiologic agent of anthrax, which is the most pathogenic and feared bacterium of the group. This could offer a critical time advantage for the diagnosis and for the clinical management of human anthrax even in case of bioterror attacks.
Introduction
The Bacillus cereus group is comprised of Gram-positive, rod-shaped and sporeforming aerobic bacteria, genetically closely related. Currently this group includes several species, phylogenetically organized in three broad clades: Bacillus cereus sensu stricto and Bacillus thuringiensis, both found in all clades; Bacillus anthracis and Bacillus wiedmannii, in Clade 1; Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus toyonensis, Bacillus cytotoxicus, Bacillus bingmayongensis, Bacillus gaemokensis and Bacillus manliponensis, in Clade 3 [1,2]. Clades are divided into seven different subgroups, defined according to multiple and combined genetic analyses [2]. Despite being extremely similar and related, only some of these microorganisms have an important impact on human and animal health, agriculture and the food industry [3].
B. cereus sensu stricto is a ubiquitous bacterium, widespread in the terrestrial and marine environment. Thanks to ability to produce spores, it survives pasteurization [4] and the various thermal processes used in food industries [5]; therefore, it is not uncommon to isolate it from both raw and cooked foods. It can produce toxins, causing two types of gastrointestinal illness: the emetic syndrome (associated with nausea and vomiting) and the diarrhoeal syndrome [6,7].
B. anthracis is the etiological agent of anthrax, an acute fatal zoonotic disease that affects primarily herbivores, but that may be transmitted also to humans. It is also known for its potential use as biological weapon. Compared to the other members of the B. cereus group, the genome consists of the chromosome and of two plasmids, pXO1 and pXO2, which carry genes coding for the toxins and the capsule, respectively, and on which its high virulence depends [8].
In the past few decades, two atypical B. cereus strains (called Bacillus anthracis-like bacteria) have been described; they possess B. cereus s.s. chromosomal DNA and virulence plasmids that are highly comparable to the anthrax virulence plasmids (pXO1 and pXO2). As they have the same pathogenicity as B. anthracis with the production of the tripartite toxin, these strains cause fatal anthrax-like disease in humans and animals [2].
Being genetically and phenotypically very similar to each other since they probably have common evolutionary relationships [1], the discrimination of members belonging to the B. cereus group is complicated.
The classic methods for microorganism identification are primarily based on biochemical tests, which are very time-consuming with results that are often ambiguous and not exhaustive, and biomolecular tests that are faster but requiring the use of specific reagents (as a set of suitable primers), not always available for routine laboratories diagnostics.
A powerful, sensitive and reliable technique now increasingly used for microbial identification and clinical diagnosis is MALDI-TOF mass spectrometry. Through this valuable diagnostic tool, the discrimination of microbial species different from the genetic and protein point of view is relatively simple but the distinction of closely related species is the real challenge.
Several studies have been conducted already on the applicability of MALDI-TOF MS in the identification/discrimination of related species, as for members of the B. cereus group [9][10][11][12].
In this study, a collection of 160 strains belonging to the Bacillus cereus group was analyzed with the aim to define the characteristic ion peaks for the species B. cereus, B. anthracis, B. mycoides, B. thuringiensis and B. weihenstephanensis, and create an algorithm to identify the species after the classical library matching approach. This method is already used by the additional software module MALDI Biotyper (MBT) Compass Subtyping for the correct species identification for Listeria [13]. The Listeria species have very similar spectra profile, and the classical identification approach allows the genus identification, but a new algorithm developed by Bruker Daltonik is able to identify correctly the species using a few characteristic peaks [14].
In addition, the study has been extended also to other species of the Bacillus cereus group such as B. toyonensis and B. wiedmannii, since, presently, they are not included in the Bruker Daltonik (BDAL) library (Bruker Daltonik GmbH, Bremen, Germany).
Bacterial Strains and Molecular Identification
Bacterial strains used in this study included n = 103 strains isolated from food samples, of which n = 49 B. cereus, n = 1 B. mycoides, n = 18 B. wiedmannii, n = 27 B. thuringiensis, n = 7 B. toyonensis, n = 1 B. weihenstephanensis and n = 57 B. anthracis strains, different for MLVA 31-loci profile, isolated from 1984 to 2020 from animal anthrax outbreaks in Italy and belonging to the collection of the Anthrax Reference Institute of Italy (Ce.R.N.A.). The complete list of isolates used in this study and their origin are listed in Table S1.
Bacterial isolation from food was performed according to ISO 21871:2006. Briefly, 5 g or mL of sample were added to 45 mL of Buffered Peptone Water (BPW) (Biolife Italiana, Milan, Italy) in sterile bags and homogenized using a stomacher (PBI International, Milan, Italy) at 230 rpm for 30 s. Then, 45 mL of double-strength Tryptone Soy Polymyxin Broth (TSPB) (Biolife Italiana, Milan, Italy) were added to initial suspension and the bags were incubated at 30 • C for 48 h under aerobic condition.
After incubation, 10 mL of enrichment broth were streaked onto the surface of solid selective medium Mannitol Egg Yolk Polymyxin Agar (MYP) (Biolife Italiana, Milan, Italy) and the plates were incubated at 30 • C for 24-48 h under aerobic condition.
After this step, typical presumptive Bacillus cereus group colonies for each sample were picked, subcultured on Columbia Agar with 5% sheep blood and then incubated at 37 • C for 18-24 h. Bacterial DNA was extracted using the DNAeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's protocol for Gram-positive bacteria.
Biomolecular identification of B. anthracis was performed using Real-time PCR assay previously described by Wielinga et al. (2011) [15], based on the amplification of three different specific sequences: pl3 gene located on chromosome; pagA gene located on the virulence plasmid pXO1 and capB gene located on the virulence plasmid pXO2.
Two combined assays were used for the identification of B. cereus s.s., B. mycoides, B. thuringiensis and B. weihenstephanensis: a multiplex PCR based on the gyrB sequence [16] and a TaqMan assay based on the amplification of the motB gene [17].
A TaqMan assay, designed on a specific region of the ccpA gene [18], was used to identify B. toyonensis.
The manipulation of B. anthracis strains was performed in a biosafety level 3 (BSL-3) laboratory within a class II safety cabinet.
Sample Preparation for MALDI-TOF Mass Spectrometry Analysis
Each Bacillus strain was grown on Columbia blood agar for 18-24 h at 37 • C before MALDI-TOF MS analysis. For the inactivation of vegetative cells and spores of Bacillus species, trifluoroacetic acid (TFA) (Sigma-Aldrich, St Louis, MO, USA) was used, an organic solvent that effectively inactivates the vegetative and spore forms and solubilizes bacterial proteins, preserving their structural integrity [20].
Using a 10 µL loop, fresh bacterial colonies were transferred into a tube containing 1 mL of 80% TFA, resuspended and incubated for 30 min at room temperature. Subsequently, a 1:2 dilution in sterile deionized water (Carlo Erba Reagents, Cornaredo, Italy) was prepared [11].
Thus, 1 µL of this mixture was applied onto a 96-well steel target plate (Bruker Daltonics, Germany). After drying, the sample spots were overlaid with 1 µL of matrix solution, α-cyano-4-hydroxycynnamic acid (HCCA, Bruker Daltonik GmbH, Bremen, Germany) 10 mg/mL. The mass spectra were acquired using Microflex LT/SH™ mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), which was operated in linear positive mode covering a mass to charge ratio (m/z) between 2000 and 20,000. Each spot of the target plate was hit with a pulsed nitrogen laser beam operating at 337 nm, with a frequency equal to 60 Hz. After laser shot, the gas phase ions obtained were accelerated in the flight tube by an acceleration voltage with optimized values for the mass range understudy. Two hundred and forty individual laser shots were added for each spectrum. For each strain, a total of 18 mass spectra were obtained. The instrument was calibrated in the broad molecular weight range between 2 and 20 kDa using Bruker Bacterial Test Standard (BTS, Bruker Daltonik GmbH, Bremen, Germany), an extract of the Escherichia coli DH5α strain, with the addition of two proteins (RNase A of 13,683.2 Da and myoglobin of 16,952.3 Da).
At the moment, the BDAL library does not include B. toyonensis and B. wiedmannii but the MALDI Biotyper system offers the opportunity to expand the library.
About this, two strains of B. toyonensis and B. weidmanni were cultured and prepared by the full extraction protocol procedures according to the manufacturer's protocol. Four colonies were transferred in 300 µL of sterile deionized water (Carlo Erba Reagents, Cornaredo, Italy) in a tube, and 900 µL of ethanol absolute (Sigma-Aldrich, St Louis, MO, USA) were added. After vortex mixing and centrifuge, the supernatant was discarded; the dry pellet was resuspended in 25 µL of 70% formic acid (Carlo Erba Reagents, Cornaredo, Italy) and 25 µL of acetonitrile, grade HPLC (Honeywell, Charlotte, North Carolina, United States). After centrifugation, 1 µL of solution was spotted on each spot of the target plate and covered by HCCA matrix after solvent evaporation. For each extract, eight different spots were prepared on the 96-well steel target plate and three different mass spectra were acquired for each of them automatically, thanks to Automatic execute run by Flex Control software (Bruker Daltonik GmbH, Bremen, Germany).
The mass spectra obtained were manually analyzed by FlexAnalysis software (v 3.4; Bruker Daltonik GmbH, Bremen, Germany) and each spectrum was subjected to spectral preprocessing procedures, such as smoothing, baseline subtraction and intensity normalization. The used parameters were the same as the default MBT processing method, applied for the automatic identification: smoothing alghoritm SavitzkyGolay, Baseline substraction alghoritm TopHat.
Twenty-four different mass spectra for species were analyzed by Flex Analysis software to verify the accuracy in terms of mass to charge ratio and the reproducibility. All the 24 mass spectra acquired for each strain were compared to verify the presence of flatline spectra or spectra with outlier peaks. Furthermore, the mass accuracy inter-spectra were evaluated by checking the mass shift of the base peak in all the acquired mass spectra, considering as maximal tolerance 500 ppm. After the spectra quality check, in terms of reproducibility and accuracy, the new MSPs for B. toyonensis and B. weidmannii were created by MBT Compass Explorer module (Bruker Daltonik GmbH, Bremen, Germany). The MSPs were created using the Biotyper Standard Method for the spectra processing and the reference peak list creation, to obtain comparable data also with reference mass spectra of BDAL library. In particular, 3000 m/z as lower bound, 15,000 as upper bound, 25% as minimum desired peak frequency, 70% as maximum desired peak number for MSP, were set as parameters for MSP creation.
The new MSPs were tested preparing fresh subculture of the same strains, for an additional quality control step, and later measured against the newly created MSP. The strains were identified with the new MSP appearing within the 10 best matches of analysis results.
All the experimental mass spectra for all the species were processed together in ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany) in all mass range acquired (2-20 kDa) with topHat baseline substraction (10% minimal baseline width), SavitzkyGolay smoothing (2.0 width m/z) and all peaks with signal-to-noise ratio higher than 3 were evaluated. All these parameters were considered for all following statistical methods (Gel view, PCA, average spectra calculation).
The Gel View displays all mass spectra of the loaded classes arranged in a pseudo-gel like look. The x-axis records the m/z value. The left y-axis displays the running spectrum number originating from subsequent spectra loading. The peak intensity is expressed by a color code. The color bar and the right y-axis indicate the relation between the color a peak is displayed with and the peak intensity in arbitrary units. In the next figure of gel view, the peak intensities are shown as gray scaled. ClinProTools offers a statistical data analysis in terms of principal component analysis (PCA). The scaling method used was the Level Scaling, where the intensity of each peak is taken in count as well the mean intensity of the peak in the data set.
From the preprocessed individual mass spectra for each species, an average spectrum is calculated. Mass spectra are weighted with the reciprocal size of the classes to get an equal representation of classes with a quite different number of mass spectra.
The peak picking to create the average spectrum for each species were performed using the total average spectrum peak picking approach. Thereby, the peak picking is applied on the calculated total average spectrum.
The automatic detection of ion peaks is based on the analysis of a smoothed first derivative. The smoothing is determined by the Resolution parameter fixed as appropriate value. To reduce the number of ion peaks picked on the total average spectrum, and thus the average ion peak list, a Signal to Noise Threshold 3.0 was applied and a Maximal Peak Number found value was fixed to 100.
ClinProTools supports also different algorithms for generating classification models. In this study, the classification models were generated using the algorithms QC (Quick Classifiers) and SVM (Support Vector Machine). Usually, to evaluate the performance of the classification models the Recognition Capability and the Cross-Validation parameters are considered. The first is calculated as the relative number of correct classified data points by the classifier for the given model. The second is a measure for the reliability of a calculated model and can be used to predict how a model will behave in the future. Both parameters were calculated for overall data and for each class considered, in our case the different species of Bacillus.
Results and Discussion
For the MALDI-TOF MS analysis, the results are generally expressed with log(score) values between 0 and 3.0, indicative of the matching between the sample spectrum and the MSPs in the reference database. A log(score) <1.7 indicates that it could not identify the genus or species of the strain; a log(score) between 1.7 and 2.0 indicates that identification could be reliable only at the genus level, while a log(score) ≥2.0 indicates that identification could be reliable at the species level of the organism.
In this study, as expected, the identification with commercial databases proved to be inconclusive for the B. cereus group species because almost all samples mass spectra matched with B. anthracis (in the SR), in most cases, with a log(score) >2.0 (data not shown). Excluding the SR library, the mass spectra of the samples corresponded to those of the B. cereus of the library BDAL with a log(score) generally between 1.7 and 2.0 or the identification was not possible (Data not shown). Furthermore, when one of the Bacillus cereus group members is identified by the MBT Compass (Bruker Daltonik GmbH, Bremen, Germany) software with BDAL library, the comment appears: "Bacillus anthracis, cereus, mycoides, pseudomycoides, thuringiensis and weihenstephanensis are closely related and members of the Bacillus cereus group. In particular, Bacillus cereus spectra are very similar to spectra from Bacillus anthracis. Bacillus anthracis is not included in the MALDI Biotyper database. For differentiation, an adequate identification method must be selected by an experienced professional. The quality of spectra (score) depends on the degree of sporulation: Use fresh material". It means that after the identification, additional tests are mandatory to correctly discriminate and identify these species due to the high degree of similarity of the mass spectra. In addition, not all the species belonging to the Bacillus cereus group are included in the library, such as B. toyonensis and B. weidmannii. At this scope, well-characterized strains of these bacteria were used to create new MSPs to add to the BDAL library. Differently from similar previous studies, we also tested the two above mentioned bacteria, in order to implement a new library and to allow the identification of these species, not possible at the moment, even if with some limitations.
Unfortunately, the main spectra profiles for B. toyonensis and B. weidmannii are enough different from the MSPs of other species contained in the library but too much similar between them, consequently a further algorithm is needed.
The same approach used to discriminate the species B. cereus, B. anthracis, B. thuringensis and B. weihenstephanensis, was used also to discriminate B. toyonensis from B. weidmannii.
The comparison of unknown and reference mass spectra involves the analysis of the total spectrum and comparison of only distinctive peaks. The analysis of specific peaks is more selective, because it "weighs" peaks specific for a given microorganism, excluding those originating from background noise or exogenous factors. Therefore, to investigate the differences at the single peak level, it is mandatory for a detailed analysis for all peaks to be revealed in the mass spectra.
The mass spectra of these species resulted to be differentiable based on their overall peak number, consistent for each Bacillus cereus group species (Figure 1). degree of similarity of the mass spectra. In addition, not all the species belonging to the Bacillus cereus group are included in the library, such as B. toyonensis and B. weidmannii. At this scope, well-characterized strains of these bacteria were used to create new MSPs to add to the BDAL library. Differently from similar previous studies, we also tested the two above mentioned bacteria, in order to implement a new library and to allow the identification of these species, not possible at the moment, even if with some limitations.
Unfortunately, the main spectra profiles for B. toyonensis and B. weidmannii are enough different from the MSPs of other species contained in the library but too much similar between them, consequently a further algorithm is needed.
The same approach used to discriminate the species B. cereus, B. anthracis, B. thuringensis and B. weihenstephanensis, was used also to discriminate B. toyonensis from B. weidmannii.
The comparison of unknown and reference mass spectra involves the analysis of the total spectrum and comparison of only distinctive peaks. The analysis of specific peaks is more selective, because it "weighs" peaks specific for a given microorganism, excluding those originating from background noise or exogenous factors. Therefore, to investigate the differences at the single peak level, it is mandatory for a detailed analysis for all peaks to be revealed in the mass spectra.
The mass spectra of these species resulted to be differentiable based on their overall peak number, consistent for each Bacillus cereus group species (Figure 1). The mass spectra demonstrate a relatively high signal-to-noise ratio, which typically permits the detection of 50 to 100 mass peaks per spectrum with the signal-to-noise ratio higher than 3.
Mass spectra obtained from each single tested species were compared to each other and for each species a main spectrum (MSP) was created and included in an in-house reference library.
In the dendrogram generated by MSPs ( Figure S1) by MBT Compass Explorer software (v. 4.1.70, Bruker Daltonik GmbH, Bremen, Germany), B. anthracis protein mass The mass spectra demonstrate a relatively high signal-to-noise ratio, which typically permits the detection of 50 to 100 mass peaks per spectrum with the signal-to-noise ratio higher than 3.
Mass spectra obtained from each single tested species were compared to each other and for each species a main spectrum (MSP) was created and included in an in-house reference library.
In the dendrogram generated by MSPs ( Figure S1) by MBT Compass Explorer software (v. 4.1.70, Bruker Daltonik GmbH, Bremen, Germany), B. anthracis protein mass spectra were placed on a separate branch compared to the branch containing most of the protein mass spectra of the remaining Bacillus species, except for some Bacillus cereus strains that shared the same branch.
All mass spectra were processed also with ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany) in all mass range acquired (2-20 kDa) with topHat baseline substraction (10% minimal baseline width), SavitzkyGolay smoothing (2.0 width m/z) and all peaks with signal-to-noise ratio higher than 3 were evaluated.
ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany) allowed the generation of simulated gel views ( Figure 2) from preprocessed mass spectra, in order to give an overview of the analyzed microbial MALDI mass spectra. These gel views show spectral peak intensities gray scaled as function the m/z values. Analysis of the gel view demonstrated a high degree of spectral reproducibility between different mass spectra of the same species.
Microorganisms 2021, 9, x FOR PEER REVIEW 7 of 14 spectra were placed on a separate branch compared to the branch containing most of the protein mass spectra of the remaining Bacillus species, except for some Bacillus cereus strains that shared the same branch. All mass spectra were processed also with ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany) in all mass range acquired (2-20 kDa) with topHat baseline substraction (10% minimal baseline width), SavitzkyGolay smoothing (2.0 width m/z) and all peaks with signal-to-noise ratio higher than 3 were evaluated.
ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany) allowed the generation of simulated gel views ( Figure 2) from preprocessed mass spectra, in order to give an overview of the analyzed microbial MALDI mass spectra. These gel views show spectral peak intensities gray scaled as function the m/z values. Analysis of the gel view demonstrated a high degree of spectral reproducibility between different mass spectra of the same species. Mass spectra, grouped for each Bacillus cereus group species, were statistically compared using Principal Component Analysis (PCA) to determine general differences in the protein mass spectra, by ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany). Analysis by PCA revealed seven different clusters (Figure 3). Mass spectra, grouped for each Bacillus cereus group species, were statistically compared using Principal Component Analysis (PCA) to determine general differences in the protein mass spectra, by ClinProTools software (v. 3.0, Bruker Daltonik GmbH, Bremen, Germany). Analysis by PCA revealed seven different clusters (Figure 3).
The clusterization obtained by the PCA means that mass spectra for different species can be differentiated by statistical analysis, and therefore mass spectra will necessarily show spectral differences and probably characteristic peaks.
The software generated a list of the 100 ion peaks considered with their mass to charge ratio, the difference between the maximal and the minimal average peak intensity of all classes (Dave), p-value of t-test, p-value of Wilcoxon test, p-value of Anderson-Darling test, the Peak intensity average for each class and relative standard deviation and percentage coefficient of variation. To reduce the data to a shorter list, only the mass peaks with higher Dave and the lower standard deviation for the peak intensity average of the main class were considered. A shorter list of twelve ion peaks was obtained (Table 1). Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 14 The clusterization obtained by the PCA means that mass spectra for different species can be differentiated by statistical analysis, and therefore mass spectra will necessarily show spectral differences and probably characteristic peaks.
The software generated a list of the 100 ion peaks considered with their mass to charge ratio, the difference between the maximal and the minimal average peak intensity of all classes (Dave), P-value of t-test, P-value of Wilcoxon test, P-value of Anderson-Darling test, the Peak intensity average for each class and relative standard deviation and percentage coefficient of variation. To reduce the data to a shorter list, only the mass peaks with higher Dave and the lower standard deviation for the peak intensity average of the main class were considered. A shorter list of twelve ion peaks was obtained (Table 1). Table 1. List of the 12 ion peaks considered with their mass to charge ratio and the peak intensity average for each class and relative standard deviation. In bold are shown the higher intensities values for each mass peak, to be considered as characteristic ion peak for the class/species. B. anthracis B. cereus B. mycoides B. thuringensis B. toyonensis B. These twelve ion peaks seem to be characteristic for five out of seven considered species. In particular, from this peak selection, no characteristic ion peaks appear for B. cereus and B. toyonensis.
Intensity ± Stdev Mass/Charge
For B. anthracis, B. thuringensis and B. weinsthephanensis species, more than one characteristic ion peak was defined. In particular, for B. anthracis the ion peaks 3339 m/z, 3592 m/z, 4871 m/z, 9740 m/z; for B. thuringensis the ion peaks 2956 m/z, 2968 m/z, 3411 m/z; for B. weinsthephanensis the ion peaks 4637 m/z, 7324 m/z 9272, can be considered as characteristic ion peaks. This allowed to create 2D Peak Distribution View (Figure 4) showing a perfect separation of the considered species; each colorful mark represents one mass spectra and, the value for x and y, the intensities for the peak reported on the axis.
The four characteristic ion peaks for B. anthracis are reported in two different plots: in the first is shown the intensity of the 3339 m/z against the intensity of the 4871 m/z; in the second the 3592 m/z vs. 9740 m/z. For the other species, the two most intense peaks of the three were considered (Figure 4). These 2D distribution are the first confirmation of the presence of characteristic ion peaks in the mass spectra analyzed for the species considered, useful for species differentiation in the B. cereus group.
These twelve ion peaks seem to be characteristic for five out of seven considered species. In particular, from this peak selection, no characteristic ion peaks appear for B. cereus and B. toyonensis.
For B. anthracis, B. thuringensis and B. weinsthephanensis species, more than one characteristic ion peak was defined. In particular, for B. anthracis the ion peaks 3339 m/z, 3592 m/z, 4871 m/z, 9740 m/z; for B. thuringensis the ion peaks 2956 m/z, 2968 m/z, 3411 m/z; for B. weinsthephanensis the ion peaks 4637 m/z, 7324 m/z 9272, can be considered as characteristic ion peaks. This allowed to create 2D Peak Distribution View (Figure 4) showing a perfect separation of the considered species; each colorful mark represents one mass spectra and, the value for x and y, the intensities for the peak reported on the axis. (Figure 4). These 2D distribution are the first confirmation of the presence of characteristic ion peaks in the mass spectra analyzed for the species considered, useful for species differentiation in the B. cereus group.
The same statistical approach was used for B. mycoides and B. wiedmannii and only one characteristic ion peak was found, in the mass range between 5410 and 5450 as mass-to-charge ratio; the ion peaks selected well defined the two species ( Figure 5). The same statistical approach was used for B. mycoides and B. wiedmannii and only one characteristic ion peak was found, in the mass range between 5410 and 5450 as mass-tocharge ratio; the ion peaks selected well defined the two species ( Figure 5). The twelve characteristic ion peaks considered in this study were forced in statistical model to "classify" our seven different species of B. cereus sensu lato to describe the mass spectra of the model generation classes in such a way that new mass spectra can be classified afterwards.
The results for the two classifying model are summarized in Table 2. The twelve characteristic ion peaks considered in this study were forced in statistical model to "classify" our seven different species of B. cereus sensu lato to describe the mass spectra of the model generation classes in such a way that new mass spectra can be classified afterwards.
The results for the two classifying model are summarized in Table 2. The recognition capability and the cross-validation values were considered an index of algorithm functionality. The recognition capability of the QC was 68.3%, while cross validation was 66.59%. The recognition capability of the SVM was 97.14%, while cross validation was 96.02%. These values take into account two classes with no characteristic ion peaks as B. cereus and B. toyonensis, which reduces the percentage values. Nevertheless, the results of the classification model showed an extremely high reliability, especially for the species in which one or more characteristic ion peaks are considered.
All this information was used to create a dedicated algorithm in FlexAnalysis software (v. 3.4, Bruker Daltonik GmbH, Bremen, Germany) for a rapid and automatic detection of characteristic ion peaks after the acquisition for the identification purpose. This software has an interesting tool named Specification check. Two different sets of algorithms were created: one useful after the identification results as B. cereus group to discriminate B. cereus, B. anthracis, B. mycoides, B. thuringensis and B. weisthephanensis, and a second algorithm to distinguish B. toyonensis and B. weidmannii. The algorithm creates the ion peak list of the experimental spectrum acquired after the automatic identification. For each species under study, a list of characteristics ion peaks was created. The algorithms search the characteristic ion peaks with a defined error in terms of mass accuracy and report the ion peaks found in the experimental spectrum. If we find ion peaks considered characteristic for a certain species, the differentiation at species level for B. cereus group is considered satisfactory.
In Figure 6, the result obtained after the automatic algorithm run in FlexAnalysis software (v. 3.4, Bruker Daltonik GmbH, Bremen, Germany) is shown. It refers to the algorithm used for B. anthracis species, the more complex one, due to the number of the ion peaks. In the example, all the four characteristic ion peaks were found with a low deviation error in term of ppm; in fact, in the result, "Specification passed" is shown. It means that the experimental spectrum can be identified now as B. anthracis with no limitations as before.
If all the characteristic ion peaks are found in the experimental spectrum considered, the specification is passed and the correct identification at species level is possible.
When all the other algorithms were applied to the same experimental spectrum, no characteristic ion peaks for other species were found. In fact, the result appears as "NOT PASSED" in Figure 7.
It means that the experimental spectrum can be identified now as B. anthracis with no limitations as before.
In Figure 6, the result obtained after the automatic algorithm run in FlexAnalysis software (v. 3.4, Bruker Daltonik GmbH, Bremen, Germany) is shown. It refers to the algorithm used for B. anthracis species, the more complex one, due to the number of the ion peaks. In the example, all the four characteristic ion peaks were found with a low deviation error in term of ppm; in fact, in the result, "Specification passed" is shown. It means that the experimental spectrum can be identified now as B. anthracis with no limitations as before. Figure 6. Result of FlexAnalysis algorithm for B. anthracis species. If all the characteristic ion peaks are found in the experimental spectrum considered, the specification is passed and the correct and unambiguous identification at species level is possible.
If all the characteristic ion peaks are found in the experimental spectrum considered, the specification is passed and the correct identification at species level is possible.
When all the other algorithms were applied to the same experimental spectrum, no characteristic ion peaks for other species were found. In fact, the result appears as "NOT PASSED" in Figure 7. means that the experimental spectrum can be identified now as B. anthracis with no limitations as before. If all the characteristic ion peaks are found in the experimental spectrum considered, the specification is passed and the correct identification at species level is possible.
When all the other algorithms were applied to the same experimental spectrum, no characteristic ion peaks for other species were found. In fact, the result appears as "NOT PASSED" in Figure 7.
Conclusions
Members of the Bacillus cereus group are strongly genetically related [21]; therefore, their discrimination is difficult. Developing fast, extremely sensitive and reliable protocols for their detection is extremely important, since some of the species belonging to this group have an important impact on human and animal health [3]. Early identification of B. anthracis is crucial, as it could offer a critical time advantage in the management of animal anthrax outbreaks and of clinical treatment of human anthrax, including bioterrorist attacks.
MALDI-TOF mass spectrometry is a valuable tool to rapidly identify and characterize microorganisms isolated from clinical and environmental samples, with results often more reliable than classic microbiological diagnostic methods [22].
This analytical technique is based on the ionization of high molecular weight biological macromolecules and separation of the ions, based on their mass to charge ratio (m/z) [23]. The obtained mass spectra appear as a set of ion peaks of different intensity, each corresponding to the mass/charge value of a molecular ion [23].
Microbial identification is possible because each bacterial species is characterized by a specific protein pattern and therefore by a characteristic spectral profile which constitutes its "fingerprint" [24]. The proteins play an important role in laboratory diagnostics as they constitute about 50% of the dry weight of vegetative bacterial cells and are present in multiple copies on the contrary DNA [25]; moreover, they provide good signals bypassing extraction or amplification steps.
Despite the high diagnostic potential, its limit is the fact that microorganisms belonging to different genera have quite different mass spectra with few or no common ion peaks and therefore are easily identifiable [26]. At the species level, mass spectra are increasingly similar [26] and distinguishing them is more complicated, especially with closely related species, as members of the B. cereus group. To overcome this issue, in this study, we examined a collection of 160 strains belonging to the Bacillus cereus group, isolated from various clinical, food and environmental matrices. The experimental mass spectra obtained with the Flex Control The MSPs obtained were added to an in-house reference library, expanding the commercial BDAL library (Bruker Daltonik GmbH, Bremen, Germany), which currently does not include B. toyonensis and B. wiedmannii mass spectra. As described by previous studies [11,27], a richer database can remarkably increase the probability of a correct identification. In case of unambiguous or doubt identification, however, we recommend to combine this diagnostic method with others, such as biomolecular techniques.
The mass spectra grouped for each Bacillus cereus group species were statistically compared using Principal Component Analysis (PCA) to determine general differences in the protein spectra, through which seven different clusters were revealed.
From the preprocessed individual mass spectra for each species, a total average spectrum was calculated, and, for all classes/species, characteristic ion peaks have been identified. This information has been used to generate two different sets of algorithms for a rapid and automatic detection after the mass spectra acquisition. In particular, the first algorithm was useful to discriminate B. cereus, B. anthracis, B. mycoides, B. thuringensis and B. weisthephanensis, while the second one was used to distinguish B. toyonensis and B. weidmannii.
This study aims to provide an important diagnostic support in the rapid identification of species belonging to a group of highly correlated bacteria, such as the Bacillus cereus group, with particular attention to the most pathogenic and feared bacterium of the group, B. anthracis, for which the classical diagnostic methods can take a long time, often with ambiguous results.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/microorganisms9061202/s1, Table S1. List of Bacillus cereus group strains used in this study.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author. | v3-fos-license |
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} | pes2o/s2orc | Dietary Montmorillonite Improves the Intestinal Mucosal Barrier and Optimizes the Intestinal Microbial Community of Weaned Piglets
The study investigated the impact of dietary montmorillonite on the growth performance, intestinal mucosal barrier, and microbial community in weaned piglets with control group (CON) and dietary supplementation of 0.2% montmorillonite (0.2% M). Compared with the CON group, 0.2% M feed in the diet increased the average daily gain (ADG) on days 15–35 and day 1–35 and the average daily feed intake on days 1–35 (ADFI) (0.05 < P < 0.1). Besides, higher villus height of the duodenum and jejunum and lower crypt depth of duodenum and colon were revealed in the 0.2% M group than in the CON group (P < 0.05). Moreover, the V/C (ratio of the villus height and crypt depth) in the 0.2% M group was increased compared to that in the CON group both from the duodenum and ileum (P < 0.05). The relative mRNA expression of mucin-1, ITGB1 (β1-integrins), and PKC (protein kinase C) of ileum in the 0.2% M group were upregulated (P < 0.05) compared to that in the CON group. The digesta sample of ileum from piglets in the 0.2% M group contained greater (P < 0.05) intestinal bacterial diversity and abundances of probiotics, such as Streptococcus, Eubacterium_rectale_group, and Lactobacillus, which could promote the synthesis of carbon-containing biomolecules. Overall, dietary supplementation of 0.2% M was shown to have a tendency to improve the growth performance of weaned piglets and may enhance their intestinal mucosal barrier function via altering the gut microbiota.
INTRODUCTION
Ensuring that weaned piglets can grow healthily into the fattening stage is a key issue within pig breeding that has an effect on the economic benefit of the pig breeding industry. The intestinal nutrient metabolism during the weaning period undergoes a transition from liquid milk digestion to feed digestion (Wijtten et al., 2011). This process is prone to diseases, such as bacterial diarrhea (Heo et al., 2012;Huang et al., 2015) or viral gastroenteritis (Wang Q. et al., 2019), and even death, which may be caused by the impaired intestinal mucosal barrier function (Han et al., 2016;Ma et al., 2017). A healthy and efficient intestinal mucosal barrier and microbial community in weaned piglets can contribute to a safe and rapid transition for weaned piglets to the fattening stage (Heo et al., 2012). The active substances that could enhance the intestinal function and optimize intestinal microbes are therefore a powerful weapon with which to improve the gut health and growth performance of weaned piglets.
Montmorillonite, a layered silicate, possesses various physical and chemical properties, such as adsorption and swelling (Bhattacharyya and Gupta, 2008). It was also found to include anti-bacterial properties (Williams, 2008), enhancing immunity (Lee et al., 2015), and the intestinal mucosal barrier (Das et al., 2015). Recently, many studies Subramaniam and Kim, 2015) have reported that the montmorillonite has the potential to improve the intestinal barrier function, digestibility of nutrients, and growth performance of weaned piglets, which may be related to the changed gut microbiota (Xia et al., 2005;Wang et al., 2012). There has been, however, limited knowledge of the interactions between montmorillonite, the intestinal barrier, and intestinal bacteria community. In the present study, the growth performance, intestinal barrier function, and intestinal bacteria community of weaned piglets were investigated in terms of the efficacy of a supplement of 0.2% montmorillonite in order to provide a theory for rational and efficient utilization of montmorillonite as a feed supplement.
MATERIALS AND METHODS
All experimental operations and processing used in this work were approved by the Animal Care and Use Committee of China Agricultural University (Beijing, China). The montmorillonite used in this work (>95% purity) was obtained from Sanding Technology Company (Zhejiang, China).
Animal Treatment and Experimental Design
A total of 60 crossbred piglets (Duroc × Landrace × Large White) weaned at 28 ± 1 day of age, with an average initial body weight of 6.5 ± 0.5 kg, were randomly allocated to two dietary groups receiving a corn-soybean meal basal diet (CON group) or a corn-soybean meal basal diet supplemented with 0.2% montmorillonite (0.2% M) according to weight, sex, and litter with six replicates (pens) of five piglets. Feed and water were available ad libitum throughout the 35 days feeding trial. The basal diet was formulated to meet the nutrient requirements recommended by the NRC. The ingredient composition and nutrient content of basal diet was given in Table 1. Diets were mixed for sample and grinded to pass through a 0.15 mm sieve. Dry matter, gross energy, crude protein, calcium and total phosphorus, ether extract, and ingredient contents of the basal diets were calculated according to the procedures of the Association of Official Analytical Chemists (AOAC) International. The body weight (BW) of piglets and feed were measured individually at the beginning, end of first 2 weeks, and end of the whole experiment to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed:gain (F:G) ratio.
Sample Collection
At the end of the experiment, six piglets closest to the average BW from each group (one pig per pen) were slaughtered after being fasted overnight (12 h), and the duodenum, jejunum, ileum, and colon were then sampled through a sterile laparotomy and placed in neutral formalin for histological analysis or collected in centrifuge tubes and then immediately placed in liquid nitrogen and stored at the temperature of −80 • C for analysis of mRNA expression levels of β-actin, mucin-1 (MUCIN-1), β1-integrins (ITGB1), collagen, occludin, and protein kinase C (PKC) in ileal tissue. Briefly, about 1 cm length of the middle part of duodenum, jejunum, ileum, and colon were collected. The digesta of ileum and colon were obtained using centrifuge tubes and immediately placed in liquid nitrogen and then stored at the temperature of −80 • C for analysis of the bacterial community.
Intestinal Morphology Analysis, RNA Extraction, and Quantitative RT-PCR Analysis Samples from duodenal, jejunal, ileal, and colonic segment were embedded in paraffin and cut into 5 µm sections. Six non-successive sections of each sample were identified with hematoxylin and eosin staining. Six well-oriented villi and their associated crypt per section from each sample were collected. The villus height and crypt depth of duodenum, jejunum, ileum, and colon were measured and analyzed using a Leica Image Processing with Analysis System (Leica Imaging Systems Limited, Berlin, Germany). Total RNA extraction and quantitative realtime PCR analysis were conducted as described previously (Shen et al., 2014) with modifications. Briefly, TRIzol reagent (Invitrogen, Carlsbad, United States) was used to extract the total RNA of ileum after tissue homogenization and mixed with DNase I (Invitrogen, Carlsbad, United States). The obtained total RNA of IM (ileum with 0.2% montmorillonite) and INC (ileum negative control) were checked by 1% agarose gel electrophoresis and 2100 Bioanalyzer RNA Nanochip (Agilent, Palo Alto, United States). PrimeScript TM RT Reagent Kit (Takara, Dalian, China) was used to perform the reverse transcription of total RNA. Expression levels of β-actin, MUCIN-1, ITGB1, collagen, occludin, and PKC in ileal tissue were analyzed by Roche LightCyler 480 system (Roche, Basel, Switzerland). The primer sequences for these six genes were showed in Table 2.
The RT-PCR system was: 10 µL of 2 × SYBR R premix Ex TaqTM II, 0.6 µL of each forward and reverse primer (10 µmol/L), 2 µL of complementary DNA template, and 6.8 µL of double distilled water. The PCR reaction included an inactivation step at 95 • C for 5 min, and this was followed by 35 cycles of denaturation at 95 • C for 10 s, annealing at 60 • C for 10 s, and extension at 72 • C for 15 s. Each reaction was conducted to 20 µL using LightCycler 480 SYBR Green 1 Master (Roche, Basel, Switzerland). Each gene was performed with triplicate biological replicates and technical replicates. The result was calculated and represented by the 2 − CT method.
Intestinal Microbial DNA Extraction and High-Throughput Sequencing
Isolation of ileal and colonic content was performed using DNA Stool Mini Kit (Qiagen, Hilden, Germany). The quality and size of extracted DNA were checked via a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, United States) and 1% agarose gel electrophoresis. The quantified DNA was stored in −20 • C for next analysis. Amplification of V3-V4 regions of the bacterial 16S rRNA gene was performed by TransStart FastpfuR DNA Polymerase (Takara, Dalian, China). The upstream primer and the downstream primer were 5 -barcode-ACTCCTACGGGAGGCAGCA-3 and 5 -GGACTACHVGGGTWTCTAAT-3 , respectively. Amplification PCR reactions was conducted in 20 µL, with 10 ng template DNA, 1U FastPfu polymerase, 1 × FastPfu buffer, 250 µM dNTP, and 0.1 µM each of the primer. PCR was under the reaction conditions of 95 • C for 2 min, 95 • C for 30 s, 55 • C for 30 s, 72 • C for 30 s for 30 cycles, and then 72 • C for 5 min. PCR products were firstly purified using AxyPrep DNA Purification kit (Axygen Biosciences, Union City, United States) after being run through 2% agarose gel electrophoresis. On agarose gels, the PCR products were quantified by QuantiFluor-ST Fluorimeter (Promega, Wisconsin, United States) using a PicoGreen dsDNA Quantitation Kit (Invitrogen, Carlsbad, United States). Purified amplicons were gathered in equimolar ratios for 2 × 300 bp sequencing by Illumina MiSeq in Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Beijing, China) based on the standard protocols. Each treatment group was performed six replications.
Bioinformatics Analysis of Sequencing Reads QIIME (version 1.9.1) and Fastp (version 0.19.6) were used for quality control and sequence filtering with the following criteria: (1) sequencing reads were clipped with an average quality score of < 20; (2) reads shorter than 50 bp were dropped; (3) reads with two nucleotides of mismatch in primer sequences or ambiguous nucleotides were deleted; and (4) paired reads with less than 10 bp overlap were discarded. Operational taxonomic units (OTUs) with a 97% identity cutoff were gathered by Uparse (version 7.0.1090) 1 , and the analysis of taxonomy of OTUs was performed using the Silva (Release132) 2 16S rRNA database. The α-diversity Frontiers in Microbiology | www.frontiersin.org FIGURE 2 | Effects of 0.2% montmorillonite on the relative mRNA expressions of ileal barrier functional gene. Bate-actin was used as an internal standard for normalization. Values are means ± SEM for three independent biological and technical replications, n = 6. *P < 0.05. ITGB1, β1-integrin; PKC, protein kinase C.
indices, including Chao 1 and Shannon, were analyzed by Mothur v.1.30.2. PCoA tools in R language were used for principal coordinates analysis (PCoA). The histogram of linear discriminant analysis (LDA) distribution was implemented using LDA effect size analysis (LEfSe) software. The 16S rRNA gene sequencing information was analyzed by PICRUSt to predict biological functions (EggNOG database) 3 and metabolic pathways (KEGG database) 4 of the bacterial community of ileal and colonic contents samples of weaned piglets.
Statistical Analysis
The data (growth performance, intestinal morphology, and quantitative RT-PCR) analysis was performed by unpaired t-test of SPSS 19.0. Results are represented as means ± SEM. The data (α-diversity indices, predictive analysis of metabolic functions, and metabolic pathways) analysis was performed via a Wilcoxon rank-sum test. LDA analysis was performed by non-parametric factorial Kruskal-Wallis (KW) sum-rank test. P-value < 0.05 was considered as statistically significant.
Growth Performance
The results shown that there are no significantly difference in the ADG, ADFI and F:G (P > 0.05) of weaned piglets no matter what period during the whole experiment (Table 3). However, the 0.2%
Intestinal Mucosal Morphology
The mucosal morphology of the duodenum, jejunum, ileum, and colon were obtained in Figure 1. The villus height of DM (duodenum with 0.2% montmorillonite) and JM (jejunum with 0.2% montmorillonite) was significantly higher (P < 0.05) than that in DNC (duodenum negative control) and JNC (jejunum negative control), respectively (Figures 1C,D). Conversely, the crypt depth in DM and CM (colon with 0.2% montmorillonite) was significantly lower (P < 0.05) than that in DNC and CNC (colon negative control), respectively (Figures 1C,F). The differences of the villus height and crypt depth ( Figure 1E) between IM and INC were non-significant, respectively (P > 0.05). Besides, the V/C (ratio of the villus height and crypt depth) in the 0.2% M group was significantly increased (P < 0.05) in comparison to that in the CON group for both the duodenum and ileum ( Figure 1G).
Intestinal Barrier Function
The relative mRNA expression of ileal barrier function-related genes is displayed in Figure 2. A diet containing 0.2% M has been shown to upregulate (P < 0.05) the relative mRNA expression of MUCIN-1, ITGB1, and PKC in the ileum of piglets (Figure 2). However, the relative mRNA expressions of collagen and occludin have shown no significantly difference (P > 0.05) between the CON and 0.2% M groups in the ileum of piglets (Figure 2).
The Bacterial Community of Digesta From the Ileum and Colon
To investigate the effects of montmorillonite on the bacterial community structure of weaned piglets, we performed 16S rRNA gene sequencing of content samples from the ileum and colon. After sequence quality control, a total of 470445 as well as 424655 clean reads (Table 4) were obtained from the CON and 0.2% M group, respectively. The OTUs numbers of INC, CNC, IM, and CM with six biological replications are also listed in Table 4. Based on OTUs information, bioinformatics analysis was further performed. The coverage curves (Figures 3A,B) showed flat trend with the increasing of sequencing reads, indicating that the sequencing reads in this experiment were sufficient to reveal the bacterial diversity of content samples from ileum and colon. Besides, the Shannon index in IM was higher (P < 0.05) than that in INC (Figure 3D), while there were no significantly difference in the Chao 1 index of the ileum and colon and the Shannon index of the colon between the CON and 0.2% M groups (Figures 3C,D). PCoA analysis (Figures 3E,F) showed a clear differentiation (P IM −INC < 0.05, P CM −CNC < 0.05) between the CON and 0.2% M groups both in the microbiota present in ileum and colon, indicating that the addition of montmorillonite changed the bacterial community structure in ileum and colon.
All differential bacteria of the ileum and colon were demonstrated from the phylum to species level in cladogram of LEfSe between CON and 0.2% M groups (Figures 5A,B). At the phylum level, the relative abundance of Proteobacteria in the ileum and the Bacteroidetes in the colon were significantly lower (P < 0.05) in 0.2% M group than that in CON group (Figures 5A,B). In the genera level, the relative abundance of Bacillus, Paenibacillus, Enterococcus, Alkaliphilus, Cronobacter, and Lactococcus were markedly abated (P < 0.05), while the relative abundance of Streptococcus, Eubacterium_rectale_group, Lactobacillus, Faetobacterium, Subdoligranulum, Blautia, Eubacterium_coprostanoligenes_group, and Ruminococcaceae_UCG_014 were signally boosted (P < 0.05) in IM instead of INC ( Figure 5C). Moreover, the relative abundance of Streptococcus were dramatically reduced (P < 0.05), but the relative abundance of Clostridium_sensu_stricto_1, Subdoligranulum, Terrisporobacter, and Blautia were observably enhanced (P < 0.05) in CM instead of CNC ( Figure 5D).
Predicted Functional Profiles of Microbial Communities Using PICRUSt
To predict the potential function of gut bacteria on nutrient metabolism in piglets after feeding on a diet with and without the montmorillonite, biological functions and KEGG pathways were analyzed by the PICRUSt program. Seventeen notably different predicted biological functions (COG level 1) and 20 markedly different KEGG pathways between INC and IM and 14 different predicted biological functions and 14 different KEGG pathways between CNC and CM were detected in Figures 6, 7. Beyond this, bacteria with carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, cell wall/membrane/envelope biogenesis, defense mechanisms, and nucleotide transport and metabolism functions were significantly more enriched (P < 0.01) in IM than in INC (Figure 6A).
The predicted metabolic pathways involved in microorganisms, such as carbohydrate metabolism, replication and repair, translation, energy metabolism, and nucleotide metabolism, were significantly enriched (P < 0.01) in IM compared to INC (Figure 7A). In the colon, significantly increased bacteria after dietary supplementation of 0.2% montmorillonite improved (P < 0.01) the functions of transcription, amino acid transport and metabolism, replication, recombination and repair, and signal transduction mechanisms ( Figure 6B). The number of significantly changed predicted metabolic pathways between CNC and CM were reduced. In CM (Figure 7B), amino acid metabolism, cellular processes and signaling, and transcription were significantly enriched (P < 0.01).
DISCUSSION
Montmorillonite is widely used as a feed additive, and its characteristics of improving the growth performance of animals have attracted much attention (Yu et al., 2008;Duan et al., 2013;Yang et al., 2014;Jiao et al., 2015Jiao et al., , 2018. Yu et al. (2008) reported that the addition of 0.5% montmorillonite had significantly improved the weight gain, feed intake, and feed conversion ratio of pigs. However, in the present study, the ADG, ADFI, and F:G of weaned piglets were not significantly changed after the addition of 0.2% montmorillonite except for an improvement trend in ADG and ADFI during days 15-35 and 1-35, respectively. Similarly with our study, the addition of graded concentration of montmorillonite only changed the feed intake without improving the weight gain of pigs (Duan et al., 2013). It is also reported that adding montmorillonite to the feed did not improve the growth performance of pigs (Xia et al., 2005;Song et al., 2012). Interestingly, relying on the adsorption of montmorillonite, feeding montmorillonite loaded with copper and zinc ions, which are beneficial to growth, showed a significant improvement in the growth performance of pigs (Jiao et al., 2015(Jiao et al., , 2018; this is an indication that the single addition of montmorillonite has a limited effect on improving the growth performance of weaned piglets. Although the superior growth performance of weaned piglets was not achieved after adding the montmorillonite to the diet, it has the potential to improve the intestinal barrier function Subramaniam and Kim, 2015). Intestinal mucosal barriers include physical barriers, chemical barriers, immune barriers, and microbial barriers, while intestinal mucosal morphology involves the function of intestinal physical barrier. The short villi not only causes an increase in the intestinal epithelial permeability, increasing the inflammatory response and amplifying the dysfunction of the intestinal motor function, but also leads to a decrease in the intestinal absorption area, which may affect the normal intake of piglets (Collins, 2001;Liu et al., 2017). Besides, the crypt is involved in the generation and transportation of epithelial cells, and the decrease of crypt depth can increase the rate of mature cells (Clevers, 2013;Liu et al., 2014). In the present study, we found that the weaned piglets fed with the 0.2% montmorillonite diet exhibited a higher villus height in the duodenum and jejunum compared with the CON group. Meanwhile, lower crypt depth of the duodenum and colon were revealed in the 0.2% M group than in the CON group, and the V/C in the 0.2% M group was significantly increased in comparison to that in the CON group for both the duodenum and ileum. These results hinted that the montmorillonite could improve the surface area for nutrient absorption, thus increasing nutrient digestibility (Clevers, 2013;Liu et al., 2014) as well as increase the intestinal defense function by improving the intestinal mucosal morphology, which may be relate to the altering of the gut microbiota (Xia et al., 2004;Shameli et al., 2011;Placha et al., 2014).
To further confirm the effect of montmorillonite on intestinal barrier function, the relative mRNA expression of intestinal mucosal barrier-related gene (MUCIN-1, ITGB1, collagen, occluding, and PKC) from the ileum were measured by qRT-PCR. The results shown that the relative mRNA expression of ileal MUCIN-1, ITGB1, and PKC were upregulated in the 0.2% M group vs. the CON group, while the relative mRNA expression of ileac collagen and occludin have no significantly difference between the two groups. Mucins are mainly involved in maintaining the structure and function of intestinal mucosa and regulating the intestinal microorganisms (Mcguckin et al., 2011). Integrins, including ITGB1, are the major receptors for extracellular matrix, and regulate the assembly of adhesive junctions in the intestinal tract, and then play roles in the rapid renewal and digestive functions of intestinal tract (Breau et al., 2009). PKC is another important protein that is expressed by gastrointestinal cells and regulates intracellular signaling and barrier integrity (Farhadi, 2005). That the relative mRNA expression of ileal MUCIN-1, ITGB1, and PKC were upregulated by adding montmorillonite revealed that the addition of montmorillonite improved the intestinal barrier function (Hu et al., 2012;Jiao et al., 2015Jiao et al., , 2018Chen J. F. et al., 2019Chen J. F. et al., , 2020; this was caused by the swelling property of montmorillonite, which will cause the volume of intake feed to become larger, slowing down the time of passing through the intestinal tract and promoting the metabolism of intestinal epidermal cells (Subramaniam and Kim, 2015).
Gut microbiota, serving as microbial barriers in intestinal mucosal barriers, play an important role in intestinal function; these functions involve nutrient absorption, mucosal barrier homeostasis, immunomodulation, and defense against pathogens for pigs (Huang et al., 2015;Fan et al., 2017). It is reported that the montmorillonite improved the intestinal barrier function, digestibility of nutrients, and growth performance of weaned piglets by regulating the gut microbiota (Xia et al., 2005;Wang et al., 2012). In the present study, the Shannon index, one of the α-diversity indices, of ileum was more increased in the 0.2% M group compared with the CON group, which indicated that the addition of 0.2% montmorillonite to the feed promoted the growth of ileal bacteria in weaned piglets and improved the diversity of the microbial community. The bacterial diversity is an indicator of a healthy and stable gut microbial community, which is beneficial to the host (Salonen et al., 2012). Besides, the results of PCoA analyses proved that there is a significant difference in the microbial community composition between the CON and 0.2% M groups, which was demonstrated in that the structure and composition of gut microbiota on weaned piglets will be changed by the montmorillonite. At the phylum level, the 0.2% M group saw a decreased relative abundance of Proteobacteria in iluem compared to the CON group in the present study. The Proteobacteria are a minor constituent within a balanced gut-associated microbial community (Eckburg et al., 2005). Recent studies have proposed that an expansion of the Proteobacteria could be a potential diagnostic microbial signature of epithelial dysfunction as well as dysbiosis in the gut microbiota (Na-Ri et al., 2015). Besides, the relative abundance of Bacteroidetes was decreased, but the Firmicutes was increased in the colon after supplementing the montmorillonite into the diets of weaned piglets. The Firmicutes and Bacteroidetes are the dominate phylum in the gut microbiota of piglets, and the increase in Firmicutes was considered a means of enhancing the body's capacity for energy acquisition from the diet, which may improve the growth of piglets (Turnbaugh et al., 2006).
In the genera level, the relative abundance of Streptococcus, Eubacterium_rectale_group, and Lactobacillus in the ileum was increased by adding the montmorillonite to the diets. Streptococcus is considered to be involved in the process of intestinal nutrition metabolism, such as amino acid utilization (Neis et al., 2015) and the production of short-chain fatty acids (Corr et al., 2009); and butyrate belonged to shortchain fatty acids (SCFAs) contributes to maintaining intestinal homeostasis (Iacob and Iacob, 2019). In addition, the D-alanine (Miyauchi et al., 2012) and exopolysaccharide (Chen Y. et al., 2019), produced by Streptococcus thermophilus, and the yogurt fermented by Streptococcus thermophilus 1131 (Usui et al., 2018) can improve the intestinal barrier mucosal function and alleviate intestinal inflammation. Moreover, the Eubacterium rectale participated in the butyrogenic effect of dietary fermentation (Louis et al., 2007). The growth of Eubacterium rectale was inhibited in a gut model from ulcerative colitis patients, indicting Eubacterium rectale might promote intestinal function through butyrate metabolism (Joan et al., 2012). Lactobacillus is considered to be a probiotic with the function of enhancing human and animal health (Colum et al., 2001). In addition, Lactobacillus is believed to be involved in the production of butyrate (Berni Canani et al., 2016). As an immunoregulatory factor, butyrate maintains the intestinal barrier and immune homeostasis; meanwhile, it also promotes the release of antimicrobial peptides and de-represses microbial virulence factors against pathogen invasion (Iacob and Iacob, 2019). The increase in Lactobacillus might therefore further improve the intestinal metabolism and growth performance of weaned piglets in the manner of butyrate metabolism. In the colon, meanwhile, the microflora was changed slightly. For instance, the relative abundance of Clostridium_sensu_stricto_1 and Subdoligranulum, which can produce SCFAs, was more enhanced in the 0.2% M group compared with the CON group, which may be due to that the montmorillonite mainly contribute to the foregut rather than hindgut . Furthermore, using the PICRUSt program to predict functional profiles of microbial communities of the ileum and colon, we found the number of gene tags involved in the synthesis of carbon-containing biomolecules in lieum and the number of gene tags involved in the energy production and conversion and the amino acid metabolism in colon were markedly more enhanced in the 0.2% M group compared with the CON group. The result suggests that (Xu et al., 2017;Lee et al., 2018) the montmorillonite supplementation may be involved in carbohydrate metabolism and amino acid metabolism by altering the gut microbiota that can product the SCFAs, but it is necessary to conduct further study into the relationship between montmorillonite supplementation and carbohydrate and amino acid metabolism.
CONCLUSION
Overall, dietary supplementation of 0.2% montmorillonite has displayed a slight influence on the growth performance of weaned piglets. However, 0.2% montmorillonite supplementation improved the intestinal mucosal morphology and increased the relative mRNA expression of intestinal mucosal barrier functionrelated genes by altering gut microbiota. These findings will contribute to a better understanding of how montmorillonite modulates gut health through nutrient intervention.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih. gov/PRJNA667820.
ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Care and Use Committee of China Agricultural University.
AUTHOR CONTRIBUTIONS
XM conceived and designed the research. HL, CW, and XG conducted the research. HL wrote the manuscript and analyzed the data. JZ and XG wrote a part of manuscript and assisted in analysis of data. XG and XM critically reviewed the manuscript. XG contributed to language review. All authors read and approved the final manuscript. | v3-fos-license |
2019-06-26T13:02:18.267Z | 2019-06-01T00:00:00.000 | 195352522 | {
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} | pes2o/s2orc | Effects of CO32− and OH− on the solubility, metastable zone width and nucleation kinetics of borax decahydrate
Measurements of the solubility and metastable zone width (MZW) of borax decahydrate in sodium carbonate and sodium hydroxide aqueous were obtained. The onsets of nucleation were detected by the turbidity technique with the temperature range from 285 to 315 K. The results showed that the solubility of borax gradually decreased and the MZW broadened with the mass percentage of sodium carbonate increasing from 0% up to 9.22%. Correspondingly, the solubility and MZW had the same trend with the addition of sodium hydroxide. Meanwhile, the nucleation parameters of borax were determined and analysed to explain the trends obtained. Applying the classical three-dimensional nucleation theory approach, it was found that the addition of carbonate and hydroxide ions led to the values of solid–liquid interfacial energy (γ) increasing, which indicated the CO32− and OH− ions adsorbed on the nuclei but suppressed nucleation rate.
Introduction
Boron compounds have unique advantages in their porosity, density and thermal stability, leading to potential applications for hydrogen storage, filtration, catalysis and optoelectronics [1][2][3]. In geological formations, boron compounds have different kinds of existing forms, such as sassoline, borax, ulexite and colemanite [1]. There is a growing interest in the crystallization of liquid and solid boron due to their long-term exploitation [4]. , CO 3 22 and OH 2 [15]. CO 3 22 ion is one of the main components of carbonate-type brine which always contains a large amount of boron. A typical example is Zabuye salt lake [16]. Different concentrations of CO 3 22 lead to different pH values, thus affecting the solubility and MZW of borax in brine. Although there are several reports on the solubility and MZW of borax, the results are still inadequate and roughly compared with cations. Therefore, the aim of this study was to investigate the influence of CO 3 22 and pH on solubility and the MZW of borax using the polythermal method. Then the experimental MZW data of different cooling/heating rates R were analysed and discussed by three-dimensional nucleation theory.
Experiment section 2.1. Material and methods
All of the chemical reagents used in this study are listed in table 1. Na 2 B 4 O 7 . 10H 2 O was recrystallized with a purity more than 99.99%. Water (resistivity, 18.25 MV cm 21 ) was deionized from a water purification system (UPT-II-20T, Chengdu Ultrapure Technology Co., Ltd) before experiments. The experimental set-up is shown in figure 1. A turbidity meter was employed to detect nucleation/ dissolution. The temperature of prepared solution was measured using the digital thermometer with precision of +0.18C. Cooling rates control was accomplished using a Crystal SCAN with four parallel reactors (E1061, HEL, UK) containing systems for temperature control and computer processing as well as a crystallizer assisted with programmable thermostatic bath (FP50-ME, Julabo, Germany). The crystallizer was a 100 ml glass vessel with an internal overhead stirrer, temperature sensor and turbidity sensor. Besides, the crystallizer was made air-tight so that the loss of solvent due to evaporation could be minimized. The X-ray diffraction (XRD) analysis (X'Pert PRO, 2006 PANalytical) was used to confirm the identity of the solid phase crystallizing from the solutions. The pH was measured by pH meter (S470 Seven Excellence, Mettler Toledo) with precision of +0.05.
Solubility and MZW determination
The determination of the solubility and MZW of the borax in sodium carbonate solutions was carried out with a temperature range from 285 to 315 K according to the conventional polythermal method. Firstly, 60 g of mixture was placed into a 100 ml crystallizer. Then, the mixture was heated with a given rate above the saturation temperature for 10 min to ensure complete dissolution of the solid phase. Finally, the solution was cooled down with the same constant rate until the first visible nucleus appeared, which can be detected by a sudden increase in turbidity. The corresponding temperature at the point of nucleation and dissolution was recorded as T 1 and T 2, respectively. The above steps were repeated at five cooling/heating rates of 55, 45, 35, 25 and 15 K h 21 and at constant impeller speed of 300 r.p.m. The concentration of borax and sodium carbonate in the solution was obtained by titration. The faster the heating rates were, the higher the measured dissolution temperature was. Therefore, the saturation temperature T 0 of borax can be obtained by extrapolating the T 2 -R curve to a virtual heating rate of 'zero'. The metastable zone width (MZW) of borax is represented by the maximum undercooling DT max (DT max ¼ T 0 2 T 1 ).
Chemical analysis
The boron content was determined by mannitol conversion acid2base titration. The CO 3 22 ion concentration was determined by adding 0.05 mol l 21 HCl, and using methyl red-bromcresol green as indicator. The accuracy of these analyses was about 0.1%. All of the estimated uncertainties of the research are listed in table 2.
XRD analysis
The XRD patterns of borax obtained from pure water, sodium hydroxide and sodium carbonate were investigated, respectively, shown in figure 2. It manifests the XRD patterns are identical and well indexed to borax without any impurities, according to the reference data JCPDS 75-1078.
Solubility
The solubility of borax in different mass percentages of sodium carbonate (0.0 -9.22%) in aqueous solution was determined. The obtained experimental solubility data are demonstrated graphically in figure 3. It can be observed in figure 3a that the solubility of borax increased with temperature, which can be attributed to the thermal motion of molecule. Besides, the addition of sodium carbonate leads to the solubility decrease. It could be the common ion effect that the addition of sodium carbonate releases Na þ into the solution, making dissolution2precipitate equilibrium move towards the precipitate. Therefore, the solubility of borax decreases. It also can be seen that the solubility curves are roughly paralleled to each other, indicating that the increment of sodium carbonate causes a gradual decrement of borax solubility. Furthermore, it can be seen that pH increases with the addition of Na 2 CO 3 (from 0 to 9.22%), based on figure 3a. Therefore, pH range from 9.9 to 10.5 was selected to investigate by the addition of Na 2 CO 3 and NaOH, respectively. As shown in figure 3a, the solubility of borax at Na 2 CO 3 concentration of 9.22% (pH ¼ 10.5) was lower than pH ¼ 10.5 adjusted by NaOH, seen in figure 3b. It suggests that CO 3 22 has more prominent effects on the decrement of borax solubility than OH 2 . (1, low constant temperature bath; 2, temperature control system; 3, a computer processing system; 4, crystallizer; 5, turbidity sensor; 6, overhead stirring; 7, temperature sensor.)
Thermodynamic properties of borax
Dissolution enthalpy, D dis H, and dissolution entropy, D dis S, are important to investigate the dissolution behaviour of the solute in different solvents. When the solubility of borax in sodium carbonate and sodium hydroxide solution at different temperatures is available, then the values of D dis H and D dis S can be determined from the van't Hoff equation as follows: Where D dis H and D dis S are the dissolution enthalpy and entropy, respectively, R G is gas constant (8.314 J mol 21 K 21 ) and x is the mole fraction of borax. The van't Hoff plots shown in figure 4 are obtained from the linear fit of ln x versus 1/T 0 . Then the dissolution enthalpy and entropy of borax which are shown in table 3 can be calculated from the slope and the interception of these plots. The values of dissolution enthalpy and entropy of borax are 34.11 kJ mol 21 and 61.14 J mol 21 K 21 [17] in the literature, which are in good agreement with our experimental data. It can be found from table 3 that the dissolution enthalpy and entropy are positive, which indicates that the dissolution is always endothermic and entropy driven. It also can be found that the mixture with more sodium carbonate and sodium hydroxide has lower solubility but higher values of D dis H and D dis S, which is consistent with general thermodynamic principles [18].
Metastable zone width
The MZW data of borax against saturation temperature at different mass percentages of sodium carbonate are given in figure 5a. It is clear that the MZW becomes broad with the increase of sodium carbonate. The effects are more significant at higher mass percentages, but have little effect with mass percentages of sodium carbonate below 5.31%. The effects could have two possible explanations. One is that carbonate ions with large size may block the active growth sites of the nuclei forming in bulk solution due to steric effect, which might depend on concentrations. When presented in relatively small concentrations, these ions suppress nucleation slightly. Therefore, it might be seen that the higher mass percentages of carbonate ions, the more powerful the inhibiting effects are. Another possible reason is that carbonate ions might act as surface active agents, rendering the nuclei inactive [19]. The results of the influences of pH on MZW are also given in figure 5b. It should be noted that the MZW of borax is larger at higher pH. This could be explained by the relationship between the pH and polyborate speciation [2]. Based on the reports [20,21], the speciation of boron strongly depends on
Classical three-dimensional nucleation theory approach
The solid -liquid interfacial energy g is an important thermodynamic parameter that indicates the ability of the solute to crystallize from solution [22]. According to the classical three-dimensional nucleation theory, the relationship between MZW and cooling/heating rate R can be represented as equation (3.2) [23,24]. Where k B is the Boltzmann's constant equal to R G /N A (N A is the Avogadro number); where A is a kinetic constant associated with the media of nucleation; f is a constant expressing the number of nuclei in certain volume; V is the molecular volume, calculated by the density. Figures 6 and 7 present plots of (T 0 /DT max ) 2 against ln R for borax in different percentage fractions of sodium carbonate and sodium hydroxide solutions according to equation (3.2). The values of g and A can be calculated from the slope and the intercept by equations (3.3) -(3.5), respectively where F, Z and B represent slope, intercept and nucleation parameter, values are calculated by equations ð3:5Þ As shown in table 4, the estimated solid -liquid interfacial energy g in pure water is 1.81 mJ 23 m 22 , which agrees well with the data 1.7 in the previously published literature [25]. It should be noted that the values of solid -liquid interfacial energy g decrease with an increase in saturation temperature T 0 , but increase with the addition of Na 2 CO 3 , seen from table 4. Based on the reported articles, it is known that the increase in the value g will suppress nucleation rate and broaden the MZW. Basically, the higher interfacial energy means the bigger nuclear barrier which leads to the harder nucleation process. According to the data from table 4, it could be concluded that the adsorption of CO 3 22 on the nucleus surface leads to the increase in g [26]. Furthermore, the solid -liquid interfacial energy g was less in NaOH-NaB 4 O 7 -H 2 O system than that of Na 2 CO 3 -NaB 4 O 7 -H 2 O system, seen from table 5. It can be explained by the fact that the charge of OH 2 is less than CO 3 22 so that the adsorption is weaker.
Conclusion
The effects of sodium carbonate and sodium hydroxide on the solubility and MZW of borax have been studied at temperature ranging from 285 to 315 K using turbidity technique. A salting-out effect was observed under the larger mass percentages of the sodium carbonate and sodium hydroxide, which resulted in the lower solubility of borax. It was found that the addition of sodium carbonate broadened the MZW significantly, and the influence depended on concentration. It was believed that the addition of sodium carbonate adsorbed on nuclei and suppressed the activities of nuclei in the solution which enabled the larger MZW. In addition, the pH had an effect on polyborate species. The increasing of pH could make the concentration of the tetraborate decrease, which led to the broadening of MZW. Finally, the obtained MZW data were analysed with the classical three-dimensional nucleation theory approach. The value of solute -solvent interfacial energy g increased as the mass percentage of sodium carbonate was larger. The investigation of these parameters is very useful for the design and development of a crystallization process.
Data accessibility. This article does not contain any additional data. | v3-fos-license |
2018-04-03T03:03:59.223Z | 1978-02-01T00:00:00.000 | 332143 | {
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} | pes2o/s2orc | Potentially carcinogenic species emitted to the atmosphere by fossil-fueled power plants.
The identities and physicochemical characteristics of potentially carcinogenic species emitted to the atmosphere by fossil-fueled power plants are presented and discussed. It is pointed out that many so-called carcinogens are preferentially concentrated on the surface of respirable fly ash particles thus enabling them to come into intimate contact with lung tissues when inhaled. Relatively little information is available about the identities of particulate polycyclic organic compounds whose emission from coal fired power plants may well be substantially greater than hitherto supposed. The importance of chemical changes, which several species may undergo following emission (but prior to inhalation) in determining their potential carcinogenic impact, is stressed.
Introduction
Production of electric power from the combustion and conversion of fossil fuels represents a ubiquitous and increasing means of obtaining energy in most countries throughout the world. It is now well established that such power plants emit substantial quantities of many carcinogenic and potentially carcinogenic chemical species to the atmosphere. Consequently, it is of considerable importance to establish whether these materials are active in promoting the occurrence of lung cancer in populations resident in the vicinity of fossil-fueled power plants.
In order to make any assessment of risk it is necessary to have knowledge of the nature, concentrations, and physicochemical characteristics of potentially carcinogenic material emitted from the various types of fossil fueled power plants. This paper, therefore, presents a brief survey of the information currently available. Special emphasis is placed on what is known about the physical and chemical characteristics and behavior of each species since these properties may have a profound influence on the inhalation toxicology of individual species (1,2 Fossil-fueled power plants are considered to be those utilizing gases, liquids, or solids as primary fuels derived, respectively, from natural gas, oil, or coal. Some difficulty is encountered in specifying individual pollutant species since definitive data on carcinogenicity are sparse. For the purpose of this paper, therefore, compounds are classified as known carcinogens, suspected carcinogens, and reactants. Compounds classified as reactants are those which are considered likely to be involved in chemical reactions which may result in the production or removal of carcinogenic species or which may interact synergistically with known carcinogens. In Table 1 are given examples of concentrations of known and suspected carcinogens in urban and rural atmospheres.
Gaseous Emissions
Gaseous emissions from fossil-fueled power plants generally contribute more material to the atmosphere than do particulate emissions (except in the now rare case of uncontrolled coal combustion). The major emissions, in terms of mass, involve carbon monoxide (CO), hydrocarbons (HC), nitrogen oxides (NO,), sulfur oxides (SO.), and oxygenated species often classified as formaldehyde (HCOH). Representative contributions are indicated in Table 2 (13). In addition, minor emissions of mercury occur, and it has been suggested that bromine (Br2), February 1978 200-3000 (2,4, 7) aThe substances listed include both known and suspected carcinogens for which reasonably reliable atmospheric concentration data are available. Also listed are several compounds which are considered to be capable of promoting carcinogenic activity in noncarcinogenic compounds or modifying that of carcinogens as a result of chemical reaction. bMost values represent 24-hr averages established over periods ranging from several days to one or more years. cApproximate averages values have been estimated for urban air noting that individual areas may exhibit atmospheric concentrations which differ considerably from the average. Due to paucity of data it is considered inappropriate to estimate similar averages for rural atmospheres.
dFluorine is present in the atmosphere as both fluorine gas and particulate fluorides. The values listed refer to the sum of both forms.
eValues listed for lead refer to concentrations measured in countries utilizing lead alkyl gasoline additives. Significantly lower values are encountered in countries which do not use leaded gasoline.
'Selenium is present in the atmosphere in both gaseous and particulate form. The values listed refer to the sum of both forms. Also, selenium has not been implicated as a causative agent of bronchial carcinoma but only of liver and kidney cancers.
"The two sets of values listed for vanadium refer, respectively, to urban areas where considerable use is made of fuel oil for power generation and domestic heating, and to urban areas where oil burning is minimal.
hA very large number of organic compounds have been implicated as causative agents for bronchial carcinoma. Only a few of these are listed here, however, since reliable atmospheric concentration data are unavailable. In general, compounds are listed by class with specific examples being given where data are available.
'These hydrocarbons are not in themselves considered to be carcinogenic. They may, however, promote formation of photochemical smog which contains several carcinogenic components.
JThe data for nitrosamines are very tenuous; they are, however, included because of the considerable current interest in these compounds.
kSeveral noncarcinogenic polycyclic compounds are listed, since some of these are known to react photochemically to produce oxygenated derivatives (such as quinones, phthalates, and endoperoxides) which are suspected carcinogens. It will be noted that some very wide concentration ranges are listed for the polyaromatic hydrocarbons. The upper ends of these ranges correspond to values measured in European cities where extensive coal burning is practiced.
Sulfur Oxides
Sulfur oxides are not, in themselves, thought to be carcinogenic. They are, however, quite reactive and are known to react with, for example, polycyclic aromatic species (2) and to promote lung damage when associated with airborne particles. In the ab-sence of controls the amounts of sulfur oxides emitted from a fossil-fueled power plant are directly related to the sulfur content of the fuel burned ( Table 2). In this case, typical SO, emissions lie in the range 500-3000 ppm with 1000-2000 ppm being most commonly encountered (15). Nowadays, however, most major installations utilize control equipment which typically achieves 85-90 percent removal of SO,. Generally, about 1-2% of the emitted sulfur oxides are in the form of SO3, which reacts rapidly with water vapor to produce sulfuric acid mist. A small amount of the SO2 is also chemisorbed by fly ash particles to form metallic sulfates (primarily calcium sulfate and alkali iron trisulfates) (16).
The rate and extent of sulfur dioxide conversion to sulfuric acid mist and solid particulate sulfate in a power plant plume are unknown; however, current thinking is that these processes occur fairly extensively, so that a significant proportion of the gaseous sulfur oxides produced actually occur in urban atmospheres as sulfuric acid mists or as particulate sulfate (4). This is an important consideration, since it means that the health hazard presented by gaseous sulfur oxides may be partly manifest through inhalation of sulfuric acid and sulfate particles.
February 1978
Nitrogen Oxides As in the case of sulfur oxides, the oxides of nitrogen are not carcinogenic but may produce carcinogenic materials as a result of chemical reactions such as those involved in photochemical smog formation. By contrast with sulfur oxides, which are derived from sulfur present in the fuel, nitrogen oxides are derived primarily from fixation (oxidation) of atmospheric nitrogen present in the combustion feed air. Consequently, nitrogen oxides cannot be effectively controlled by selection or pretreatment of the fuel.
Representative emission factors for nitric oxide (NO) and nitrogen dioxide (NO2) combined are given in Table 2, although it should be recognized that NO, emissions are not directly related to the amount of fuel consumed. Rather, they depend on the feed rate of air supporting combustion, the temperature, and the fuel-air mixing characteristics.
Actual concentrations of NOX are normally in the range 300-1300 ppm in stack emissions. Significantly higher concentrations are produced during natural gas combustion than during combustion of coal or oil ( Table 2).
The amount of NO2 formed during combustion is generally much less than that of NO; however, conversion of NO to NO2 occurs fairly rapidly in a power plant plume following emission. From the standpoint of human health, therefore, it is reasonable to presume that the primary direct exposure will be to NO2.
Organic Gases
The amounts of organic material emitted as gases or vapors from fossil-fueled power plants are quite small (2.5%) when compared with those from other anthropogenic sources. For example, transportation accounts for some 53% of the gaseous organics emitted in the United States. Consequently, rather little work has been done to determine the identities and amounts of individual organic gases emitted from power plants. Primary emissions consist of hydrocarbons, aldehydes, and organic acids for which representative emission factors are given in Table 3. Of these materials only certain aldehydes (e.g. formaldehyde and acrolein) are suspected carcinogens. Hydrocarbons and organic acids are, however, quite reactive (hydrocarbons in photochemical smog production) and are thus worthy of consideration in the present context (3).
Other Gaseous Emissions
With the exception of elemental mercury vapor, very little is known about gaseous emissions other than those already discussed. Mercury is, however, a suspected carcinogen.
Mercury levels in coal and fuel oil average about 1 ug/g and 0.1 ,ug/g, respectively. When the coal is burned, about 90-95% of the mercury present is emitted to the atmosphere as elemental vapor. The remainder is associated with fly ash. There is no apparent tendency for mercury vapor to become adsorbed or otherwise associated with fly ash or atmospheric aerosols. Consequently, mercury is transported long distances from a coal fired power plant. This behavior is exactly opposite to that of other, particulate associated, metals.
The actual concentrations of mercury vapor emitted from a given power plant will depend on the type and origin of the fuel burned; however, because of the almost quantitative release of mercury to the atmosphere, stack concentrations can be readily calculated where stack gas flow rates are known. Typical stack exit concentrations are around 1 ,ug/m3 for a coal fired power plant. Plume concentrations depend, of course, on atmospheric conditions but concentrations around 80 ng/m3 have been measured (17) at ground level on the plume center line 1.2 miles downwind of a coal fired power plant. Representative concentrations in urban atmospheres lie in the range 2-100 ng/m3, most of which is present as mercury vapor (18).
Fossil fuel combustion also results in the release to the atmosphere of several radioactive species including the gas radon (222Rn), which, with its daughter products, is a known carcinogen. The few available measurements indicate that natural gas contains 10-20 pCi/I of 222Rn and that coal contains 0.1-0.4 pCi/g. While quantitative release of radon to the atmosphere will occur, present estimates suggest that fossil fuel combustion does not contribute significantly to the natural 222Rn background even in the vicinity of power plants (9).
Particulate Emissions
Fossil-fueled power plants contribute approximately 25% of the anthropogenic particulate matter emitted to the atmosphere in the United States. In Environmental Health Perspectives many countries the proportion is even higher. As indicated by the data in Table 2, particulate emissions from coal-fired power plants are much greater than those derived from oil or natural gas combustion. Some idea of particle mass emission factors can be obtained by noting that modern electrostatic precipitation equipment usually operates with mass removal efficiencies in excess of 98%.
Assessment of the carcinogenic hazard associated with airborne particulate material such as fly ash is very much more difficult than is the case for a gaseous pollutant. This is because particles contain a large number of potentially carcinogenic chemical species including both organic and inorganic compounds. The relative amounts of these species, and thus their net carcinogenicity, can vary significantly with the type and origin of the fuel burned and even with the operating characteristics of individual power plants. Furthermore, the way in which a given chemical species is distributed among different particles and even within a single particle can strongly influence its potential health impact. Finally, it must be recognized that, although many potentially carcinogenic compounds may be associated with solid fly ash particles these compounds are unlikely to constitute a hazard to health unless they can be mobilized into solution, e.g., body fluids.
The extent to which information is available about each of the above factors is discussed in the following sections. For convenience, different classes of chemical compounds are considered separately even though all may be present together. In this regard it is useful to note that a single particle effectively concentrates many chemical species in a localized microregion so that its influence is likely to be exerted over a very localized area of lung tissue when inhaled. This is in contrast to the more generalized influence of inhaled gases.
Particle Morphology, Size Distribution, and Matrix Composition Particles emitted to the atmosphere from fossil fueled power plants are more or less spherical. In the case of coal combustion both solid and hollow spheres occur and some of the latter have small respirable spheres encapsulated inside them (15,19). Particles derived from oil and natural gas combustion have a highly porous structure rather like that of a sponge (20).
The aerodynamic size of a particle is a major factor in determining the efficiency with which it can be collected by control equipment, its atmospheric transport characteristics and lifetime, and its deposition and clearance behavior when inhaled (1). In addition, the size of a particle determines the specific surface area which can come into intimate contact with body fluids and tissues. The size distributions of particles produced by different power plants exhibit considerable variation; however, a typical size distribution of fly ash emitted from a coal fired power plant equipped with an electrostatic precipitator is presented in Figure 1 (21). It is apparent from this figure that much of the emitted fly ash falls in the respirable size range. Relatively few measurements have been made of particle size distributions in power plant plumes. As a rough indication, however, particulate material collected at a distance of 5 miles downwind from a coal-fired power plant plume under stable plume conditions has an aerodynamic mass median diameter in the range 0.08-0.25 ,tm. Such samples usually exhibit a bimodal distribution, with the two modes being centered around 0.04 ,tm and 0.3 um. The smaller modal particles are thought to represent a secondary aerosol consisting primarily of sulfate particles. Comparable information is not, to our knowledge, available for oil or natural gas-fired power plants, although similar general behavior would be expected.
The major matrix elements present in coal fly ash are Al, Si, and Fe, with minor amounts of Ca, Mg, K, Na, Ti, and S. Some typical composition ranges, expressed as weight percent as the oxides, are presented in Table 4 (22). The matrix elements in oil fly ash are C, Ca, Fe, S, Si, Ti, and V (23), whose relative proportions vary considerably in individual particles. Coal fly ash consist § primarily of a semitransparent aluminosilicate glass with small amounts February 1978 10.0 F of microcrystalline hematite (Fe2O3) magnetite (Fe3Ol), a-quartz (SiO2), mullite (3AI203 . 2SiO2), anhydrite (CaSO.,), and lime (CaO). In addition some elemental carbon (soot) particles are present. All these compounds have low solubility in water which accounts for the low bulk solubility of fly ash. The compounds present in fly ashes derived from oil and natural gas combustion have not been established although it is known that such particles are highly carbonaceous in nature.
Trace Elements
As a result of their geological origins coal and petroleum oil contain essentially all known stable elements in minor or trace amounts. Of these, the elements As, Be, Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb, Se, and V are regarded as either known or suspected carcinogens. It must be strongly emphasized, however, that the chemical and toxicological properties of any element depend upon the nature of the chemical compound in which that element is present. Unfortunately very little is known about the identities of metal compounds emitted in particulate form from fossil fueled power plants. Consequently it is accepted, though strictly incorrect, practice to refer to the metallic elements themselves.
The specific concentrations (,ug/g) of individual trace elements found in coal and oil fly ashes depend primarily on the trace element content of the original fuel. However, the relative concentrations of elements differ significantly between fly ash and fuel due to the different partitioning characteristics of individual elements between bottom ash and fly ash. Thus, in the case of coal fly ash, it is not reasonable to assume that because a given fraction of one element ends up in emitted fly ash that the same fraction of other elements will do likewise. This assumption is, however, reasonable in the case of an oil fired power plant which produces very little combustion residue. Some representative specific (,g/g) and volume (,ug/m3) concentrations of trace elements emitted from coal and oil fired power plants are presented in Table 5. It should be noted that volume concentrations will be highly dependent on individual plant operating conditions. It should also be noted that vanadium, V, is emitted in substantial amounts from the combustion of fuel oil. This is because the element is concentrated in the form of several vanadium porphyrins in the original fuel (24).
At this point it is appropriate to comment on the partitioning of different elements in coal-fired (and probably also oil-fired) power plants. The most important aspect in this regard is that several potentially carcinogenic elements or their compounds are apparently volatilized at the combustion temperatures (1400-1600°C) encountered. These elements then condense or absorb onto the surface of coentrained fly ash particles as both particles and vapors leave the combustion region. Since small particles have a greater specific surface area than do large particles this phenomenon results in the volatile elements becoming preferentially associated with small particles (14). Table 6 presents data showing how the specific concentrations of several potentially carcinogenic elements depend on aerodynamic particle size in fly ash both emitted and retained by a representative coal-fired power plant.
This dependence of trace element concentration on particle size has the net effect of decreasing the aerodynamic equivalent mass median diameters of volatilizable trace elements with respect to that of the bulk fly ash with the following important results: (1) many potentially carcinogenic trace elements are most concentrated in the small, pulmonary depositing, fly ash particles which are least effectively collected by existing particle control devices; (2) the concentrations of volatilizable trace elements determined by analyzing fly ash collected by control devices are very much lower than the concentrations of those elements actually emitted. Trace element emission factors cannot, therefore, be obtained by multiplying the specific concentration of an element measured in retained fly ash by the bulk particle emission factor.
It should be pointed out that, although the specific concentrations of volatilizable elements increase more or less linearly with inverse particle diameter (14,25), the same is not true when volume concentrations are employed. This is because volume concentrations depend upon the way in which the bulk particulate mass is distributed with respect to aerodynamic particle size. Some typical elemental size distributions determined in the stack gas of a coal fired power plant are presented in terms of volume concentration (,ug/m3) in Figure 2 (23). As mentioned previously, coal combustion results in the emission of several carcinogenic radionuclides in particulate form. Specific concentrations of 210Pb, 226Ra, 228Ra, 228Th, 238Th, and 238U have been measured in coal fly ash (25)(26)(27)(28); however, only 210Pb and 238U are enriched with respect to the levels found in soil. Measurements of 226Ra, 232Th, and 238U in the plume 6 km downwind from a coal fired power plant show that these elements are enriched over normal background levels by factors of 9, 4, and 28, respectively (29). These authors have assessed the lung doses from a 1000 MW coal fired power plant to be approximately 10 Effective Cutoff Diameter (kLm) FIGURE 2. Representative aerodynamic particle size dependences of the elements As, Pb, Se, and Zn in fly ash emitted from a coal fired power plant. F refers to the final filter employed with the cascade impactor used in sampling (23).
Surface Association of Trace Elements
Recent results (30) have established that a number of trace elements, including several potential carcinogens are more highly concentrated on the surfaces of coal fly ash particles than in their interior. This phenomenon is probably due to parti-cle surface deposition of elements volatilized during combustion and is found to occur for particles derived from a variety of high temperature combustion or smelting operations, e.g., automobile exhaust particulates and blast furnace dusts (31).
It is difficult to make quantitative measurements of surface concentrations. However, some semiquantitative estimates of specific concentrations of several potentially carcinogenic elements present in a shell 300 A thick at the surface of coal fly ash particles are compared to bulk concentrations in Table 7 (31). It should be stressed that these data are presented primarily for the purposes of illustration and should not be regarded as definitive. This surface association is considered to be of considerable importance in determining the toxicity of trace elements in coal fly ash. The following reasons are cited. (1) Since it is the surface of a particle which comes into immediate contact with the external environment (e.g. body fluids and tissues), the surface predominance of toxic trace elements ensures their (2) Conventional bulk analyses of particulates provide a poor measure of the actual concentrations of toxic trace elements to which the external environment is exposed. This fact must be considered in designing toxicity studies using synthetic particulates. (3) Since the surface layer contains an increasing fraction of the total particle mass with decreasing size, small, lung depositing particles will have a greater proportion of their associated toxic species in immediate contact with lung tissues than will large particles, i.e., as indicated earlier, lung-depositing particles definitely constitute the most potentially carcinogenic fraction of all fly ash particles.
Solubility
Probably one of the most important properties of particulate matter emitted by fossil fueled power plants is its solubility. Indeed, unless the associated toxic chemical species can be extracted by lung fluids their ability to act as chemical carcinogens is probably negligible. Surprisingly, this point is frequently overlooked.
It is now well established that only about 2-3% of the mass of both coal and oil fly ash is soluble in water. Very little more is soluble in most dilute acids or bases. However, while the fly ash matrix is effectively insoluble, the so-called surface layer, in which many potentially carcinogenic elements are highly concentrated, is quite soluble. This is illustrated for the case of Pb in Figure 3 the dependence of concentration on radial depth into coal fly ash particles before and after leaching with water (16).
The factors controlling the rate and extent of solubility of individual elements associated with fly ash are complex (32); however, it is apparent that a substantial fraction (probably-50%) of most potentially carcinogenic elements is extractable from respirable particles.
It is appropriate here to draw attention to the distinction between the concentration and amount of a species extracted from a particle. Thus, the total amount of a given species may be quite small and unlikely to constitute a hazard. On the other hand, the localized concentration of that species may be very high (due to its surface predominance) and quite capable of causing damage in a microregion surrounding each particle. The question is whether or not such local effects are important. If not, then the surface predominance of carcinogenic trace elements may be of little consequence.
Particulate Organic Compounds
Particulate associated organic material emitted from fossil fueled power plants is known to contain both aliphatic and aromatic compounds. To date essentially all studies have been directed towards the latter class of compounds with special emphasis being given to polycyclic aromatic species which include many well established carcinogens (2). Even within this group, primary emphasis has been placed on hydrocarbons and little attention has been paid to heterocyclic compounds containing oxygen, nitrogen, or sulfur. Similarly, derivatives containing substituents such as carboxylic, nitro, sulfonic acid, or phenolic groups (if, indeed, they occur) have received little attention. At this time, therefore, the only polycyclic organic compounds which have been uniquely identified as being associated with fly ash emitted by fossil fuel power plants are listed in Table 8 (15,16,33). It should be noted that many more compounds have been tentatively identified but have not yet received full confirmation. A number of studies of particulate polycyclic organic matter (POM) emitted by fossil fueled power plants have concluded that total emissions are negligibly small compared with those from other sources (2). A summary of reported emission factors for several coal combustion operations is presented in Table 9. These figures translate to a total emission of 1 ton of benzo[a]pyrene from all coalfired power plants in the United States. The much higher emission factors associated with hand stoked furnaces are attributed to inefficient combustion.
There is now substantial evidence indicating that most, if not all, organic material remains in the vapor phase so long as the stack gases are within a power plant stack system (15,32,33). With the temperature decrease which occurs following emission to the atmosphere, however, rapid adsorption of organics onto the surfaces of co-entrained fly ash particles takes place. What this means is that fly ash retained by control equipment or collected within a power plant stack contains only a small fraction of the total organic material emitted. Conversely, emitted fly ash contains much higher specific concentrations (,ug/g) of organics than the same fly ash prior to emission. In establishing POM emission factors, therefore, it is vitally important to ensure that material present in both vapor and particulate form be included when samples are collected from within a plant.
In view of the high carcinogenic potential of POM (2), this vapor-to-particle conversion process is of more than academic interest since it has the following ramifications.
Since polycyclic organic compounds appear to associate with fly ash by adsorption they will be present primarily on particle surfaces which can make intimate contact with lung tissues and fluids. Furthermore, preliminary indications are that extraction into solution is quite facile (31).
Since adsorption depends upon the available surface area of particulate adsorbent the highest specific concentrations of POM will be found associated with small particles in the respirable range. In fact size distribution studies indicate that the aerodynamic mass median diameter of benzo[a]pyrene in fly ash emitted from a coal fired power plant is around 0.1 ,um (1,33). In short, it is reasonable to assume that essentially all POM derived from fossil fueled power plants is capable of pulmonary deposition.
Since particulate association of POM apparently occurs primarily following emission, analysis of particulate material collected inside a power plant stack may provide a gross underestimate of POM emissions (16). This point is illustrated by the data in Table 8, which show that when account is taken of dilution, significantly (possibly several orders of magnitude) higher concentrations of POM are found in emitted fly ash than in that collected in a power plant stack (33). While there is no reason, at this time, to disbelieve the mission estimates presented in Table 9, it is of considerable importance that they be fully substantiated by additional measurements relating to modern fossil fueled power plants.
Environmental Health Perspectives
Chemical Conversion of POM A number of studies have shown that particulate polycyclic organic species can be modified in the atmosphere as a result of photochemical decomposition or reaction with sulfur or nitrogen oxides (2). This is of considerable importance, since such reactions may significantly alter the carcinogenic potential of POM. Indeed, the chemical compounds actually inhaled may be quite different from those originally emitted to the atmosphere.
Recent studies (34) of the photochemical decomposition of several polycyclic aromatic compounds adsorbed onto the surface of coal fly ash indicate that some compounds, e.g., phenanthrene and pyrene, do not decompose appreciably under the influence of solar radiation. A second group, e.g., anthracene and benzo[a]pyrene, decompose with half lives of several hours, giving the corresponding quinone as the major product. Interesting behavior is observed in the case of fluorene, which decomposes to fluorenone in the absence of light.
Data such as these illustrate the point that estimates of the carcinogenic potential of POM emitted from fossil fueled power plants must necessarily be based on analyses of particulate material collected from the plant plume at some distance from its origin. Until the results of such analyses are available, very little can be inferred about the nature and amounts of potentially carcinogenic organic species likely to be present.
Conclusions
It is apparent from the foregoing remarks that the identities and amounts of most air pollutants emitted by fossil fueled power plants are reasonably well established. The major gap in knowledge of this type concerns the emission of particulate polycyclic organic matter (POM) which probably includes the most potentially carcinogenic species.
It is also apparent that simple knowledge of the identity of a toxic substance is scarcely adequate to enable assessment of its significance as a health hazard. This is of primary importance in the case of particulate matter for which such factors as aerodynamic size distribution and surface predominance may play a major role in determining toxicity. In this regard, the information which would be of most value is a quantitative measure of the availability of carcinogenic species associated with particles.
While there is considerable information about potentially carcinogenic species which are actually emitted from fossil fueled power plants only rudimentary knowledge is available about the changes that these species undergo prior to inhalation. Consequently, contemporary estimates of human hazards must, of necessity, be based on what is known about emitted species plus what can be inferred or guessed about the ways in which their carcinogenicity may be modified prior to inhalation. Part of the research described herein was supported by grants ERT-74-24276, MPS-74-05745, and DMR-73-030206 from the United States National Science Foundation and by grant R-803950-01 from the United States Environmental Protection Agency. | v3-fos-license |
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} | pes2o/s2orc | Modeled and observed ozone sensitivity to mobile-source emissions in Mexico City
The emission characteristics of mobile sources in the Mexico City Metropolitan Area (MCMA) have changed significantly over the past few decades in response to emission control policies, advancements in vehicle technologies and improvements in fuel quality, among others. Along with these changes, concurrent non-linear changes in photochemical levels and criteria pollutants have been observed, providing a unique opportunity to understand the effects of perturbations of mobile emission levels on the photochemistry in the region using observational and modeling approaches. The observed historical trends of ozone (O 3), carbon monoxide (CO) and nitrogen oxides (NO x) suggest that ozone production in the MCMA has changed from a low to a high VOC-sensitive regime over a period of 20 years. Comparison of the historical emission trends of CO, NO x and hydrocarbons derived from mobile-source emission studies in the MCMA from 1991 to 2006 with the trends of the concentrations of CO, NOx, and the CO/NOx ratio during peak traffic hours also indicates that fuel-based fleet average emission factors have significantly decreased for CO and VOCs during this period whereas NO x emission factors do not show any strong trend, effectively reducing the ambient VOC/NO x ratio. This study presents the results of model analyses on the sensitivity of the observed ozone levels to the estimated historical changes in its precursors. The model sensitivity analyses used a well-validated base case simulation of a high pollution episode in the MCMA with the mathematical Decoupled Direct Method (DDM) and the standard Brute Force Method (BFM) in the 3-D CAMx chemical transport model. Correspondence to: M. Zavala ([email protected]) The model reproduces adequately the observed historical trends and current photochemical levels. Comparison of the BFM and the DDM sensitivity techniques indicates that the model yields ozone values that increase linearly with NO x emission reductions and decrease linearly with VOC emission reductions only up to 30% from the base case. We further performed emissions perturbations from the gasoline fleet, diesel fleet, all mobile (gasoline plus diesel) and all emission sources (anthropogenic plus biogenic). The results suggest that although large ozone reductions obtained in the past were from changes in emissions from gasoline vehicles, currently significant benefits could be achieved with additional emission control policies directed to regulation of VOC emissions from diesel and area sources that are high emitters of alkenes, aromatics and aldehydes.
Abstract. The emission characteristics of mobile sources in the Mexico City Metropolitan Area (MCMA) have changed significantly over the past few decades in response to emission control policies, advancements in vehicle technologies and improvements in fuel quality, among others. Along with these changes, concurrent non-linear changes in photochemical levels and criteria pollutants have been observed, providing a unique opportunity to understand the effects of perturbations of mobile emission levels on the photochemistry in the region using observational and modeling approaches. The observed historical trends of ozone (O 3 ), carbon monoxide (CO) and nitrogen oxides (NO x ) suggest that ozone production in the MCMA has changed from a low to a high VOC-sensitive regime over a period of 20 years. Comparison of the historical emission trends of CO, NO x and hydrocarbons derived from mobile-source emission studies in the MCMA from 1991 to 2006 with the trends of the concentrations of CO, NO x , and the CO/NO x ratio during peak traffic hours also indicates that fuel-based fleet average emission factors have significantly decreased for CO and VOCs during this period whereas NO x emission factors do not show any strong trend, effectively reducing the ambient VOC/NO x ratio.
This study presents the results of model analyses on the sensitivity of the observed ozone levels to the estimated historical changes in its precursors. The model sensitivity analyses used a well-validated base case simulation of a high pollution episode in the MCMA with the mathematical Decoupled Direct Method (DDM) and the standard Brute Force Method (BFM) in the 3-D CAMx chemical transport model.
Correspondence to: M. Zavala ([email protected]) The model reproduces adequately the observed historical trends and current photochemical levels. Comparison of the BFM and the DDM sensitivity techniques indicates that the model yields ozone values that increase linearly with NO x emission reductions and decrease linearly with VOC emission reductions only up to 30% from the base case. We further performed emissions perturbations from the gasoline fleet, diesel fleet, all mobile (gasoline plus diesel) and all emission sources (anthropogenic plus biogenic). The results suggest that although large ozone reductions obtained in the past were from changes in emissions from gasoline vehicles, currently significant benefits could be achieved with additional emission control policies directed to regulation of VOC emissions from diesel and area sources that are high emitters of alkenes, aromatics and aldehydes. they are generated. In addition to the impacts on NO x and VOCs, mobile source emissions are also significant contributors to toxic species. Therefore, substantial benefits for human health, ecosystem vitality and climate can be achieved by policies that result in reduced mobile-source emissions.
Past and recent field campaigns held in the MCMA have shown a strong influence of mobile-source emissions on the photochemical pollutant levels within the basin . Moreover, the emission characteristics of mobile sources in the MCMA have changed significantly over the course of the past few decades in response to emission control policies: advancements in emission control technologies, improvements in fuel quality, and vehicle inspection and maintenance programs (Molina and Molina, 2004). Fleet turnover with introduction of better technologies in new vehicles has reduced emissions from individual gasoline fuelled vehicles. Along with these changes, concurrent non-linear changes in photochemical pollutant levels as well as PM, toxic and criteria pollutants have been observed . These pollutant changes indicate that the ambient VOC/NO x ratio in the city has been modified, which should impact future control policy emphases.
The large number of measurements of both mobile-source emissions and air pollutant levels during the past two decades in the MCMA provides an opportunity to understand the effects of perturbations of mobile emission levels on the photochemistry in this region. By comparing concurrent changes in photochemical levels to mobile-source emission changes, it is possible to investigate this "observed" sensitivity and compare it to "modeled" sensitivity tests using air quality models.
To carry out the model sensitivity analyses we selected a base case simulation of a high pollution episode on 13-16 of April during the MCMA-2003 Campaign using the 3-D CAMx chemical transport model (ENVIRON, 2006), which simulates the emission, transport, chemistry, and deposition processes of pollutants in the troposphere. The evaluation of the model performance is provided in Lei et al. (2007). The sensitivity studies that estimate model responses to changes in the model inputs were carried out using the mathematical Decoupled Direct Method (DDM) and the results are validated using the standard Brute Force Method (BFM).
The BFM is conceptually the simplest sensitivity analysis method since each model input parameter is perturbed separately and the model output is evaluated for each control run. The change in model output is compared between the control run and the base case; this process is repeated for each input parameter investigated. In air quality modeling (AQM) applications this process can rapidly become time consuming and computationally expensive. Besides being computationally inefficient for most AQM applications, the magnitude and direction of the sensitivities estimated with the BFM depend upon the magnitude of the perturbation if the model response is nonlinear. In addition, for small model input perturbations the estimated sensitivities may contain significant levels of uncertainties introduced by numerical noise. Nevertheless, since the method is in principle applicable to any model input parameter and the results are relatively easy to interpret, the BFM is often used for the validation of more complex mathematical-based sensitivity analyses (Hamby, 1994).
The DDM was first used in the calculation of sensitivity coefficients in a three-dimensional air quality model (the Urban Airshed Model, UAM) by Alan Dunker in the early 1980s (Dunker, 1981). The DDM was also applied to the California/Carnegie Institute of Technology (CIT) airshed model in an application termed DDM-3-D (Yang et al., 1997). More recently, the implementation of the DDM in CAMx to calculate first-order sensitivities with respect to emissions and initial and boundary concentrations was reported by Dunker et al. (2002), who compared the accuracy of the DDM sensitivity analyses with those estimated using the BFM. The DDM was found to be highly accurate for calculating the sensitivity of the 3-D-model for perturbations up to 40% in reductions from the base case. The DDM has been used also in the CMAQ model to estimate PM sensitivities (Napelenok et al., 2006) and for PM source apportionment of linear systems (Yarwood et al., 2004).
In this paper we investigate the observed and modeled effects on the photochemical pollutant levels from perturbations of mobile-source emissions. We first describe the historically observed effects on ozone, CO and NO x trends in the urban area using data from the local ambient monitoring network. We also compare the historical trends of CO, NO x and hydrocarbons (HCs) derived from mobile-source emissions measurements in the MCMA from 1991 to 2006 with pollutant trends of hourly data of CO, NO x , and the CO/NO x ratio from the monitoring network during peak traffic hours. We use this approach to estimate the magnitude of the historical perturbations in mobile-source emissions in the urban area. The observed effects are the basis for evaluating the sensitivity analyses obtained from the DDM and BFM modeling techniques.
Observed sensitivity effects
To investigate the observed effects on the photochemical levels in the urban area from perturbations of mobile-source emissions, we have analyzed the concurrent changes in ozone, CO and NO x using a 21-year (1986-2006) data set from the MCMA ambient air quality monitoring network, RAMA (for Red Automatica de Monitoreo Atmosferico), which has continuously measured criteria pollutants since 1986. The network consists of 34 stations located in the metropolitan area and is maintained by the Environment Secretary of the Government of the Federal District. The data is reported hourly and is publicly available at http://www.sma. df.gob.mx/simat/. We have included all hourly data with a valid reading for each day only from March to April. By doing this, we minimize the effects of inter-annual seasonal variability of key meteorological parameters on ozone formation in our analysis. These months, which typically register high pollution levels, correspond also to the periods in which the MCMA-2003 and MCMA-2006/MILAGRO (Molina et al., 2008 field campaigns were carried out. To minimize data noise induced by the "weekend effect" (e.g., Altshuler et al., 1995) on the analysis of the ozone trends, we have excluded all data from Saturdays and Sundays. In addition, we have excluded the days for the Holy Week and all other major holidays when traffic patterns are different and mobile emissions are reduced.
Ozone trends
We focus our analysis on the trends of the daily ozone peak (that is, the city-wide maximum of all the daily ozone maximums over all the stations in a given day), the daily ozone maximums (the maximum ozone concentration for each station per day), and the ozone mean daily profile (averaged over all stations). Interesting trends on the magnitude, temporal and spatial allocation of ozone are clearly discernible from the 21-year data. Figure 1a shows that the peak ozone levels have significantly decreased (by more than 100 ppb) whereas a moderate decrease in the mean and maximum ozone diurnal levels has occurred. These changes are concurrent with a decrease in the spatial variability of the ozone fields (that is, the differences between the ozone levels measured at all the stations), mainly due to smaller ozone peaks in recent years.
The results indicate significant changes in the diurnal profiles of ozone, particularly before and after 1992. Figure 1b shows that mean ozone concentrations initially increased from 1986 to 1991 and then started a steady downward trend. In general, the night-time ozone levels are lower in recent years and the levels in the early hours before sunrise have decreased also. Overall, the mean ozone concentrations during the peak photochemical periods have reduced by about 25 ppb since 1991. As shown later, this is probably linked to diurnal changes in NO x emissions. Figure 1c also indicates that a rapid increase in maximum concentrations occurred during the first 5 years of data with an alarming rate of 12±1.3 ppb/year, followed by a steady decrease of about 2.7±0.5 ppb/yr since 1992. As will be discussed later, these changes are strongly related to changes in anthropogenic emissions, with mobile sources playing a significant role in both the increase and decrease. The observed inter-annual variability of the maximum ozone concentrations trends has not significantly changed (Fig. 1b), indicating that high concentrations are still common in the city. Figure 1d shows that the reduction of the daily ozone peak levels has been accompanied with a "delay" in its local time occurrence by 2 to 3 h with respect to earlier years. This effect has been observed also in maximum ozone concentrations for all stations with a delay from about 11:00 a.m. in the early years to about 01:00 p.m. in recent years. Because the data have been filtered to minimize seasonal effects, this delaying effect on the ozone-peak timing is most probably caused by changes in ozone precursor levels, rather than to changes in meteorological conditions. As a consequence of the observed time delay in high ozone concentrations, the spatial allocation of the peak ozone concentrations may change significantly since air masses containing the city emissions may travel longer distances by the time the ozone peak occurs. The delay suggests that some reductions in high ozone levels in fact may not be as large as indicated by the monitoring network because the network could be "blinded" to the actual (basin-wide) daily ozone peak, which may be occurring downwind at places where there are no monitoring stations. This idea is explored further in the modeling sensitivity analysis section below.
CO and NO x trends
CO and NO x levels are dominated by anthropogenic emission sources in urban areas. In Mexico City, mobile sources are the predominant sources accounting directly for about 99% and 84% of total CO and NO x emissions respectively (CAM, 2006). Therefore, the observed variability of CO and NO x levels is likely to be significantly influenced by changes in the emission characteristics of mobile sources. Figure 2 shows that CO mean concentrations have decreased significantly whereas mean NO x concentrations have stayed about the same over time. The spatial variability of the CO concentration fields (as indicated by the changes between the 90th and 10th percentiles, not shown) has also been reduced, but NO x variability has not. Similarly, large reductions are observed for the high CO concentrations in early morning hours whereas the corresponding high NO x concentrations have persisted. The CO mean concentration profiles in Fig. 2 show a much larger relative reduction than the reductions in mean ozone concentrations shown in Fig. 1, because ozone is a secondary pollutant and is not linearly dependent on its primary precursors. The variability of the mean NO x concentration profiles in recent years is similar to the early years. This suggests that the balance between sources and sinks of NO x has not changed significantly over this period.
Important changes in the temporal emission patterns of mobile emissions in the city appear to have happened. Figure 3 shows the normalized temporal profiles for mean CO in which each hourly value represents the percentage contribution to the total accumulated daily concentration. The figure shows a negative shift in time of 1 to 2 h of the median concentration patterns during the early morning hours. The same effect is observed in the corresponding analysis of NO x levels (not shown). Due to the filtering criteria used we expect similar mixing characteristics over these years, thus this effect is most likely due to changes in the temporal emission patterns in the city. One possible explanation is that, as congestion increases due to large increase of vehicle fleet size with little changes in road infrastructure, residents need to leave home earlier to arrive to work on time. Fresh NO x emissions would remove ozone carried over from night time chemistry and transport, in agreement with the decrease in ozone levels observed in Fig. 1b at this period. The massive injection of emissions in "dark" conditions into a shallow boundary layer has profound implications on the chemistry in subsequent hours.
Estimating historical changes in mobile-source emissions
Mobile-source emissions in the MCMA have been studied since the early 1990's using several measurement techniques including remote sensing (Bishop et al., 1997;Schifter et al., 2003a;GDF, 2006), dynamometer studies (Schifter et al., 2003b), tunnel studies (Mugica et al., 1998), and more recently in 2003 and 2006 with on-road sampling techniques using a mobile laboratory (Zavala et al., 2006). Fleet-average mobile-source emission estimates were also obtained in 2003 using long-path measurement techniques by analyzing the build-up of emitted species/CO 2 ratios during early morning rush hours (Volkamer et al., 2005). Cross-validation and inter-comparison of these techniques are intrinsically difficult to perform due to differences in sampling times and frequencies, pollutant measurements instrumentation, sampling size and analysis assumptions for each measurement type. Nevertheless, since these techniques have been applied in the MCMA at different times in the past two decades, it is worthwhile to make what comparisons are possible. Figures 4a and c show the emission factors of CO, HCs, and NO x measured in the MCMA during the past two decades. In this figure, the units reported from different studies were homogenized to fuel-based estimates assuming a fuel density of 0.75 kg/l, a fuel economy of 10 km/l, and 70.3 moles of C per kg of fuel for gasoline. The results indicate that fleet average emission factors have drastically de-creased for CO and HC over the past two decades (Fig. 4a), whereas NO x emission factors have not shown any strong trend ( Fig. 4c), keeping in mind that the vehicle fleet has increased greatly during these years. Figure 4b also shows that the renewal rate of the vehicle fleet (the difference between the cumulative vehicle sales and the number of vehicles between two given periods) has significantly increased in recent years and that gasoline sales have increased almost linearly. The reducing variability bars of Fig. 4a indicate that the age of the fleet is homogenizing faster: not only are many new vehicles being added to the fleet, but older vehicles appear to be rapidly taken out of the fleet. The observed reduction of CO and HC variability is, in turn, in good agreement with the observed downward trends in variability for ambient CO concentration profiles shown in Fig. 2. The effect of greater reductions in CO and HC than in NO x emissions is not surprising. Gasoline cars and light trucks are the major mobile sources for CO and HC, while almost half of the mobilesource NO x emissions are from diesel vehicles. The introduction of improved gasoline emission control devices over this time period would affect total mobile-source CO and HC more than NO x . Figure 4c indicates that there is no strong trend for the NO x emission factor and that a larger variability is observed for NO x than for CO and HCs emission factors. Unfortunately, the earliest fleet-average NO x emission measurements were performed in the year 2000; therefore there are simply not sufficient measurements in this time span to observe any trend in NO x . In order to obtain a better estimate of changes in NO x emissions over the past two decades, we used a second, indirect, approach by analyzing changes in the 06:00 to 09:00 a.m. values of the CO/NO x ratio measured by the ambient monitoring stations in the city. Mobile sources are the major emission source during the 06:00 to 09:00 a.m. time period because high emissions levels are generated by rushhour traffic (e.g. Zavala et al., 2006) and they are not rapidly (Volkamer et al., 2005), and on-road sampling from the Aerodyne mobile lab in , 2003(Zavala et al., 2006. dispersed at this time because the mixing height is still at a developing stage (Shaw et al., 2007). Furthermore, the use of the ratio of concurrent CO to NO x concentrations accounts for dilution effects -due to vertical mixing and transportduring the analysis. Because of the filtering criteria applied, the 06:00 to 09:00 a.m. CO/NO x ratio trend is more likely to be the result of long-term variability of mobile sources. Also, at this time of the day the pool of emitted NO x molecules is not yet significantly catalytically active (as compared to the rest of the diurnal period) in the photochemical cycle because NO 2 photolysis peaks around noon. The observed NO x trend has a stronger signal of its primary component (NO) during this period (the measured NO 2 /NO x ratio is at its lowest, not shown).
A significant decrease in the early morning ambient CO/NO x ratio of about 4.6 is observed in Fig. 4d, which also shows that the decrease has been driven by reductions in CO. These results are similar to those found by Stephens et al. (2008), who examined day-of-the-week patterns of air pollutants in the MCMA during the same two-decade period. The concurrent reduction in the magnitude and variability of ambient CO concentrations with the trends of CO and HC mobile emission factors, the early morning CO/NO x ratio, as well as the high percentage of mobile-source emissions in the emission inventory, indicate that emissions from mobile sources, rather than point and area sources, are largely responsible for the observed downward concentration trends.
Base case simulation
The formulation and evaluation of the model's performance for the 4-day base case simulation period (13-16 April 2003) used here are based on the findings of Lei et al. (2007). The 3-D CAMx model (Environ, 2003) is driven by hourly meteorological output fields from the Penn State/National Center for Atmospheric Research (PSU/NCAR) Mesoscale Modeling System (MM5) (Grell et al., 1995) configured to three one-way nesting with grid resolutions of 36, 12 and 3 km and 23 sigma levels in the vertical (de Foy et al., 2006). The model was run using the SAPRC99 gas phase chemical mechanism (Carter, 2000) and the photolysis rate frequencies for clear sky were pre-computed with the Tropospheric Ultraviolet and Visible radiation model (TUV) (Madronich and Flocke, 1998) (CAM, 2006). A summary of the total basin-wide initial emissions by source category and pollutant type is given in Table 1. Interpolated total annually emitted masses of VOCs, CO and NO x were distributed across mobile-, point-and area-source emissions categories and were transformed into spatially and temporally resolved and chemically speciated emission fields.
The performance of the MM5 model for simulating the meteorological patterns of the selected period has been described in detail elsewhere (de Foy et al., 2006. See also other MM5 modeling studies of Mexico City, e.g., Jazcilevich et al., 2005). The main features of the Mexico City basin flow are well represented by the model including the northerly flow from the Mexican Plateau during the day, upslope flows on the basin edges and the southerly gap flow from the southeast passage in the late afternoon and early evening. Weak synoptic forcing leads to surface winds that are weak and variable and are particularly challenging to model. The performance of CAMx was evaluated by comparing near surface ozone, NO y and CO concentrations with data from the RAMA monitoring network and the extensive VOC data sets obtained during the MCMA-2003 campaign . Simulated O 3 , CO and NO y agree particularly well with the measurements from the monitoring network within the modeled and experimental variability, and there are no systematic biases between modeled and observed daytime CO and NO y (Lei et al., 2007(Lei et al., , 2008. The results give us confidence in the model performance and in the accuracy of the emissions of CO and NO x used in the model. As described in Lei et al. (2007), the predicted concentrations of alkenes and speciated alkanes agree very well with measured concentrations from canister samples. Similarly, there is good agreement between predicted and observed concentrations of aromatics and formaldehyde, particularly for 15 April with no systematic bias. We have selected the April 15th simulation event as our base case for further sensitivity analyses because of the good performance of the model in simulating observed meteorological parameters and reproducing observed ozone concentrations simultaneously with measured VOCs, CO and NO x levels.
Brute force sensitivity method
We analyze the sensitivity of the model to perturbations of VOC and NO x emissions from the gasoline fleet, diesel fleet, all mobile (gasoline plus diesel) and all emission sources (anthropogenic plus biogenic). In Fig. 5 (right panel) each cross represents a model control run with a given perturbation in emissions (VOC (i) , NO x(i) ) to the base case (VOC (base) , NO x(base) ). To further investigate the spatial response of the model's sensitivity we have analyzed the changes of ozone concentrations with respect to the three spatial domains indicated in Fig. 5: AD refers to all cells of the model domain, CD refers to the cells corresponding only to the urban area, and ED corresponds to the cell containing the supersite location during the MCMA-2003 campaign. In addition, we have also analyzed the changes of the position and the occurrence of the local timing of the ozone peak as a result of perturbations in emissions. Figures 6 and 7 show the corresponding isopleths for the average and peak ozone concentrations along with the resulting changes in the position and local timing of the ozone peak. The shape of the peak and average concentration isopleths are quite similar for each corresponding emission source category, indicating that the direction of the sensitivity of the model is similar for both the ozone peak and average concentrations to the perturbations of a given emission source. However, the magnitudes of the relative changes for the peak and average ozone concentrations are significantly different. As an illustration, for a perturbation VOC (i) /VOC (base) =7 and NO x(i) /NO x(base) =1.25 of the gasoline fleet emissions, the domain-wide peak ozone concentrations increase more than 120 ppb with respect to the base case. For the same emission perturbation, the city-wide ozone diurnal average concentrations increase only by about 30 ppb. Despite the large sensitivity for the ozone peak concentrations in the AD domain, the magnitude of the changes of the diurnal average concentrations in the AD domain is much smaller than the corresponding changes in the city wide (CD) domain. This is because in the AD domain, that contains many non-urban areas, there is a larger number of cells with relatively low ozone concentrations, thus significantly biasing the spatial averaging of ozone concentrations.
Although directionally equal, the sensitivity of the model is not linearly additive: the sensitivity of the model to changes in all mobile emissions is not equal to the sum of the individual sensitivities to perturbations in gasoline and diesel emissions. This is because these source categories have different VOC/NO x emission ratios and therefore contribute differently to the propagation of radicals in the ozone formation process. For example, isopleths obtained from the perturbations in the diesel fleet emissions show a predominant NO x sensitive regime since these sources contribute strongly to NO x levels. The model also predicts sensitivities three to four times higher with respect to the ozone peak in the city than the average concentrations when perturbing mobile emissions. The sensitivity of the model is in general higher for perturbations from all sources than from mobile sources alone.
The longitudinal (x (i) ) and latitudinal (y (i) ) changes for the position of the ozone peak were estimated using the difference in the grid cell index (where the ozone peak registers) between a control run and the base case (x (i) -x (base) and y (i) -y (base) ) multiplied by the length of the cell (3 km). Thus, changes in the position of the ozone peak were estimated as the difference between the cell locations of the ozone peak of the control run and the base case. Similarly, changes in the local timing occurrence of the ozone peak were estimated using the difference in the hours of the occurrence of the local ozone peak for a control run and the local timing of the ozone peak for the base case. Figure 7 indicates that the model reproduces the observed lag in the local timing occurrence of the ozone peak (see Fig. 1d) by about 2 h with respect to a past VOC-rich scenario. In addition to the observed changes in ozone concentrations, the model reproduces the observed temporal delay in ozone peak formation. Nevertheless, although the model reproduces the time delay effect, clearly the changes in the geographical allocation of the ozone peak are only illustrative because we are limited to one meteorological scenario.
DDM sensitivity
The detailed mathematical basis for the implementation of the DDM in CAMx is presented in detail elsewhere (e.g., Dunker et al., 2002). Briefly, the DDM calculates the sensitivity coefficient, S i (x,t), defined as: with respect to the scalar parameter λ i by approximating the output concentration variable c with a Taylor series expanded around the base case λ 0 . As the magnitude of the input perturbation approaches zero, the output response will become dominated by the first-order sensitivity element: To obtain the first order sensitivity coefficients to the perturbation λ i by the DDM, the Advection-Diffusion Equation (ADE) for trace pollutants in the atmosphere: is differentiated with respect to λ i : In Eqs. (3) and (4), K is the turbulent diffusion coefficient vector, u is the average horizontal wind speed considered in the advection term, E[x, t] is the emission input fields and E , denoting the differentiation of the numerical algorithm for the emission process, is the unperturbed emission rate of the sensitivity parameter. Including photolytic reactions, all chemical reactions are aggregated into the volume term R, whereas J is the Jacobian matrix J =(∂R/∂c) of the reaction rates. An important advantage of the DDM method is that Eqs. (3) and (4) are solved with the same spatial grid and time steps in the program's model. Figure 8 shows the ozone sensitivity coefficients to perturbations in VOC, NO x and combined VOC and NO x for all emission sources obtained with the application of the DDM in CAMx for our base-case simulation. The effect on the magnitude of the ozone concentrations at first-order is given by Eq. (2) for a given perturbation level of the emissions. Similar to the results obtained with the BFM, the sensitivities depicted in Fig. 8 indicate that ozone is reduced when reductions in VOC emissions alone are implemented, while reductions in NO x emissions alone lead to ozone increases. Another major advantage of applying the DDM over the BFM is that the sensitivity coefficients of multiple input parameters can be estimated simultaneously during the same simulation. We obtained the sensitivity coefficients for each VOC emission group included in the VOC speciation of the SAPRC99 chemical mechanism in the model. Figure 9a indicates that ozone levels in the current base-case episode are highly sensitive to emissions of aromatics and alkenes. These two VOC groups, in addition to formaldehyde emissions, have large contributions from mobile sources (both gasoline and diesel sources), in agreement with the results from the application of the BFM. These results suggest significant benefits in ozone abatement in the city can be achieved by controlling sources that are high emitters of alkenes, aromatics and aldehydes. However, Fig. 9b also indicates that combined diesel and area sources contribute significantly to these highly reactive VOC groups, suggesting potential benefits in reducing ozone levels by controlling these emission sources.
Discussion
To discern the effects of changes of the anthropogenic emissions on the photochemical levels in an urban area is a complex task because ambient concentrations also strongly depend on meteorological conditions (e.g. Jazcilevich et al., 2005). Figure 1 shows that overall the ozone peak has been reduced significantly, but high median and maximum concentrations still persist. However, the figure also indicates that the day-to-day variability of the city-wide ozone peak is very large, even considering only the data of a couple of months each year, which indicates the large effect of daily meteorological conditions on ozone levels. Such large (but short-term) variability presupposes that properly-filtered long-term data sets have to be used for estimating the effects of changes of anthropogenic emissions on the photochemical levels.
We explored the influence of temperature variability on the observed ozone trends. The results indicate that the dailydomain-wide peak temperature variability does not have a discernible trend in the data period (not shown), suggesting that the observed trend in ozone is not influenced by the long-term variability of temperature. Short-term variability of temperature is likely to be correlated with ozone levels (warmer/cooler days versus daily ozone levels), but will not affect the results from the long-term analysis. In addition, some of the variability in the observed values during the first years may be due to differences in data acquisition protocols or problems with instrument calibration rather than to changes in the rate of the ozone formation process. Apparently, during the initial five years of its operation, the network suffered from poor quality assurance protocols for some instrumentation, which may be responsible for some of the large data variability during this period (Armando Retama, RAMA director, personal communication). After that period, the network has maintained the highest standards and quality assurance protocols.
Changes in vehicle technology and the introduction of emission control devices, as well as changes in motorization and deterioration rates, have decisively influenced the overall Fig. 9. Semi-normalized sensitivity coefficients for various lumped classes of VOCs according to the SAPRC99 a chemical mechanism. a The detailed species composition of each VOC lumped class is given elsewhere (Carter, 2000). Briefly, as the number specification of each group grows (e.g. ALK1 to ALK5), the magnitude of the rate constant of the species with OH increases. The number of individual VOC species in each group can be significant. As an approximation in molar weighting, however, we can consider that ALK1 is primarily ethane, ALK2 is primarily propane and acetylene, ALK3 is mostly n-butane and isobutane, ALK4 is mostly iso-pentane, n-pentane and 2-methyl pentane, ALK5 is a large mixture of pentanes (others than those in ALK4) to decanes, ARO1 is primarily toluene, benzene, n-propylbenzene and ethylbenzene, ARO2 is primarily m, p, and o xylenes, OLE1 are primarily terminal alkenes, OLE2 are mostly internal alkenes, CCHO are acetaldehyde and glycolaldehyde, RCHO are lumped C3+ aldehydes and TERP are biogenic alkenes other than isoprene. Explicitly emitted compounds include ETHE as ethene, HCHO (formaldehyde), ISOP as isoprene and ACET as acetone. emission levels from mobile sources in an urban area. Other indirect factors include changes in the intensity of ridership of public transportation and traffic congestion, both linked to urban infrastructure development. A detailed description of the major air quality management programs that have been implemented since 1986 in the MCMA can be found in CAM (2002) and in Molina and Molina (2002). Some of the most relevant control policies implemented included: 1) the introduction of two-way catalytic converters for new vehicles in 1991 and three-way catalytic converters in 1993; 2) the "Hoy No Circula" ("A day without a car during weekdays") program, first started in 1989 only during winter time and then continued as mandatory throughout the year; and 3) a vehicle emissions verification program that mandated the inspection and maintenance (I/M) of all vehicles twice a year. These and other control policies have significantly reduced the levels of primary pollutants, particularly those emitted from gasoline vehicles.
Measured fleet-average CO and HC emission factors show a significant decrease over time concurrently with long-term decreases of CO levels, indicating that technology improvements have overcome the effects of increases in fuel consumption and vehicle fleet (see Fig. 4b) on the overall CO (and likely HC) emissions. Normally, the contributions from non-combustion sources of hydrocarbons (such as painting, solvent consumption, cleaners, liquid petroleum gas (LPG) leakages, etc.) tend to increase over time as population increases. However, CO and HC emissions are both byproducts of incomplete mobile source combustion and, there-fore, the reductions in ambient concentrations of CO should be accompanied by reductions in ambient HC concentrations if non-combustion HC sources increase more slowly than the mobile source combustion-related emission reduction rates.
Semi-continuous canister measurements conducted between 1992 and 2001 for the C2-C12 hydrocarbons showed a slight but statistically significant decrease for that period in a small number of sites in Mexico City (Arriaga-Colina et al., 2004). However, there are no long-term continuous measurements of ambient HC levels in Mexico City and detailed HC information has been only obtained during intensive field campaigns. Molina and Molina (2002) and Molina et al. (2007) present a description of the major field campaigns in Mexico City. Trend assessments using the ambient levels measured in these studies would be difficult to perform due to all the intrinsic short-term variability that results from differences in the locations, time of the year (therefore different meteorological conditions), and techniques for the sampling. However, examination of the reported maximum measured concentrations in these studies shows a consistent decrease in HC ambient levels that is hard to explain without taking into account changes in anthropogenic emissions (Molina and Molina, 2002). More recently, measurements from the MCMA-2003 field campaign showed that vehicle exhaust is the main source of VOCs in Mexico City and that diurnal patterns strongly depend on vehicular traffic in addition to meteorological processes (Velasco et al., 2007).
The results also suggest that overall NO x emissions from mobile sources have not experienced a constant, long term, downward trend as CO and HCs have shown in the past two decades. As noted before, there are no fleet-average NO x emission measurements available prior to 2000; it is therefore possible that there are simply not sufficient measurements in the time span of Fig. 4c to observe any trend in NO x emissions. However, analysis of the 06:00 to 09:00 a.m. CO/NO x ratios for the 21-year period shows that the decrease has been mainly the result of reduction in CO. Additionally, individual analysis of these ratios for each monitoring station showed essentially the same behaviour suggesting that the effect is spatially homogenous.
Comparison of the variability bars in Fig. 4a and c indicates that the effect of homogenization of the vehicle fleet in its CO and HC emission characteristics is not being observed in the NO x emissions. Some possible factors that may contribute to such effect are: 1) vehicle technology, which is in turn directed by the establishment of emission standards, has been more effective in controlling CO and HC than NO x (comparison of emission standards of in-use and new vehicle, not shown, indicates that CO and HC emissions have been historically more aggressively addressed than NO x ); 2) faster deterioration in the efficiency (shorter lifetime) of the catalytic converters for reducing NO x than for oxidizing HC and CO emissions; 3) faster growth rates of the diesel fleet than the gasoline fleet. A comparison of fuel sales data between 2000 and 2006 from the latest emissions inventory shows that gasoline fuel sales increased about 1.8% annually on average whereas diesel sales increased about 4% annually on average over that period. The greater reduction in HC and CO than for NO x is not surprising since most of the emission reduction policies and technology advancements over this time period were aimed at gasoline vehicles, which are responsible for most of the CO and HC mobile-source emissions.
Analyses of the results from the BFM indicate that for both the peak and diurnal average ozone concentrations, the shape of the ozone isopleths for each emission source category perturbed is remarkably similar among the three spatial domains studied. This suggests that a given emission control policy directed towards the reduction of ozone will be effective in both the urban area and its surroundings. The sensitivity of the model with respect to the ozone peak concentrations is larger in the AD (domain-wide) domain, independent of the emission source category. However, that is not the case when assessing the changes in the diurnal average concentrations, in which the model is most sensitive within the CD (city-wide) domain, indicating that significant benefits can be achieved simultaneously inside the city (CD domain) and outside the city (AD domain) as a result of controlling anthropogenic emissions, particularly VOCs.
The figures demonstrate that the model reproduces the corresponding relative changes in historical observations of ozone peak and diurnal average concentrations. As inferred from the historical changes of mobile emissions, the isopleths of Figs. 6 and 7 also suggest that ozone production in the MCMA has changed from a low to a currently high VOC-sensitive regime over a period of 20 years. Comparison of the isopleths between the different emission source categories also suggests that mobile-source emissions, particularly the gasoline vehicle fleet, have largely contributed to the transition.
In the current VOC-sensitive regime, the ozone concentrations are more sensitive to perturbations of all emissions others than mobile sources alone (Fig. 6). This suggests that larger benefits could be achieved with emission control policies directed to the regulation of VOC emissions from area sources along with diesel emission sources. As indicated in Table 1, solvent consumption is possibly a good candidate for such reductions given their large contribution to VOC emissions.
The isopleths of the changes of the position and local timing occurrence of the ozone peak suggest significant changes in the ozone formation rates in the city as a result of changes in anthropogenic emissions. Holding the same meteorological scenario in the model, during the transition from a low to high VOC-sensitive regime the position of the ozone peak moves significantly with respect to the base case. Considering again the illustrative perturbation of VOC (i) /VOC (base) =7 and NO x(i) /NO x(base) =1.25, the position of the ozone peak moved about 10 km. An analysis of x (i) and y (i) indicates that such change occurred mainly southward in the model domain. Nevertheless, the magnitude and direction of this effect is merely qualitative here because the actual spatial changes in ozone formation as a result of historical changes in emissions depend on the particular meteorological conditions of every daily ozone peak event.
Similar to the BFM results, the sensitivity analyses using the DDM also indicate that ozone is reduced when reductions in VOC emissions alone are implemented and that reductions in NO x emissions alone lead to ozone increases. Simultaneous reductions of all emissions will also lead to (albeit smaller) ozone reductions only during later morning and afternoon (11:00 a.m. to 16:00 p.m.). The transition between the two regimes between 11:00 a.m. to 12:00 p.m. suggests changes on the fate of radicals and NO x in the ozone formation process. During the period from 08:00 a.m. to about 11:00 a.m. the removal of radicals may be largely dominated by the magnitude of the HO x -NO x sink pathways (formation of HNO 3 , PANs, and other organic nitrates). In contrast, after midday the production of radicals would largely depend on the sources of radicals (VOC oxidation and photolysis) and the subsequent ozone formation from the NO 2 photolysis dominate over the HO x -NO x termination reactions. As the cycling of radicals during the ozone formation process depends on the specific reactivity of the air masses, both the magnitude of the regimes and the timing of their transition observed in Fig. 8 are the results of the characteristic VOC/NO x ratios of the city. Night-time ozone levels are entirely linked to NO x levels in the model as the titration pathway represents the main sink of ozone at night.
We have compared the sensitivities of the model using the BFM and the DDM methods for perturbations of NO x , VOC and all emission sources. The comparison may be used to test the consistency of the two techniques and gives an estimate of the first order accuracy of the sensitivities provided by the DDM. For the comparison we investigated the magnitude and the temporal distributions of the sensitivity coefficients obtained by the two techniques. While Eq. (4) is used to obtain the sensitivity coefficients with the DDM, in the case of the BFM the sensitivity coefficients S BFM i are obtained by the two-side finite difference approximation: With this approximation, the quadratic dependency of c on λ cancels and the sensitivity coefficients are accurate through first-order in the perturbation δ. Figure 10 shows the comparisons between the BFM and the DDM for various magnitudes of the perturbation of VOC, NO x and all emission sources. The figure shows that the BFM sensitivities converge to the DDM sensitivities as the magnitude of the perturbation decreases in all three cases. Such convergent behaviour between the two techniques has been observed in other air quality modeling applications (e.g., Dunker et al., 2002;Hakami et al., 2003;Cohan et al., 2005). The results suggest that the model sensitivity to these emissions types behaves relatively linear for perturbations of up to 30% of the base case emissions. The model sensitivity becomes rapidly non-linear, particularly for NO x after that magnitude. The response of ozone to simultaneous perturbations of NO x and VOC emissions is more nearly linear than perturbations of any of these two alone.
Comparison of the spatial distributions of ozone concentrations predicted by the two sensitivity techniques is illustrated in Fig. 11. Whereas the spatial distribution of ozone is predicted directly in the BFM, for the DDM it was constructed using the first-order approximation (Eq. 2). Figure 11 shows that both sensitivity analyses predict similar spatial effects on ozone distribution at remarkable precision when increasing the corresponding input emission parameters by 25% from the base case. Nevertheless, there are some important differences in the magnitude of the ozone predictions. The DDM seems to predict slightly lower ozone concentrations than the BFM when considering VOC sensitivity alone. The opposite holds for NO x sensitivities: the DDM predicts higher ozone concentrations compared to the BFM. The magnitude and spatial allocation of ozone predicted by the two techniques is remarkably similar for simultaneous reductions of VOC and NO x . Although the ozone spatial distributions shown in Fig. 11 were obtained only at 14:00 h local time, the effects of the model sensitivity to emission perturbations agrees with the corresponding temporal distributions comparison obtained in Fig. 10. We note that model sensitivity studies tend to be local parametric analyses that rely on the model being structurally correct; the validity of such assumption is partially addressed (but not completely) when a thorough evaluation of the model performance is satisfactory against key simultaneously-measured chemical and meteorological parameters. Other factors such as the effects of the assumptions in the models' formulation of emission processes and the model sensitivity to meteorological and chemical parameters also affect the sensitivity of the model ozone predictions and deserve further investigation. Similarly, model sensitivities to concurrent perturbations of various VOC emission sources are not explored in this work and should be evaluated. Cross-sensitivities and higher order sensitivities (e.g. Hakami et al., 2003Hakami et al., , 2004 and their associated uncertainties of various VOC sources are also not investigated in this work. The inclusion of second-order sensitivities in an air pollution modeling episode in the South-eastern United States provided accurate characterization of the response to large perturbations in emissions (Cohan et al., 2005). These types of sensitivities may be important when evaluating mitigation strategies that simultaneously target specific VOC emissions sources (such as solvent, paint and dry cleaning emissions, etc.), where more than one VOC emission species is affected.
Conclusions
The analyses of the concentration trends of CO, NO x and ozone over the past 21 years in the MCMA show that significant changes have occurred for the variability and the diurnal temporal profiles of air pollutants in the city. The changes at night, early morning and peak concentrations are particularly evident for CO and ozone. Notably is the observed ozone-titration effect during the early morning that is probably linked to changes in the traffic patterns in the city as residents need to leave earlier to arrive to work on time. Overall peak ozone concentrations have been significantly reduced in the city while high median concentrations still persist. This observation is particularly important because these two metrics have very different implications when assessing the impacts of air quality management policies from the perspective of human exposure to ozone levels.
Since 1992 a reduction rate of 2.7±0.5 ppb/yr has been observed in maximum ozone concentrations after a growth rate of 12±1.3 ppb/yr from the start of the monitoring in 1986 to 1992. Using direct measurements of emissions and an indirect approach using CO/NO x ratios during rush hours, we further showed that these changes are strongly linked to concurrent perturbations of anthropogenic emissions, particularly from mobile sources. Fuel-based fleet average emission factors have substantially decreased for CO and HC over the past two decades whereas NO x emission factors have not shown a strong long-term trend. Reductions in CO and HC are likely due to improvement in gasoline vehicles and emission control technologies, which are in turn related to regulatory policies and the implementation of more stringent emission standards for new and in-use vehicles.
Using the BFM, the CTM model reproduces successfully the corresponding relative changes in historical observations of ozone peak and diurnal average concentrations. This provides further evidence that ozone production in the MCMA has changed from a low to a high VOC-sensitive regime over a period of 20 years. Sensitivity analysis of the model predicts that significant benefits can be achieved simultaneously within the city and its surroundings by controlling anthropogenic emissions, particularly VOCs. Further, comparisons of the VOC and NO x sensitivities for the various emission group categories studied indicate that large benefits may be achieved also with emission control policies directed to the regulation of VOC emissions from diesel and area sources along with traditional controls of the gasoline fleet.
Comparison of the BFM and the DDM sensitivity analyses indicates that, under the base-case emission/meteorological scenario studied, the model is equally sensitive (albeit with opposite sign) to NO x and VOC emission reductions up to 30% from the base case. Beyond those perturbation levels, the sensitivity of the model is no longer linear and the accuracy of the first-order approximation decreases significantly. Using the DDM, we have modeled sensitivity coefficients to individual perturbations of VOC species lumped in groups as described by the SAPRC99 chemical mechanism. We showed that the model is particularly sensitive to aromatics, higher alkenes, and formaldehyde emissions. | v3-fos-license |
2018-12-31T20:17:51.597Z | 2017-10-22T00:00:00.000 | 67798397 | {
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} | pes2o/s2orc | Controlled Assembly of Nanorod TiO 2 Crystals via a Sintering Process : Photoanode Properties in Dye-Sensitized Solar Cells
We present for the first time a synthetic method of obtaining 1D TiO2 nanorods with sintering methods using bundle-shaped 3D rutile TiO2 particles (3D BR-TiO2) with the dimensions of around 100 nm. The purpose of this research is (i) to control crystallization of the mixture of two kinds of TiO2 semiconductor nanocrystals, that is, 3D BR-TiO2 and spherical anatase TiO2 (SA-TiO2) on FTO substrate via sintering process and (ii) to establish a new method to create photoanodes in dye-sensitized solar cells (DSSCs). In addition, we focus on the preparation of low-cost and environmentally friendly titania electrode by adopting the “water-based” nanofluids. Our results provide useful guidance on how to improve the photovoltaic performance by reshaping the numerous 3D TiO2 particles to 1D TiO2-based electrodes with sintering technique.
Introduction
Organic molecular-based optoelectronic devices have been extensively studied over the last decade, such as in electroluminescent (EL) displays and photovoltaics [1].In particular, low-cost fabrication is one of the fundamental issues in order to realize a large-scale commercial production.In this regard, a solution processibility of these devices has significant advantages over inorganic ones that require a high-cost vacuum process.Among many emerging technologies, dyesensitized solar cells (DSSCs) represent opportunities for the creation of many advanced materials and device structures both for indoor and outdoor applications.Grätzel's group for the first time demonstrated the use of highly dispersed TiO 2 nanoparticles as anode materials for high-efficiency DSSCs [2].The synthesis of highly crystallized TiO 2 nanoparticles is generally required in order to develop high-performance photoelectrode materials in DSSCs [3].Besides, it is necessary to engineer the porous layer of deposited TiO 2 nanoparticles to (a) enhance the monolayer dye adsorption to the surface of TiO 2 nanoparticles, (b) efficiently collect the generated electrons and conduct them out, and (c) suppress the recombination phenomenon to enable efficient electron transport inside TiO 2 films which plays a crucial role in the solar cell's performance.A number of investigations have demonstrated how DSSCs' performance will be affected by characteristics of TiO 2 nanoparticles; many groups have shown the better electron transport of 1D morphologies of TiO 2 particles over nanoparticulate materials [4].
So far, most of the 1D TiO 2 particles including nanowires, nanotubes, and nanorods have been synthesized by a bottom-up method via wet-chemical approach such as hydrothermal reactions and anodic electrodeposition of suitable Ti(IV) precursors [5,6].We present for the first time an alternative synthetic method of obtaining 1D TiO 2 nanorods with sintering methods using bundle-shaped 3D rutile TiO 2 particles (BR-TiO 2 ) with the dimensions of around 100 nm (Figure 1).Interestingly, it was found that the cocrystallization with tiny spherical anatase TiO 2 crystals (SA-TiO 2 ) 2 International Journal of Photoenergy produced better photoanode performance, that is, improved light-to-electricity conversion efficiency in DSSCs.
There are several cost analyses of DSSCs that have shown a high material cost including transparent conducting glasses, dye molecules, and TiO 2 pastes (precursors as photoanodes) [7].Therefore, we also focused on the preparation of the low-cost and environmentally friendly titania precursors for creating porous TiO 2 electrodes in DSSCs by the development of "water-based" nanofluids.Here, we combined a standard metal-free indoline dye molecule, D205, as a sensitizer with the newly processed TiO 2 anodes in DSSCs, since the light absorption by such organic dyes can cover a wide range of visible light (with high molar extinction coefficient).Accordingly, the porous TiO 2 semiconductor film with indoline dyes can significantly reduce the TiO 2 thickness, leading to the reduced recombination reactions at the material interface of dyes and electrolytes in DSSCs.We report here the design and preparation of TiO 2 photoelectrode films as a generalized method to develop 1D morphology metal oxides using a sintering method.In addition, a preliminary assessment of the photoanode performance of DSSCs is provided by the creation of cocrystallized TiO 2 assembly using two different TiO 2 particles.
Experimental
A solar cell has several main elements, including FTO substrate, dye molecules, electrolytes, and counter electrodes.The main components of the dye solar cells have explained briefly as follows.
2.1.Substrate.F-doped SnO 2 -coated glass substrates (FTO, 13Ω/sq.)were used to prepare dyed TiO 2 electrodes.A mixture solution of 8 ml ethanol (99.5%), 0.07 g HCl (35-37%), and 0.71 g titanium tetraisopropoxide (95%) was deposited on the top of FTO by a spin coating.The purpose of the coating was to form a sublayer for deposited TiO 2 nanofluids.The coated layer would enhance the wettability and stability of deposited BR-TiO 2 particles.The coating was conducted by using two-step method: step one, the substrate was coated using 1500 rpm for 30 sec, and step two, the substrate was dried, using 1000 rpm for 60 sec.Figure 1(a) shows the deposited BR-TiO 2 nanoparticles on top of FTO 2.2.Deposited Porous Layer of TiO 2 Nanoparticles.We have recently reported on synthesis and crystal growth mechanism of BR-TiO 2 [8].Practically, the BR-TiO 2 were mixed with deionized water to produce nanofluids with a concentration of 40 wt%.It was observed that BR-TiO 2 were dispersed uniformly inside the water which provide low-cost and environmentally friendly nanofluid.To see the effects of the combination of particles with different geometries, small anatase nanoparticles with the average crystallite size of 6 nm (commercial, AMT100, TAYKA, represented as A-TiO 2 ) (Figures 1(c) and 1(d)) were mixed with the BR-TiO 2 .The mass percentage of SA-TiO 2 (m A /m A + m NR ) was 35%, 50%, and 85%.m A is the mass of SA-TiO 2 , and m NR is the mass of BR-TiO 2 .Figure 1(b) shows the TEM image of BR-TiO 2 .
The nanoparticles were mixed uniformly, using ultrasonicator and rotational agitator.To form a layer of TiO 2 nanoparticles on substrate, a layer of nanofluid was deposited on the coated FTO substrate, using spin coating technique.The thickness of deposited nanofluid was controlled by speed and period of spin coating.In this study, two-step and onestep strategies were used to coat the FTO substrate.(Two steps: substrate was coated, using 1500 rpm for 30 sec, and then it dried at 1000 rpm for 60 sec.One step: substrate was coated and dried, using 500 rpm for 250 sec.)Surface wettability has an important role on thickness of nanofluid on substrate [9][10][11][12][13][14][15][16].The thickness of nanofluid decreases, as affinity of liquid for substrate increases.The base liquid, surfactant, concentration, and characteristics of nanoparticle have a significant role on surface wettability.After depositing nanoparticles, the sintering method was applied to join the nanoparticles together and form tiny porous layer of deposited nanoparticles on FTO substrate.Typically, the deposited nanoparticles were sintered at 500 °C for 60 minutes, using an oven.The temperature was raised with a speed of 100 rpm per hour to achieve 500 °C.Similarly, the temperature was reduced to ambient temperature.
Device Assembly.
The deposited TiO 2 porous layer on FTO substrate was immersed in the D205 dye solution.The solvent was the combination of acetonitrile (super dehydrated) and t-butyl alcohol with a volume ratio of 1 : 1.The solution was kept in the dark at 25 °C.The dye adsorption time for D205 dye was 4 hours, and dye concentration was 0.2 mM.Chenodeoxycholic acid was used as a coadsorbent (0.4 mM).Pt-sputtered FTO glasses were used as counter electrodes.The dyed electrode and counter electrode were assembled into a sandwich-type cell.The regeneration of the oxidized dye (formed by light irradiation) is possible by accepting electrons from an electrolyte (a redox system).The electrolyte solution was a combination of 0.6 M 1,2-dimethyl-3-propylimidazolium iodide, 0.1 M LiI, 0.05 M I 2 , and 0.05 M 4-t-butylpyridine using 3-methoxypropionitrile as a solvent.The electrolyte was filled into the assembled cell with a vacuum backfilling method [17].
Evaluation of Electrode Films and Device Performance.
Structures of porous TiO 2 electrodes were characterized by scanning electron microscopy (SEM) using HITACHI S-4800, HITACHI SU-8000, KEYENCE VE-7800, and X-ray diffraction (Rint-Ultma/PC).Film thickness was estimated with KEYENCE VE-7800.The performance of photon-toelectricity conversions was examined by fabricating DSSCs in a similar manner to the reported method [18].Photocurrentvoltage curves were obtained under AM 1.5-simulated sunlight (100 mW/cm 2 ) by using Yamashita Denso YSS-80A
Results and Discussion
The BR-TiO 2 were mixed with deionized water to produce TiO 2 nanofluid.The TiO 2 nanofluid was dispersed on substrate, using a spin coating technique.The deposited nanoparticles were sintered to enhance the electron transport in the semiconductor, using an oven.Figure 2 place resulting in the reshaped 1D-shaped nanorods.In order to characterize the obtained particles, X-ray diffraction (XRD) measurement which is often used to analyze polycrystalline compounds was performed.The powder XRD pattern of the same sample (see Figure 3) matches well with the database of rutile TiO 2 crystals.Previously, we reported that as-prepared BR-TiO 2 has a rutile crystal phase.Accordingly, it was found that the rutile crystal type of TiO 2 is retained after sintering.
We anticipated that such a surface reactivity would enhance the interparticle's connection of BR-TiO 2 , especially when BR-TiO 2 are surrounded by small SA-TiO 2 .It is plausible that the SA-TiO 2 nanoparticles (anatase) have a tendency to agglomerate and produce bigger clustered particles in the nanofluid.Figure 4 shows the porosity of semiconductor (concentrations of SA-TiO 2 and BR-TiO 2 were 20 wt%) after sintering.It was observed that SA-TiO 2 clusters at the surface of BR-TiO 2 and produces larger crystals after sintering.This simultaneously results in the joining of TiO 2 interparticles in the film.Figure 5 shows the porosity of semiconductor for different concentration of SA-TiO 2 .After sintering, SEM images of TiO 2 films (porous photoanodes) in Figure 5 suggest that as nanoparticle concentration increases, the possibility of nanoparticle collision increases and consequently nanoparticles have more chance to agglomerate and form bigger clusters on substrate.Similar phenomenon has been observed during nanofluid pool and flow boiling [19][20][21][22].
Figure 6 shows XRD pattern of mixed TiO 2 particles deposited on FTO substrates.XRD pattern shows that more intense peaks at around 25 °(2θ) assigned to added SA-TiO 2 are observed with increasing the TiO 2 concentration.From these peaks, we estimated crystallite sizes of the anatase TiO 2 particles using the Scherrer equation, D = Kλ/βcosθ, where a peak width β is inversely proportional to crystallite size (D).The values of K, λ, and θ indicate Scherrer constant (=0.90),X-ray wavelength (=1.54 Å), and Bragg angle, respectively.It turned out that the values of crystallite sizes of the anatase TiO 2 particles are 11 nm, 17 nm, and 19 nm for 35 wt%, 50 wt%, and 85 wt%.HR-TEM analysis was performed for the sintered film to investigate the crystallization of anatase TiO 2 .As shown in Figure 7, the 50 wt% sintered sample indicated the lattice fringe of d = 3 5 Å, which is assigned to an anatase phase of TiO 2 .It was found that sintering process of clustered SA-TiO 2 produces larger anatase crystals on the rod-shaped particle, as suggested from TEM image in Figure 7(a).The TEM image in Figure 7(b) shows the formation of 1D nanorod TiO 2 due to a sintering process.Importantly, it is postulated that the number of grain boundary of sintered rutile BR-TiO 2 particles is reduced by the addition of SA-TiO 2 to form chain-like TiO 2 crystals in the TiO 2 film as shown in Figure 6.
In this study, the effects of nanoparticle concentration were investigated on performance of deposited TiO 2 semiconductor by comparing photovoltaic performance of solar cells.Figure 8 shows typical I-V curves, and Table 1 summarizes the photovoltaic performance of devices made by different nanofluid concentrations, exhibiting TiO 2 thickness, shortcircuit current density J sc (mA/cm 2 ), open-circuit voltage V oc (mV), FF, and percentage of photovoltaic performance.Solar cells were made, using coated FTO substrate and D205 dye which is a well-known indoline sensitizer.
SA-TiO 2 were mixed with BR-TiO 2 to prepare nanofluids with 40 wt% TiO 2 and 60 wt% deionized water.The effects of the concentration of SA-TiO 2 were investigated on performance of semiconductor while the total concentration of TiO 2 was kept constant at 40 wt%.The nanofluid was deposited on FTO substrate, and then the deposited nanoparticles were sintered to produce the semiconductor.It was observed that the efficiency of solar cell increased with concentration of SA-TiO 2 .The solar cell had a maximum efficiency at concentration of 20 wt% SA-TiO 2 and 20 wt% BR-TiO 2 .As concentration of SA-TiO 2 increased further, the efficiency of solar cell decreased.For all above cases, two-step method was used to coat the FTO substrate.Substrate was spin coated, using 1500 rpm for 30 sec, and then the dispersed nanofluid was dried at 1000 rpm for 60 sec subsequently.
To understand the effects of coating strategy, a mixture of 60 wt% water, 20 wt% SA-TiO 2 , and 20 wt% BR-TiO 2 was used to build the semiconductor on FTO substrate using two different coating strategies.The photovoltaic performance in Table 2 clearly shows that coating strategy has a significant effect on solar cell efficiency.The experimental results indicated that the solar cell efficiency enhances with thickness of the semiconductor for given conditions.As thickness of semiconductor increases, the interfacial area for dye adsorption increases to improve the photocurrent.
Table 3 shows the photovoltaic performance of DSSCs compared with two different sintering strategies.The longer sintering time (5 hours for the raising temperature) leads to the V oc increase (~29 mV).It is likely that a better diffusion between nanoparticles and more crystal growth leads to the reduction of grain boundaries of TiO 2 and consequently suppression of recombination phenomenon which can be seen with reduction of V oc .It is also important that the electrode with 20 wt% SA-TiO 2 and 20% BR-TiO 2 can show relatively higher V oc .These results indicated that the recombination phenomenon is not significant.In principle, the recombination phenomenon is suppressed with less grain boundary of TiO 2 particle [23].Therefore, such an unwanted reaction can be reduced at an optimal TiO 2 mixture that enables to decrease the grain boundary of TiO 2 domain.The sintering of the SA-TiO 2 (anatase) accelerates the interparticle crystal growth.This may decrease the grain boundary, and consequently, the electron transport efficiency and V oc increase.Contrarily, the 85 wt% of SA-TiO 2 had lower V oc .This is probably due to the increased recombination reaction sites with increasing the surface area of nanostructured anatase TiO 2 .
It was also found that the efficiency of solar cell is enhanced up to 4.86%, by modifying characteristics of nanofluid, spin coating, and sintering strategies.The conversion efficiency achieved by using D205 dye molecules is comparable with indoline dyes (3.39-5.46%),reported by our group, in which a high-performance TiO 2 paste (PST-18NR) was adapted for the device assembly [17].Furthermore, such a respectable performance was achieved without a commonly used TiCl 4 treatment (for making better contacts between TiO 2 particles and/or, TiO 2 particle, and FTO) in this work, suggesting that our submicron-scale TiO 2 particles can offer favorable electron transport in indoline-based DSSCs.
Conclusions
We present the effects of water-based TiO 2 nanofluids on the improved photoanode performance of indoline-based DSSCs.A mixture of SA-TiO 2 and 3D BR-TiO 2 particles was used to optimize the DSSC performance.It was suggested that the overall solution deposition process of TiO 2 photoelectrodes has significant potential in further development of highefficiency DSSCs, in which numerous 3D TiO 2 particle morphologies can be applied to boost the power conversion efficiency.Most importantly, our water-based nanofluid is expected to open up new synthetic routes of the semiconducting porous photoelectrodes with anisotropic rod-shaped morphologies, whose environmentally friendly production is beneficial for realizing cost-effective DSSCs.
Figure 1 :
Figure 1: (a) Schematics of BR-TiO 2 and SA-TiO 2 deposited on FTO substrate above the sublayer and subsequent sintering process.(b) TEM images of BR-TiO 2 .(c) A TEM image of SA-TiO 2 .(d) A SEM image of SA-TiO 2 .
Figure 2 :Figure 3 :
Figure 2: SEM images of the porous layer of deposited BR-TiO 2 particles (33 wt%) on FTO substrate, after sintering.(a) A cross-sectional image of 5 μm thick TiO 2 film.(b) Top side image.(c) A SEM image of 3.5 μm thick TiO 2 film before sintering is shown.
Figure 4 :
Figure 4: SEM images of the TiO 2 film after sintering.The TiO 2 nanofluid was spread on substrate, using spin coating method.The concentrations of SA-TiO 2 and BR-TiO 2 were, respectively, 20 wt% and 20 wt%.
Figure 6 :
Figure 6: XRD pattern of mixed TiO 2 particles of (a) 35 wt%, (b) 50 wt%, and (c) 85 wt% after sintering.The measurements were performed using TiO 2 films deposited on FTO substrates.The triangle indicates SnO 2 peaks coming from the substrate.
Figure 7 :
Figure 7: (a) HR-TEM image showing the lattice spacing of anatase TiO 2 crystals (yellow lines) formed by successive sintering.The red line shows zigzag-shaped edges of an anatase phase TiO 2 exposing 101 crystal facets.(b) A TEM image showing the formation of 1D nanorod.
Table 1 :
Photovoltaic performance of DSSCs using water-based TiO 2 nanofluids.Two-step method was used to manufacture the semiconductor on FTO substrate.Step one, the substrate was coated at 1500 rpm for 30 sec.Step two, the substrate was dried at 1000 rpm for 60 sec.
Table 2 :
Photovoltaic performance of DSSCs using water-based TiO 2 nanofluids.Two different spin coating strategies were used to manufacture the semiconductor on FTO substrate.
Table 3 :
Photovoltaic performance of DSSCs using water-based TiO 2 nanofluids.Two different sintering strategies were used to manufacture semiconductor on FTO substrate.Sintering strategy: (i) sintered at 500 °C for 60 minutes.The temperature raised and decreased to 500 °C with speed of 100 rpm per hour and (ii) the temperature raised to 500 °C with speed of 100 rpm per 6 min and decreased to ambient temp.with speed of 100 rpm per hour. | v3-fos-license |
2017-06-21T23:12:08.341Z | 2010-05-12T00:00:00.000 | 286533 | {
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"year": 2010
} | pes2o/s2orc | Protein binding hot spots and the residue-residue pairing preference: a water exclusion perspective
Background A protein binding hot spot is a small cluster of residues tightly packed at the center of the interface between two interacting proteins. Though a hot spot constitutes a small fraction of the interface, it is vital to the stability of protein complexes. Recently, there are a series of hypotheses proposed to characterize binding hot spots, including the pioneering O-ring theory, the insightful 'coupling' and 'hot region' principle, and our 'double water exclusion' (DWE) hypothesis. As the perspective changes from the O-ring theory to the DWE hypothesis, we examine the physicochemical properties of the binding hot spots under the new hypothesis and compare with those under the O-ring theory. Results The requirements for a cluster of residues to form a hot spot under the DWE hypothesis can be mathematically satisfied by a biclique subgraph if a vertex is used to represent a residue, an edge to indicate a close distance between two residues, and a bipartite graph to represent a pair of interacting proteins. We term these hot spots as DWE bicliques. We identified DWE bicliques from crystal packing contacts, obligate and non-obligate interactions. Our comparative study revealed that there are abundant unique bicliques to the biological interactions, indicating specific biological binding behaviors in contrast to crystal packing. The two sub-types of biological interactions also have their own signature bicliques. In our analysis on residue compositions and residue pairing preferences in DWE bicliques, the focus was on interaction-preferred residues (ipRs) and interaction-preferred residue pairs (ipRPs). It is observed that hydrophobic residues are heavily involved in the ipRs and ipRPs of the obligate interactions; and that aromatic residues are in favor in the ipRs and ipRPs of the biological interactions, especially in those of the non-obligate interactions. In contrast, the ipRs and ipRPs in crystal packing are dominated by hydrophilic residues, and most of the anti-ipRs of crystal packing are the ipRs of the obligate or non-obligate interactions. Conclusions These ipRs and ipRPs in our DWE bicliques describe a diverse binding features among the three types of interactions. They also highlight the specific binding behaviors of the biological interactions, sharply differing from the artifact interfaces in the crystal packing. It can be noted that DWE bicliques, especially the unique bicliques, can capture deep insights into the binding characteristics of protein interfaces.
Background
A protein binding hot spot is a small cluster of residues [1] tightly packed at the center of the interface between two interacting proteins. Though a hot spot constitutes a small fraction of the interface, it contributes most to the binding stability and free energy. A hot spot of binding energy was initially conceptualized by Clackson and Wells (1995), supported by an important finding that a 'functional epitope' (a hot spot) between human growth hormone and its receptor accounts for more than threequarters of the binding free energy [2]. This hot spot was also found to be geometrically surrounded by less important contact residues that are generally hydrophilic and partially hydrated [2]. On the basis of these pioneering observations and studies, Bogan and Thorn (1998) formalized more intuitively a hypothesis named O-ring theory to characterize the topological shape of the surrounding residues [1]. The O-ring theory points out that the residues of the O-ring likely function a role to occlude bulk water molecules from the hot spots. Thus, the O-ring theory is also known as 'water exclusion' hypothesis [1,[3][4][5][6][7][8]. This theory is profound and influential. However, the organizational topology of the ringinside, energetically more important hot spot residues is not specified by the O-ring theory. Recently, we investigated the spatial adjacency and vicinity of these hot spot residues and proposed a hypothesis called 'double water exclusion' (DWE) [9] to refine the O-ring theory. At one hand, the DWE hypothesis agrees with the O-ring principle that there should exist a ring of residues surrounding the hot spot for avoiding the invasion of water molecules after the complex formation; on the other hand, the DWE hypothesis affirms that the hot spot itself is water-free, having a zero-tolerance to water molecules. In fact, the DWE hypothesis shares a light with the 'coupling' proposition [10] which is another insightful theory about hot spot residues, and it also theoretically strengthens the influential 'hot region' principle [11]. The requirements for a cluster of residues to form a hot spot under the DWE hypothesis can be mathematically satisfied by a biclique subgraph [9] if a vertex is used to represent a residue, an edge to indicate a close distance between two residues, and a bipartite graph to represent a pair of interacting proteins. We term these hot spots as DWE bicliques, and note that in a DWE biclique, residues from one chain all have full connection with the residues from the other chain. In our latest evaluation [9] by applying to the ASEdb repository [4] and the Hotsprint database [12], we found that these DWE bicliques are rich of true hot spot residues.
With the perspective change from the O-ring theory to the DWE hypothesis, it is interesting to study the physicochemical properties of DWE biclique hot spots, and to compare with those [1,3,13] under the O-ring theory. Specifically in this paper, we examine those DWE bicliques that are unique to crystal packing contacts, or unique to biological interactions including obligate and non-obligate interactions. Crystal packing are enforced by crystallographic packing environments and formed during the crystallization process, but they do not occur in solution or in their physiological states [14]. On contrast, obligate interactions are stable, but their protein chains have no stable tertiary structures in vivo and they function only in the complex form [15]. However, the protomers in non-obligate interactions may disassociate after the accomplishment of a particular function [15]. Clearly, obligate and non-obligate interactions depend on various factors for promoting complex formation although some factors are common, while the non-specific crystal packing should have different properties from these two kinds of biological interactions. So at the residue level, it is of our primary interests to see which DWE bicliques are signature binding hot spots of the different types of protein interactions, and which are common for demonstrating their such difference. Given two types of interactions (e.g., biological interactions and crystal packing contacts), a unique DWE biclique is defined as a DWE biclique that frequently occurs in one type of interaction (e.g., biological interactions) but is absent in the other type (e.g., crystal packing). We also examine the residue composition of DWE bicliques, and the distribution of residue pairs and their pairing preference within DWE bicliques.
Residue composition and residue pairing preference are two fundamental physicochemical properties for protein folding [16,17] and protein interactions [18,19]. Both of them have been broadly used in comparative analyses based on protein structure data. One aspect of this comparative analyses is on different segments within protein complex structures, such as comparison between intraproteins and protein interfaces [19], or interdomain comparison [20]; another angle is on different types of protein interactions [13,21,22]. De et al. [23] and Lukman et al. [24] analyzed residue contacts of obligate complexes and non-obligatory/transient protein complexes, and reported that the interaction patterns of these two types of complexes were different. Ofran and Rost [20] studied the preferences of residue contacts in a more complicate way for six types of protein interfaces. One finding is that the preferences differed remarkably between the six types of interfaces. However, in almost all previous works, the individual residues and the residue pairs were dissected based on the whole interfaces. In this work, we analyze the composition of residues and residue pairs in DWE bicliques, the energetically important part of each interface.
We propose to use interaction-preferred residues (ipRs) and interaction-preferred residue pairs (ipRPs) to describe the binding specificity for different types of interactions. Our important findings include: (i) in the obligate interactions, hydrophobic ipRs and ipRP contacts involving only hydrophobic residues are widely conserved, no anti-ipRs are hydrophobic, and the contacts involving only hydrophilic residues are depleted in ipRPs; (ii) aromatic ipRs and their ipRPs much prefer to the biological interactions, especially to the non-obligate interactions; (iii) hydrophilic ipRs and ipRPs involving only hydrophilic residues seem to be rich in crystal packing contacts; the anti-ipRs of crystal packing, such as Met, Trp and Cys, are just the ipRs of the obligate or non-obligate interactions. So, these ipRs and ipRPs in DWE bicliques provide a clear distinction for the specific binding behaviors of biological interactions as well as crystal packing contacts.
Results and Discussion
The data used for our evaluation is a nonredundant data set consisting of 291 crystal packing contacts, 289 non-obligate interactions and 287 obligate interactions. This data set is combined from 4 previously compiled datasets [22,[25][26][27] after a redundancy-removal process shown in Methods.
We identified 1580, 725 and 208 DWE bicliques from these obligate, non-obligate and crystal packing contacts, respectively. All of these DWE bicliques have at least two residues in the smaller side and at least three residues in the bigger side, and occur in at least two interactions. The detailed computational steps are presented in the Methods section for how to detect DWE bicliques from a pair of interaction chains. There are 26 obligate interactions, 75 non-obligate interactions and 159 crystal packing that do not contain any DWE bicliques. Please note that by 'containing no DWE bicliques', we mean that these interactions do not contain any DWE bicliques of high frequency or of big size; they may contain some bicliques with low support (only one occurrence) or with a small size (for example, those bicliques with only one residue from each protein chain).
The remaining results are organized into four parts in this section. Firstly, we show that a binding hot spot is usually a very small area in comparison to its binding interface. Then, we present the distribution of DWE bicliques among the three types of protein interactions to provide an intuitive way for understanding the protein interfaces. After that, we conduct statistical analysis on the composition of the residues and on the pairing preference of residue pairs in our DWE bicliques.
A. Binding Hot Spots are Small in Binding Interfaces
We calculated the fractions of residues in DWE bicliques over interface residues. As expected, we found that binding hot spots are small in binding interfaces as shown in Table 1. In this table, all the DWE bicliques are from the biological interactions; in the first column, the numbers split by '-' mean that the residue number of the smaller partite in DWE bicliques must be not less than the left number, and that the residue number of the larger partite must be not less than the right number; in the following two columns, C means that the fraction is over the whole contact residues, and I means that the fraction is over the whole interface residues. Here, interface residues comprise contact residues and their nearby residues [28]. A pair of contact residues are two residues each from one chain that have at least two atoms contacting to each other. (Two atoms are considered to contact to each other if their distance is below the sum of their van der Waals radii plus 2.75 Å.) The nearby residues refer to those residues that are from the same chain as the contact residues, and their CA atoms also contact with the CA atoms of contact residues. Here, two CA atoms are considered to contact if their distance is less than 6 Å [28].
B. Common and Unique DWE Bicliques in Different Types of Interactions
In this work, unique DWE bicliques are defined based on bicliques' support information. Given a set of interactions containing one or more types of interactions, a DWE biclique may have multiple occurrence in these interactions. We call the number of occurrence the support level of this biclique. We also use support ratio r to help the comparisons of DWE bicliques in different types of interactions. Suppose that a biclique has a higher support S h in one type c h and a lower support S l in the contrasting type c l , r = S h /S l indicates the preference of this biclique between these two types. In the extreme cases when the support ratio r is infinite (i.e., S l = 0), this biclique is more interesting and it is defined as a unique biclique. In other words, this kind of unique bicliques have zero occurrence in one entire type of protein interactions but have multiple, perhaps very high, occurrence in the other type. To avoid some possible noise patterns, we require that S h + S l should be not less than 2 which is consistent with the biclique filtering constraint presented in Methods. Thus for unique bicliques, S h is always equal to or greater than 2 since S l = 0. Obligate and non-obligate interactions are two types of distinct biological interactions, each possessing their specific binding behaviors. However, crystal packing contacts are randomly formed by chance. We present DWE bicliques that are unique to biological interactions (c p+ ) when compared to crystal packing contacts (c n-), and those unique to crystal packing when compared to biological interactions. Meanwhile, the common bicliques, those with support ratio around 1.0, between biological interactions and crystal packing are also presented. Then we report common and unique DWE bicliques between obligate (c p+ ) and non-obligate interactions (c n-) to demonstrate their distinct binding specificity. After that, a case study is followed to examine deep structural details of unique DWE bicliques. Here, the symbol c p+ stands for a 'positive' set, while the symbol c n-stands for a 'negative' set. If the higher support S h of a biclique is in the positive set, then we denote the support ratio r as S h /S l ; otherwise if the higher support S h is in the negative set, we denote r as -S h /S l . The distribution of DWE bicliques on the range of their support ratios from -INF to +INF is shown in Table 2.
It can be noted that the type of crystal packing contacts contains much fewer favorite DWE bicliques than the type of biological interactions (columns 2 and 3 vs columns 5-8). In particular, there are only 13 DWE bicliques unique to crystal packing, counting to only 0.79% of the total DWE bicliques occurring in the biological and crystal packing interactions. In contrast, the type of biological interactions is rich of DWE bicliques, including unique bicliques. Table 2 shows that about 86.2% bicliques are unique bicliques to biological interactions. Few DWE bicliques contained by crystal packing but abundant DWE bicliques in biological interactions are interpretable with the following reasons: (i) crystal packing are constructed randomly based on protein surfaces during the crystallization process, and their artifact interfaces cannot form much repeatable DWE bicliques; (ii) residues in biological interfaces are found to be more conserved than crystal packing and than the rest of protein surfaces [26,29], and thus biological interactions can easily form stable and repeatable biclique structures. Table 3 shows the number of common and unique bicliques for the obligate and non-obligate interactions.
Common and Unique DWE Bicliques: Obligate vs Nonobligate Interactions
About 43.5% (641) of them are unique bicliques for the type of obligate interactions, and 16.6% (244) of them are for the type of non-obligate interactions. These unique bicliques can demonstrate the intuitive difference of binding behaviors in the two types of biological interactions, although there exist overlapping bicliques occurring in both obligate and non-obligate interactions. Detailed information of important unique DWE bicliques will be provided in the next subsection.
Related Evidence for Unique Bicliques
The existence of unique bicliques in the three types of interactions is in agreement with the observations in our another work [30] for classifying three types of interactions (i.e. crystal packing, obligate and non-obligate interactions). In that work, we compared classification performance between our propensity vectors, binary vectors and frequency vectors on three literature datasets under three evaluation frameworks. In [30], all of the propensity vectors, binary vectors and frequency vectors contain 3-dimensional summary information of protein interactions and another 210 dimensions for residue pairs. The difference was that the values of 210dimensional residue pairs in binary vectors indicated whether a certain residue pair occurs or not in a certain corresponding protein interaction, and those in frequency vectors were the frequency of corresponding residue pairs in protein interactions, while those in propensity vectors were the propensity value of corresponding residue pairs in protein interactions. Table 2 Common and unique DWE bicliques in biological interactions and crystal packing. Table 3 Common and unique DWE bicliques in obligate and non-obligate interactions. Our comparison results of these three vectors in [30] suggested following two evidences to support our concept of unique bicliques. (i) In almost all cases, the performances under the DWE hypothesis are better than those on all interface residues. That is, the whole interfaces may cover more noise features among different types of interactions, while DWE bicliques can remove some noise patterns and pinpoint distinct features. (ii) Binary vectors had a similar high classification performance to, sometimes a bit higher than, the frequency vectors. That similar performance maybe implied that certain combination patterns of residue pairs rather than their frequency are signature features for different types of protein interactions. When DWE bicliques are detected from DWE bipartites of protein interactions, these DWE bicliques are likely to be this kind of the combination patterns. In fact, the union of bicliques, possibly not maximal bicliques, might also be one kind of the combination patterns.
Unique Bicliques: A Case Study
We present four unique DWE bicliques and study their structural properties. They are: (i) a unique biclique = 〈{GLY, GLY, LYS}, {GLN, THR}〉, which occurs only in two crystal packing contacts (Table 4); (ii) a unique biclique 2 = 〈{LEU, THR, VAL}, {LEU, VAL}〉, which occurs only in five obligate interactions as shown in Table 5; a DWE biclique 3 = 〈{ALA, LEU, VAL}, {LEU, TYR}〉 which is a unique biclique to the type of biological interactions occurring in two obligate and three non-obligate interactions (Table 6), and (iv) a unique biclique 4 = 〈{GLN, GLY, SER, SER, TYP}, {CYS, LYS}〉 contained in only 6 non-obligate interactions (Table 7). At the first column of these four tables, the first four letters represent PDB entry identifiers, if necessary, followed by '-' and two interaction protein chains which are separated by ':'. At the columns 2 and 3 of these four tables, the strings split by '-' are residue types followed by their corresponding positions in the amino acid sequences, representing specific residues from the two interacting protein chains.
We take these examples to highlight that the uniqueness of DWE bicliques matches to different interfacial properties of the three types of protein interactions in terms of polarity, hydrophobicity, the composition of residues and residue pairs in protein interfaces. As mentioned, the unique biclique (Table 4) has a support of only 2. In fact, the maximum support of the unique bicliques that occur in the type of crystal packing contacts is 2. While the maximum support of the unique bicliques in the biological interactions is larger, 7 for the obligate bicliques and 6 for the non-obligate bicliques. Thus, the biclique structures in the biological interactions are more stable and repeatable than those in the crystal packing contacts. The four examples of unique bicliques also give a glance at residue composition in the three types of interactions. As shown in Table 4, the unique biclique of crystal packing consists of more polar and hydrophilic residues, such as GLY/LYS and GLN/THR, while the unique biclique 2 to the biological interactions comprises more hydrophobic residues, such as LEU and VAL as shown in Table 5. Table 5 also indicates that the contacts of identical residues easily occur in obligate interactions. However, this is found less in non-obligate interactions as shown in Table 6 and Table 7.
We would also like to present the conservation scores and the residues' ASA (accessible surface area) of unique bicliques in specific PDB entries. We take in the crystal packing 2ACY in Table 8 and 4 in the transient interaction 2PTC in Table 9 as example. In these two tables, the conservation score is taken from the website of rate4site [31], while ASA is calculated by NACCESS [32]. The location of these two bicliques at the protein interfaces are displayed in Figure 1.
It can be seen from Table 9 that the biclique residues in 4 have relatively small ASA in the 2PTC complex and larger ASA change upon complex formation. This can be easily understood from Figure 1: Figure 1(a) and 1(b) clearly show that the biclique residues in the biological interface 2PTC are buried. The ASA of LYS15 in chain I decreases from 201.71 Å to 0.6 Å, indicating that this biclique is closer to the interface center than to the rim of interface in Figure 1(a). However in the crystal packing 2ACY, both partites of have residues with relatively larger ASA, more than 50 Å. So, the biclique residues in chain B in Figure 1(c) are a little away from the interface center.
As shown in Table 8 and Table 9, both bicliques contain residues with high conservation scores. However, three of the seven residues in 4 have a conservation score less than 5. For example, the conservation score of LYS15 in chain I of 2PTC is 3, but this residue contributes greatly to the formation of the complex 2PTC -its mutation results in a big binding free energy change (10 kcal/mol) according to ASEdb (Alanine Scanning Energetics database) [4]. This observation might give a hint that although residue conservation is one of major factors contributing to frequent bicliques, frequent unique bicliques to biological interactions can capture more specific evidence for understanding complex formation than the conservation alone, such as ASA, residue physicochemical properties, and tightly packing residue contact. Next, we present our sequence and structural analysis results on the unique biclique 4 . As shown in Table 7, 4 occurs only in six non-obligate interactions in six different PDB protein complexes. These six interactions are all about trypsins/trypsinogen interacting with different types of inhibitors in different organisms. For example, 1TGS is about 'three-dimensional structure of the complex between pancreatic secretory inhibitor (kazal type) and trypsinogen', and 2BTC is about 'bovine trypsin in complex with squash seed inhibitor (cucurbita pepo trypsin inhibitor II)'. The sequence similarities of the six interacting chain pairs are as follows. Chain E of 1TAB, chain Z of 1TGS, chain E of 2BTC and chain E of 2PTC are identical chains. In comparison to this identical chain, chain A of 1EJA has only 83% sequence similarity and chain H of 1BTH possesses only about 36% sequence similarity. The sequence similarity among the other chains of these interactions is very low except two identical chains P in 1BTH and I in 2PTC.
Overall, there are no two pairs of interactions whose sequence similarity is larger than 40%. That is, there is no pair sequence redundancy in these six non-obligate interactions. We also note that although the two chain Es in 2BTC and in 2PTC are identical chains, the specific residues involved in 4 are not the same due to the low similarity between their partner chains. Residue SER in 2BTC is in the position 217, while it is in the position 195 in 2PTC. So, both bicliques are interesting to show. The details of these sequence similarities are provided in Table 10. The computational steps for determining the sequence similarity between two sequence pairs can be found in Methods.
The 3D structures of this DWE biclique in the six different PDB protein complexes are displayed and compared in Figure 2. The 3D shape of these structures looks highly similar to each other with a common lockand-key topology [33]. Since this stable topology is repetitive in six non-obligate interactions, it is worthy of further investigation to see whether this group of residues in this biclique is closely related to or involved in the above mentioned protein functions.
Another interesting question is: which residues in this DWE biclique are energetically outstanding. As mentioned, Lys in the sequence position 15 of chain I in 2PTC is a wet-lab confirmed hot spot residue with an extremely high energy (10 kcal/mol) according to ASEdb [4]. This may suggest that the Lys residue is also a hot spot residue in the other 5 interacting chain pairs.
C. Residue Composition of the DWE Bicliques for the Three Types of Protein Interfaces
The residue composition of protein binding interfaces or binding sites has been intensively studied previously [13,[21][22][23][24]34]. The composition of residues and residue pairs in DWE bicliques are studied by the current work in order to understand whether protein binding hot spots change their residue composition under the constraint of 'double water exclusion' hypothesis. We focus on the preference and tendency of residues to the specific types of interactions, as well as the preference and tendency of residue pairs. We would like to note that the composition of residues and their pairs in unique bicliques may be more interesting than those in DWE bicliques. But our investigation shows that there is no significant change for the composition of residues and their pairs in going from DWE bicliques to unique bicliques. This situation may be due to (i) that unique bicliques dominate DWE bicliques, and/or (ii) that common bicliques among the different types of interactions, especially those with larger support ratios, may also cover useful patterns for understanding protein binding behaviors. Therefore in the following two subsections, our analysis on the residues and their pairs in DWE bicliques is not on unique bicliques alone. We begin our analysis on the interaction-dominated residues (short for idRs) and interaction-preferred residues (short for ipRs). A residue is an idR in a type of interactions if its percent frequency in a set of DWE bicliques for this type of interactions is high; while a residue is an ipR in a type of interactions if its frequency ratio over the background is high. See Methods for the detailed definitions of idRs, ipRs and anti-ipRs. Figure 3(a) shows the frequency information of the twenty amino acids in our DWE bicliques for the three types of interactions, and Figure 3(b) displays the frequency ratio information of the twenty amino acids with reference to their background frequencies.
From Figure 3(a), we can see that the idRs for the obligate interactions are Leu (14.4%), Ala (8.20%), Val (7.63%), Gly (6.78%), Ile (6.68%), Arg (6.68%) and Phe (6.38%) ordered by their frequencies. Five of these residues are hydrophobic except Gly and Arg. However, Arg is broadly considered to be the richest in hot spots [1]. Gly's frequency ratio over its background percentage is 0.96, near to 1. That is, the abundance of Gly in nature makes Gly become an idR. Thus, we can make a conjecture that the binding hot spots of obligate interactions are dominated by hydrophobic residues. This point agrees to the frequency ratio trend of the ipRs of the obligate interactions as shown in Figure 3 12) and Thr(1.07) according to the ranking of their frequency ratios. Six of these ipRs are hydrophobic except Tyr, Arg and Thr. However, Tyr is an aromatic residue which can form π-π/cation-π interactions to stabilize protein binding. This is also why all aromatic The italic half is for the sequence similarity among these inhibitors, while the bold-face half is for the sequence similarity among trypsins/trypsinogen; '-' means no significant sequence alignment.
residues, Tyr, Phe and Trp, are ipRs of obligate interactions. Another interesting observation from Figure 3(b) is that the anti-ipRs are Cys, Gln, Ser, Glu, Asp and Lys-none of them is hydrophobic. Therefore, all these observations are consistent, and indicate that the binding hot spots of obligate interactions are hydrophobic and stable. Different from the obligate interactions, the binding hot spots of the non-obligate interactions contain only three hydrophobic idRs (Leu, Val and Ile), in addition to three hydrophilic idRs (Gly, Tyr and Ser) and one basic idR (Arg). It seems that non-obligate interactions are generally less hydrophobic than obligate interactions. The ipRs of these non-obligate interactions have a similar composition to their idRs, including three hydrophilic residues (Trp, Phe and Leu), three hydrophobic residues (Tyr, Cys and Gly), and two basic residues (His and Arg). Three aromatic residues, especially Trp and Tyr, seem to have a higher propensity to non-obligate interactions.
Quantifying the Difference of Residue Composition for the Three Types of Protein Interactions
We take two ways to quantify the residue composition difference between different interaction types. One is a Euclidean distance Δf [3,13] as described by Equation 1 in Methods to measure the difference of residue percent composition in the three types of protein interactions; the other is a correlation coefficient CC [20] as described by Equation 2 in Methods mainly to compare different residue ratio composition. The comparison result is presented in Table 11.
It is not surprised to see that the residue composition of all the three types is highly correlated to the background residue composition in the Swiss-Prot database with CC > 0. 7 [20]; however, biological interactions have larger Euclidean distance from the background residue composition with Δf = 3.7% for the obligate interactions and Δf = 2.922% for the non-obligate interactions. The Euclidean distance of residue percent composition in the three types of interactions is also large. For example, this Euclidean distance between the obligate and non-obligate interactions is 2.56%, while that between the cores of protein-protein complexes and homodimers is 2.0% [13]. We can understand from Table 11 that frequency ratio of residue composition in the three types of protein interactions has very low correlation coefficient, especially between non-obligate interactions and crystal packing with CC = 0.0561. The exceptionally larger correlation coefficient but larger Euclidean distance (Δf = 2.87%) between crystal packing and obligate interactions is partly, if not mainly, due to that most of crystal packing in the analyzed dataset are based on identical chains while most of obligate interactions are homodimers.
Comparison on Residue Composition Between the O-ring and DWE Hypothesis
Our analysis result on the residue composition of our DWE bicliques is in agreement with the influential study by Bogan and Thorn [1] who investigated the binding hot spots of protein interfaces under the O-ring hypothesis. Bogan and Thorn [1] found that hot spots are abundant with Trp, Tyr and Arg. We also found that these three residues are actually ipRs for both obligate and non-obligate interactions. Similar to Bogan and Thorn's method, Janin and her colleagues had a study for identifying the core and rim from a protein interface. They found that aromatic residues have high propensity values in the core of protein-protein recognition sites [3]. For homodimeric proteins, their another work [13] pointed out that aliphatic and aromatic residues are very where rows and columns represent different amino acids, and the residues are ordered according to their hydrophobicity with I as the most hydrophobic and R as the least hydrophobic. In these figures, the colors from blue to red mean the values from smallest to largest, and the similar colors mean the similar value in the second row. rich in the binding hot spots. All these results are consistent with ours.
Therefore, we can see that when protein binding hot spots are refined from the O-ring theory to the double water exclusion hypothesis, the composition properties are inherited and some properties are more enlightened and sharpened.
D. Residue Pairing Preference in DWE Bicliques for the Three Types of Protein Interfaces
A DWE biclique can contain many residue pairs. We are interested in those residue pairs that dominate, with high frequency, the binding hot spots of a type of protein interactions. We term this kind of residue pairs as interaction-dominated residue pairs (or idRPs for short). Meanwhile, we also examine interaction-preferred residue pairs (ipRPs). (See exact definitions for idRPs and ipRPs at the Methods section.) The composition information of all possible 210 residue pairs in our DWE bicliques is displayed in Figure 4. It can be seen that the obligate interactions are dominated by the contacts of hydrophobic residues. Taking the idRP group of I-V-L-F as example, the total frequencies of ten idRP contacts within this group are 18.22%, 10.74% and 11.74% for the obligate interactions, nonobligate interactions and crystal packing, respectively. The contact frequency of this most hydrophobic group in the obligate interactions is much higher than those in the other two types of interactions.
The ipRPs sharpen the difference of residue pairs in the three types of interactions. In the obligate interactions, most ipRPs are from four groups: (i) the contacts of identical residues, especially the residues with hydrophobicity not less than Tyr (Y)-this observation agrees with the discussion in [24] where identical residues more likely contact themselves in obligate interactions; (ii) the interacting pairs between aliphatic residues, Ile (I), Val (V) and Leu (L)-all these residues are most hydrophobic; (iii) the contacts between aromatic residues, Tyr (Y), Trp (W) and Phe (F); and (iv) the contact pairs between aromatic residues and Arg(A)/aliphatic residues. Aromatic residues are much involved in ipRPs due to that they easily form π-π/cation-π contacts which are vital to the stability of biological interactions. Besides the above ipRPs, another three ipRPs of obligate interactions are residue pairs Met(M)-Leu(L), Ala(A)-Leu and Met-Phe(F).
All these ipRPs shape an interesting distribution as outlined in Figure 4(f). Most of the ipRPs are located at the top-left corner of Figure 4(f), an area on the top of and at the left of Tyr (Y) included. We call this area ipRP area. There are fewer ipRPs outside this area, while those ipRP exceptions outside the ipRP area are the identical contacts of Asn (N) and Arg (R), and the interactions between Arg (R) and aromatic residues. That is, when taking the aromatic residue Tyr (Y) as the dividing line for the columns and also for the rows in Figure 4(f), ipRPs are depleted in the top-right corner (and also the bottom-left corner due to the symmetry of Figure 4(f)). The bottom-right corner also has rare ipRPs where the least hydrophilic residues are solely involved in the contacts. Such an ipRP distribution suggests that it is the very hydrophobic contacts that much prefer to the obligate interactions.
Similarly, in the non-obligate interactions, there are also very fewer ipRPs in the bottom-right corner of Figure 4(e) except the contact between Arg (R) and Asp (D). In the non-obligate interactions, the ipRPs are mainly from the contacts involving Trp (W), Tyr (Y), Phe (F), Cys (C), His (H) and Leu (L), specially the contacts involving W and Y. Of these contact residues, three are aromatic residues (Y, W and F), and two are nonpolar (hydrophilic C and hydrophobic L). Cysinvolved ipRPs are expected due to that Cys contains a sulfate atom and can form disulfate bridges to stabilize the protein interactions. The reason why H is also involved in the ipRPs may be that H is sometimes categorized into aromatic residues [35] and likely possesses some properties of aromatic residues in certain environments. In conclusion, residue pairs involved by hydrophobic and aromatic residues are abundant in the two types of biological interactions, indicating the importance of these ipRPs in specific binding behaviors.
In contrast to biological interactions, the ipRP distribution for the crystal packing contacts is completely opposite. Crystal packing contacts have more ipRPs of hydrophilic contacts (at the bottom-right corner of Figure 4(d)) and fewer ipRPs at the top-left corner. The top-right corner (and also the bottom-left corner due to the symmetry) of Figure 4(d) has more ipRPs than the top-left corner does.
Conclusions
With the integration of the influential O-ring theory and the insightful 'coupling proposition', DWE (double water exclusion) is a more comprehensive hypothesis for modeling protein binding hot spots. In this work, we constructed DWE bipartites from interacting protein chains under the constraints of both residue contacts and residue accessibility. Biclique patterns were then detected for each type of protein interactions. Our comparative analysis on DWE bicliques suggested that there do exist unique bicliques in the three types of interactions. Compared to crystal packing, those unique bicliques only occurring to biological interactions made it much clear that the biological binding behaviors have strong specificity. The unique bicliques in the obligate and non-obligate interactions also confirmed the different binding behaviors in these two types of biological interactions. Therefore, the idea of DWE bicliques provides a new way to the study on protein interfaces.
The composition of residues and the composition of residue pairs, in particular ipRs and ipRPs, did reveal the deep characteristics of these types of interactions. The protomers of obligate interactions fold and bind at the same time. Obligate interfaces need hydrophobic residues to form their interior cores, similar to the cores of protein tertiary structures in the same folding-binding process. Therefore in the obligate interactions, hydrophobic residues were greatly involved in ipRs and ipRPs, while none of the anti-ipRs of the obligate interactions was hydrophobic. Also in this process of protein folding and binding in a solvent environment, hydrophilic and polar residues prefer protein solvent surface than hydrophobic core, and the contacts involved by hydrophilic residues of obligate interactions were thus depleted in ipRPs.
On the other hand, two protomers in non-obligate interactions fold separately. They then come together to bind upon a specific molecular stimulus, and may dissociate after that. In a unbound form of non-obligate protomers, their interface surfaces have to contact with the solvent, and less hydrophobic residues are necessary [23] to keep the stability of unbound non-obligate protomers. So, the hydrophobic ipRPs in non-obligate interactions are much less than in obligate interactions. To compensate for the decrease of hydrophobic ipRPs in non-obligate interactions, aromatic residues are rich in non-obligate interfaces. Aromatic residues, such as Trp, Tyr and His sometimes, can contribute protein binding through the hydrophobic effect. Meanwhile, aromatic residues do not result in a large entropic penalty because they have few rotatable bonds [1]. This is why aromatic ipRs and their ipRPs were observed to be abundant in biological interactions, especially in nonobligate interfaces.
In contrast, hydrophilic ipRs and ipRPs were affluent in crystal packing to stabilize monomers in crystal packing without interactions. The anti-ipRs of crystal packing, such as Met, Trp and Cys, were just the ipRs of the biological interactions, indicating they can form significant atom contacts to greatly increase the probability of biological interactions, such as π involving contacts and disulphide bridges.
In summary, these ipRs and ipRPs in DWE bicliques are excellent indicators for the specificity analysis of biological binding behaviors. They can be used to identify biological interactions from crystal packing and classify different types of biological interactions, such as obligate and nonobligate interactions [30]. The identification of obligate or non-obligate interactions can help docking algorithm to remove the noise of produced crystal packing.
A. Compiling a Nonredundant Dataset
The data used in this paper contains three types of protein interactions: obligate interactions, non-obligate interactions and crystal packing contacts. All of them are obtained from previously published literature works. (i) The obligate interactions are from the obligate interactions used in [26,27], as well as the homodimers used in [22,25]; (ii) the non-obligate interactions comprise the non-obligate interactions used by [26,27], and the protein complexes used in [22]; and (iii) the crystal packing contacts are those from [22,26] and the monomers used in [25].
To get rid of the redundancy within each type of protein interactions, we remove those redundant interaction pairs with high similarity. Let C i 1 and C i 2 , and C j 1 and C j 2 , i ≠ j, be two pairs of protein interaction chain pairs in one type of interactions, these two pairs are redundant if score ( , ) C C s i j 1 1 ≥ and score ( , 1 ≥ , where the score function is a sequence similarity score of two protein sequences and it can be produced by the BLAST software (downloadable from NCBI http://www.ncbi. nlm.nih.gov/BLAST/download.shtml) without filtering of low compositional complexity, and s = 90% here. This redundancy removing process resulted in a non-redundant data set comprising 291 crystal packing contacts, 289 non-obligate interactions and 287 obligate interactions. The distribution of these interactions under different s value ranges is shown in Table 12. At one hand, it is clear in Table 12 that most of them have a low similarity of s = 40% or below. On the other hand, the detected bicliques from chain pairs with high similarity are actually different. For example, in Table 7, although chain E of 2PTC and chain E of 1TAB are the identical, the occurring bicliques are involved with residues of different positions in the interaction partner chains.
B. Constructing DWE Bipartites for Protein Interactions
Given two interacting polypeptide chains C 1 and C 2 , according to the DWE hypothesis, we define its DWE bipartite as a bipartite graph G = 〈V 1 , V 2 , E〉, where (i) the vertices in V 1 and in V 2 represent the amino acids from C 1 and C 2 respectively; (ii) the relative accessibility of all residues in V i , i = 1, 2, is less than a certain threshold t ra ; and (iii) E represents all residue contacts between V 1 and V 2 , and every residue in V i must contact at least one residue in V j , i, j = 1,2 and i ≠ j. We take two steps to construct DWE bipartites: (i) constructing bipartite graphs from protein interactions; (ii) filtering out those residues in the bipartite graphs by using the constraint of residue accessible surface area.
Constructing Bipartite Graphs
Each pair of chains can be transformed into a bipartite graph according to the contact requirement of the DWE bipartites above. In this work, two amino acids from V 1 and V 2 are considered as contact if the minimum of the distances of atoms from these two amino acids is less than the sum of van der Waals radii of the corresponding atoms plus a certain threshold. To ascertain that there is no water between interacting residue pairs, this threshold, denoted as d water , is set to van der Waals diameter of water molecules (2.75 Å).
In other words, residue r ik of C i and residue r jl of C j , i, j = 1, 2 and i ≠ j, contact if and only if the minimal distance among those distances between the atoms of r ik and the atoms of r jl is less than d water . Here, all heavy atoms in backbone and sidechains of amino acids are used. The distance between a pair of atoms a i' from r ik and a j' from r jl is calculated by: d = d(a i' , a j' )-r(a i' )-r(a j' ) where d(a i' , a j' ) is the spatial distance of a i' and a j' , and r (a k' ) is van der Waals radius of a k' , k' = i' or j'. Suppose we are given m p number of protein interactions, the bipartite graph database can be denoted by
Accessibility Filtering
The constructed bipartite graphs P are further processed by using the constraint of water accessible surface area of residues. We take NACCESS [32] to produce the relative accessible surface area for each residue in a protein interaction. The remaining ones are only those residues whose relative accessible surface area is less than a certain threshold t ra . In this work, t ra is set as 36% as recommended by [9]. [36] to mine maximal DWE bicliques. The LCM-MBC algorithm needs two parameters: p and q, p ≤ q. Suppose that V i is with less vertices than V j in a maximal DWE biclique , i, j = 1 or 2 and i ≠ j, p is the minimum size of V i and q is the minimum size of V j . That is, the LCM-MBC algorithm filters out those in which the minimum size of V i is less than p or the minimum size of V j is less than q. In this work, p is set to 2 and q to 3. Assume that the LCM-MBC algorithm detect n maximal bicliques from H, denoted as M = { j = 〈V 1 , V 2 〉 |j = 1, 2, ..., n} where items j are maximal bicliques with amino acids as their vertices. Not every j in M is useful for our analysis due to that some bicliques are infrequent and random.
Therefore, for each DWE biclique j , we enumerate H to get its occurrence in protein interactions. If the occurrence is not less than a threshold sup, j is considered to be interesting. Here, a biclique occurs in an interaction if all the residues in this biclique are in the DWE bipartite of this interaction and these residues also maintain the same biclique structure of full contacts. However, the space of possible bicliques is too large. Take bicliques of one partite with 2 residues and of the other partite with 3 residues for example, there are 323,400(( C 20 2 + 20) × ( C 20 3 + 20 2 )) possible bicliques and 20 5 biclique instances if each residue is considered to be independent. However, there are only 868 interactions including non-biological interactions. Thus, if each residue is with equal probability, the maximum of the expected support levels for these bicliques is 0.027 (10*868/20 5 ), much less than 1. That is, those bicliques whose support is equal to 1 also have a higher support than what they are expected. They have a lower support likely due to that there are limited sample interaction pairs and larger biclique space. However, bicliques occurring once can not show the specificity of binding behaviors in different types of protein interactions. Therefore, sup is set to 2 in this work, and the frequent maximal bicliques are referred to as DWE bicliques.
D. The Definitions Related to Our Composition Analysis on Residues and Residue Pairs
We calculate the frequencies of residues and residue pairs in DWE bicliques for each type of protein where r x and r y are frequent ratio vector for the twenty standard amino acids, and r is the mean of the corresponding r i s. | v3-fos-license |
2018-04-03T01:28:23.282Z | 2017-12-21T00:00:00.000 | 3546832 | {
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} | pes2o/s2orc | CCAAT/enhancer‐binding protein β overexpression alleviates myocardial remodelling by regulating angiotensin‐converting enzyme‐2 expression in diabetes
Abstract Diabetic cardiomyopathy, a major cardiac complication, contributes to heart remodelling and heart failure. Our previous study discovered that CCAAT/enhancer‐binding protein β (C/EBPβ), a transcription factor that belongs to a family of basic leucine zipper transcription factors, interacts with the angiotensin‐converting enzyme 2 (ACE2) promoter sequence in other disease models. Here, we aimed to determine the role of C/EBPβ in diabetes and whether ACE2 expression is regulated by C/EBPβ. A type 1 diabetic mouse model was generated by an intraperitoneal injection of streptozotocin. Diabetic mice were injected with a lentivirus expressing either C/EBPβ or sh‐C/EBPβ or treated with valsartan after 12 weeks to observe the effects of C/EBPβ. In vitro, cardiac fibroblasts and cardiomyocytes were treated with high glucose (HG) to investigate the anti‐fibrosis, anti‐apoptosis and regulatory mechanisms of C/EBPβ. C/EBPβ expression was down‐regulated in diabetic mice and HG‐induced cardiac neonatal cells. C/EBPβ overexpression significantly attenuated collagen deposition and cardiomyocyte apoptosis by up‐regulating ACE2 expression. The molecular mechanism involved the binding of C/EBPβ to the ACE2 promoter sequence. Although valsartan, a classic angiotensin receptor blocker, relieved diabetic complications, the up‐regulation of ACE2 expression by C/EBPβ overexpression may exert greater beneficial effects on patients with diabetic cardiomyopathy.
Introduction
Diabetic cardiomyopathy (DCM), which is characterized by left ventricular (LV) dilatation and systolic dysfunction, occurs independently of recognized causes, such as coronary artery disease, valve disease, arterial hypertension or other cardiovascular diseases [1][2][3]. LV remodelling is one of the major pathological mechanisms that ultimately lead to congestive heart failure. Numerous mechanisms are involved in the formation and development of LV remodelling in patients with DCM, including myocardial fibrosis, cardiac hypertrophy, mitochondrial damage, inflammatory, apoptosis and activation of the renin-angiotensin system (RAS) [4].
Homeostasis of the RAS is based on a balance between the angiotensin-converting enzyme (ACE)-angiotensin (Ang) II-angiotensin II receptor type 1 (AT1R) axis and the ACE2-Ang(1-7)-Ang1-7 receptor (MasR) axis [5]. ACE2, a homologue of ACE, has been shown to exert anti-fibrosis and anti-hypertrophy effects and to reduce LV remodelling in patients with type 1 diabetes [6,7]. ACE2 catalyses the cleavage of Ang II to Ang(1-7) and then counteracts endogenous ACE by activating MasR [8,9]. Based on the results of clinical trials and experimental studies, the activation of the RAS is associated with the development of LV remodelling in patients with DCM [10,11]. However, the exact mechanism between RAS and DCM remains poorly understood.
CCAAT/enhancer-binding protein b (C/EBPb), a transcription factor that belongs to a family of basic leucine zipper transcription factors, affects cell growth and differentiation [12,13]. Reduced C/EBPb expression up-regulates the expression of a GATA-binding protein 4, T-box transcription factor 5 (Tbx5), NK 2 homeobox 5 (Nkx2.5), a-myosin heavy chain (a-MHC), troponin I (TnI) and troponin T (TnT), all of which are hypertrophy-related genes [14]. According to our previous study, C/EBPb binds the ACE2 promoter sequence and decreases ACE2 expression in Ang II-treated cells, indicating that C/ EBPb might also regulate ACE2 expression in DCM. We suggested that C/EBPb overexpression may attenuate collagen accumulation, apoptosis and LV remodelling by regulating ACE2 synthesis and other RAS members in the mouse model of type 1 diabetes.
Animal protocol
All animal experiments were conducted in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals. The protocol was approved by the Animal Care and Use Committee of Shandong University Qilu Hospital. After 1 week of acclimation, 8-week-old male C57BL mice (Beijing Hua Fu Kang Biological Polytron Technologies Inc, Beijing, China) were randomized into the control group (n = 20) and the treatment group (n = 80). Type 1 diabetes was induced in the treatment group by the intraperitoneal injection of 50 mg/kg streptozotocin (STZ; Sigma-Aldrich, St. Louis, MO, USA) for five consecutive days. Meanwhile, the mice in the control group received intraperitoneal injections of a solvent (0.1 mol/l sodium citrate, pH 4.5). Random blood glucose measurements greater than 16.7 mmol/l in the treatment group for 3 days indicated the successful induction of type 1 diabetes (ACCU-CHEK Active; Roche, Indianapolis, IN, USA). After 12 weeks, the diabetic mice were randomized into the following four groups: (i) DM + shRNA negative control (N.C.) (n = 20), (ii) DM + C/EBPb (n = 20), (iii) DM + sh-C/EBPb (n = 20) and (iv) DM + valsartan (n = 20). Mice in the indicated groups were injected with 1 9 10 7 UT/30 ll of lentivector containing sh-N.C., C/ EBPb or sh-C/EBPb (GENECHEM, Shanghai, China) through the caudal vein. Valsartan (30 mg/kg; Novartis, Beijing, China) dissolved in normal saline was administered by gavage to the mice in the valsartan group [15]. Sixteen weeks after the first STZ injection, all the mice were killed.
Echocardiography
The heart function and dimension parameters were measured using a standard protocol after 16 weeks by transthoracic parasternal echocardiography using the VEVO770 imaging system (VisualSonics, Toronto, ON, Canada). LV parameters, including the left ventricular end-diastolic diameter (LVEDd), left ventricular posterior wall thickness (LVPWd), left ventricular ejection fraction (LVEF) and fractional shortening (FS), were measured in M-mode via the long/short axis view. The ratio of the early peak (E, mm/sec.) to the late peak (A, mm/sec.) mitral flow velocities was determined using pulsed-wave Doppler echocardiography.
Histology and immunohistochemistry
After fixation with 4% paraformaldehyde, dehydration with an alcohol gradient and embedding in paraffin, the heart tissues were cut into 4.5 lm sections. Sections were stained with haematoxylin and eosin (H&E) to measure the cardiomyocyte width and with Masson's trichrome to assess the collagen content. Immunohistochemical staining was performed on sections using a previously described method [16]. Sections were incubated with the following primary antibodies at the appropriate concentrations overnight at 4°C: anti-C/EBPb, anti-ACE2, anti-ACE, anti-transforming growth factor-b1 (TGF-b1), anti-collagen I and anti-collagen III (all from Abcam, Cambridge, MA, USA). The secondary antibodies were used according to the manufacturer's specifications. Images of the LV sections were obtained at 4009 magnification and measured using the computer software ImagePro Plus 6.0.2 (Media Cybernetics, Houston, TX, USA).
Statistical analysis
All data from at least three independent experiments were expressed as mean AE SD, and intergroup differences were analysed using a one-way ANOVA via SPSS software 18.0 (SPSS, Chicago, IL, USA). P < 0.05 was regarded as statistically significant.
Fasting blood glucose concentrations and morphometric profiles
As expected, 1 week after STZ injection, fasting blood glucose concentrations in diabetic mice showed a marked elevation that persisted until the end of the experiment (Table 1). Excessive water intake, excessive food intake and polyuria were observed in the diabetic mice, particularly in the DM + sh-N.C. and DM + sh-C/EBPb groups. Meanwhile, differences in body weight, heart weight and the ratio of heart weight to body weight were statistically significant among the five groups (Table 1). Thus, C/EBPb overexpression might reverse cardiac remodelling.
C/EBPb overexpression and the valsartan treatment ameliorated myocardial remodelling
Echocardiography was employed to evaluate cardiac function at the end of the experiment. LVEF, FS and the E/A ratio were substantially decreased, and LVEDd and LVPWd were increased in the DM + sh-N.C. group compared with those in the controls. Compared with the DM + sh-N.C. group, the DM + C/EBPb and DM + valsartan groups exhibited improvements in LVEF, FS and the E/A ratio and decreases in LVEDd and LVPWd, but the DM + sh-C/ EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 1A-F). The increased heart size and cardiomyocyte width were reduced by C/EBPb overexpression and valsartan (P < 0.05; Fig. 1G-J). Although the valsartan treatment improved cardiac function indices and attenuated heart size and cardiomyocyte width, C/EBPb overexpression had a much better effect on myocardial remodelling.
C/EBPb overexpression ameliorates extracellular matrix deposition in vitro and in vivo
Masson's trichome staining of cardiac sections revealed the elevated expression of the extracellular matrix (ECM) in the interstitial areas of diabetic mice compared to the expression in the control mice. C/EBPb overexpression and the valsartan treatment dramatically reduced collagen deposition in the intramyocardial and perivascular regions compared to diabetic mice that were transfected with sh-N.C. Additionally, the expression was much lower in the C/EBPb overexpression group than in the valsartan group. The DM + sh-C/ EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 2A).
The induction of diabetes increased the accumulation of the fibrotic markers collagen I, collagen III and TGF-b1 compared to that in healthy controls. C/EBPb overexpression and the valsartan treatment reduced the levels of collagen and TGF-b1 compared to those in the DM + sh-N.C. group, and collagen was expressed at much lower levels in the C/EBPb overexpression group than in the valsartan group. The DM + sh-C/EBPb group was not significantly different from the DM + sh-N.C. group. The effects of all groups were confirmed by immunohistochemistry and Western blotting (P < 0.05; Figs 2B,C and D and 3A,B and D).
According to the Western blot results, MMP-9 expression was not significantly different from all groups (P < 0.05; Fig. 3C). But C/EBPb overexpression and the valsartan treatment ameliorated the diabetesinduced reduction in MMP-2 levels, and MMP-2 was expressed at much higher levels in the C/EBPb overexpression group than in the valsartan group. The DM + sh-C/EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 3E). Consistent with the Western blot results, the ELISA showed that the serum MMP-9 levels were not significantly different from all groups, whereas C/EBPb overexpression and the valsartan treatment increased the serum MMP-2 levels compared to those in the DM + sh-N.C. group, and MMP-2 levels were much higher in the C/ EBPb overexpression group than in the valsartan group. The sh-C/ EBPb treatment decreased MMP-2 levels, but the difference was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 3F and G). Consistent with the alterations observed in diabetic mice, the expression of the collagen I and III and TGF-b1 proteins was increased in high glucose-treated CFs. Both C/EBPb overexpression and the valsartan treatment attenuated the up-regulation of the collagen I and III and TGF-b1 compared to expression in the HG + sh-N.C. group, and collagen was expressed at lower levels in the C/EBPb overexpression group than in the valsartan-treated group. The HG + sh-C/EBPb group was not significantly different from the HG + sh-N.C. group (P < 0.05; Fig. 3H-J). Likewise, the results of MMPs proteins expression in tissue were verified by exposing CFs to HG (P < 0.05; Fig. 3K and L). Additionally, significant differences in activity of MMP-9 were not observed among the groups, but C/EBPb overexpression and the valsartan treatment both remarkably ameliorated the HG-induced decrease in MMP-2 activity (P < 0.05; Fig. 3M and N).
C/EBPb overexpression suppresses apoptosis in the diabetic myocardium and H9C2 cardiomyocytes and decreases inflammatory factors expression in serum
The Bax/Bcl-2 ratio and AT2R expression were increased in vehicle-treated diabetic mice compared with expression in the healthy controls. C/EBPb overexpression effectively reduced the Bax/Bcl-2 ratio and AT2R expression compared to that in the DM + sh-N.C. group, whereas the valsartan treatment only exerted a significant effect on the Bax/Bcl-2 ratio. The apoptosis indices of the DM + sh-C/EBPb group were not significantly different from that of the DM + sh-N.C. group (P < 0.05; Fig. 4A and B). The results were verified by exposing H9C2 cardiomyocytes to HG (P < 0.05; Fig. 4C and D).
Serum IL-6 and MCP-1 levels were increased in the diabetic mice, but they were alleviated by C/EBPb overexpression and the valsartan treatment compared to the levels in the DM + sh-N.C. group. The DM + sh-C/EBPb group was not significantly different from the DM + sh-N.C. group (P < 0.05; Fig. 4E and F).
Diabetes and the HG treatment decrease C/EBPb expression, and C/EBPb overexpression up-regulates ACE2 expression
The C/EBPb protein was expressed at lower levels in the sh-N.C. group than in the controls, but it was up-regulated in the C/EBPb overexpression group. The sh-C/EBPb and valsartan-treated groups showed no significant differences compared with the vehicle-treated group (P < 0.05; Fig. 5A). Meanwhile, the ACE2 protein was expressed at lower levels in the sh-N.C. group than in the control group. C/EBPb overexpression increased ACE2 protein expression compared to that with the sh-N.C. treatment. The sh-C/EBPb and valsartan-treated groups were not significantly different from the vehicle group in vivo (P < 0.05; Fig. 5B). Similar effects of C/ EBPb and ACE2 expressions were verified in CFs treated with HG and in the myocardium by immunohistochemistry (P < 0.05; Fig. 5D-G).
C/EBPb overexpression decreases Ang II and increases Ang(1-7) levels in DCM
The sh-N.C. treatment, C/EBPb silencing and valsartan treatment markedly increased myocardium Ang II levels in diabetic mice compared to the levels in the controls. C/EBPb overexpression alleviated the increased Ang II content compared to the sh-N.C. treatment. The levels of Ang (1-7) were significantly increased in the C/EBPb overexpression group and slightly increased in the valsartan-treated groups compared to the sh-N.C. group. The sh-N.C. treatment and C/EBPb silencing decreased the myocardium Ang(1-7) levels, but the difference was not statistically significant (P < 0.05; Fig. 6A and B). Serum Ang II and Ang(1-7) levels are consistent with those in tissue (P < 0.05; Fig. 6C and D).
Levels of the ACE, AT1R and MasR proteins in the diabetic myocardium and CFs
Diabetes significantly elevated the expression of the ACE and AT1R proteins and decreased the expression of the MasR protein. C/EBPb overexpression remarkably reduced levels of the ACE and AT1R proteins and increased levels of the MasR protein, but the valsartan and C/EBPb silenced groups were not significantly different from the sh-N.C. group (P < 0.05; Fig. 6E and F). The results were verified in HG-treated CFs (P < 0.05; Fig. 6G and H). The expression of ACE was tested consistently by Western blot in vivo and in vitro and verified by immunohistochemistry analysis (P < 0.05; Figs 5C and 6I and J). C/EBPb overexpression alleviates fibrosis and apoptosis by up-regulating ACE2 levels Consistent with the changes described above, ACE2 was expressed at significantly higher levels in CFs in the si-N.C + C/EBPb group than in the sh-N.C. + si-N.C. group and decreased in the C/EBPb + si-ACE2 group compared to the levels in the si-N.C. + C/EBPb group. Additionally, the expression of collagen III and TGF-b1 was not reduced in the C/EBPb + si-ACE2 group compared to expression in the si-N.C + C/EBPb group, which corroborated our hypothesis regarding the C/EBPb-ACE2 pathway (P < 0.05; Fig. 7A-D).
ChIP assays were used to obtain further insights into the molecular interactions between C/EBPb and ACE2 following the HG treatment. The ACE2 promoter sequence was obviously enriched in the DNA immunoprecipitated with the anti-C/EBPb antibody, indicating that C/EBPb directly binds to the ACE2 promoter. Furthermore, the results verified that the binding of C/EBPb to the ACE2 promoter decreased following the HG treatment, further indicating that hyperglycaemia-induced ACE2 down-regulation may be directly caused by the weaker binding force between C/EBPb and the ACE2 promoter (P < 0.05; Fig. 7E). Based on these findings, C/EBPb regulates ACE2 expression by directly binding to its promoter.
Discussion
The major finding in our study was that C/EBPb overexpression ameliorated diabetes-induced myocardial remodelling by up-regulating ACE2 expression and modulating the expression of other members of the RAS.
The most important effect of C/EBPb on the successful model was the amelioration of DCM-induced fibrosis and remodelling. Enhanced fibrosis in cardiac tissue is a vital hallmark of cardiac dysfunction. Ang II, which is converted from Ang I by ACE, is one of the most important factors that contribute to the up-regulation of collagen expression in patients with diabetes [19,20]. Ang II stimulates collagen synthesis in fibroblasts and myofibroblasts via AT1R [21]. Candesartan inhibited the increased collagen I expression in HG-treated fibroblasts in a previous study, indicating that Ang II regulates HG-induced collagen deposition in CFs [22]. The up-regulation of ACE2 and Ang(1-7) expression alleviates the fibrosis and cardiac dysfunction in subjects with DCM [7,23]. In the present study, C/EBPb overexpression decreased Ang II levels and reduced collagen production and ECM deposition in HG-treated fibroblasts and diabetic mice. As shown in our previous study, diabetic mice and CFs both expressed increased levels of TGF-b1, which acts as a major mediator of cardiac remodelling by altering collagen metabolism and inducing cardiomyocyte hypertrophy [24,25]. TGF-b1 expression is induced by Ang II in cardiac cells [26,27]. Ang II inhibition has been reported to reduce ECM synthesis by modulating TGF-b1 expression in CFs and diabetic rats [28,29]. In the present study, levels of the TGF-b1 protein were significantly suppressed by C/EBPb overexpression or the valsartan treatment, whereas diabetes and sh-C/EBPb exacerbated fibrosis in vitro and in vivo by up-regulating TGF-b1 expression, indicating that C/EBPb overexpression reduces ECM deposition by decreasing Ang II-induced TGF-b1 expression.
An imbalance between ECM deposition and degradation in the heart plays a major role in fibrosis in DCM. The expression and activity of MMP-2 mediate collagen degradation and are down-regulated in diabetic mice and in CFs treated with HG or Ang II [7,25,30,31]. ACE2 overexpression reduces ECM deposition by increasing MMP-2 activity and expression [7]. MMP-2 primarily degrades collagen, which consists of fibrillary peptides and newly generated fibres. However, MMP-9, which is similar to MMP-2, also degrades collagen, although with lower proteolytic activity [25]. The sh-N.C. and sh-C/ EBPb treatment enhanced the reduced MMP-2 levels, whereas C/ EBPb overexpression increased MMP-2 expression and activity by up-regulating ACE2 expression, thus increasing the degradation of the ECM in mice with DCM. However, MMP-9 activity was not significantly altered among the groups in our study.
An increase in cardiomyocyte loss and hypertrophy underlies cardiac fibrosis and dysfunction in diabetes [32]. As expected, the Bax/ Bcl-2 ratio, which reflects the rate of apoptosis, was higher in diabetic mice and in HG-induced cardiomyocytes, consistent with previous findings [18]. Ang II triggers apoptosis and exacerbates cell growth, both of which are vital pathological features in DCM [33,34]. AT2R, which has controversial functions, was expressed at high levels in the diabetic mice and HG-induced CFs in our study. However, according to some studies, AT2R, like MasR, opposes AT1R activity and counteracts the negative effects induced by AT1R [35]. An increase in AT2R levels accompanied by a reduction in Bcl-2 levels was previously observed in DCM. Specific stimulation of AT2R after serum starvation exacerbates apoptosis [36]. In addition, AT2R exerts a proapoptotic effect on neonatal cardiomyocytes and R3T3 mouse fibroblasts [37]. Based on our results, AT2R promoted apoptosis in diabetes. C/EBPb overexpression not only decreased the Bax/Bcl-2 ratio but also reduced AT2R expression in DCM. Although the exact mechanism by which C/EBPb regulated AT2R was not investigated in the present study, our findings suggest a new method for studying apoptosis in DCM.
Based on our study and previous research, the mechanisms by which C/EBPb alleviated fibrosis and apoptosis were mainly due to the up-regulation of ACE2 expression and reduction of Ang II levels. In a previous study, ACE2 was recognized as an important modulator of DCM [38]. However, as an enzyme, the development of ACE2 as a clinical drug is very difficult due to its instability and ease of degradation. Therefore, future studies should focus on identifying a factor that induces ACE2 expression or activity. Using ChIP assays, we revealed a new regulatory mechanism in which C/EBPb binds to the ACE2 promoter sequence and enhances ACE2 production as a transcription factor. The HG treatment reduced the expression of C/ EBPb and its binding to the ACE2 promoter. Meanwhile, the expression of fibrosis markers was not reduced following treatment with C/ EBPb + ACE2-siRNA. Therefore, following up-regulation by C/EBPb, ACE2 catalyses the cleavage of Ang II to Ang(1-7) and inhibits ACE function. Ang(1-7) alleviates fibrosis and cardiac dysfunction by activating MasR, leading to reductions in ACE, AT1R and AT2R levels, as verified in previous studies [7,23]. However, we did not comprehensively examine whether the effects of C/EBPb suppression on fibrosis and apoptosis in DCM depended on ACE2 completely, and the new mechanism by which C/EBPb regulates DCM warrants further investigation.
Valsartan and other ARBs have been shown to prevent DCM. We also believe that the effects of valsartan and other ARBs on relieving DM are undeniable. However, ARBs have been shown to relieve diabetic complications by specifically blocking rather than circulating intracellular Ang II. Furthermore, intracellular Ang II is more relevant to fibroblasts in diabetic hearts, and several research studies have indicated that some effects of intracellular Ang II are not inhibited by ARBs [7,39]. In our study, C/EBPb up-regulated ACE2 expression, ACE2 catalysed the cleavage of Ang II to Ang(1-7) and decreased intracellular Ang II level and counteracted the effects of ACE on CFs and cardiomyocytes in ameliorating diabetic fibrosis and cardiac dysfunction, but valsartan had no effect on ACE2 expression [7]. As AT1R antagonists, valsartan only effects on AT1R and has no effect on AT2R and MasR according to its pharmacological action. C/EBPb up-regulated Ang(1-7), which decreased ACE, AT1R and AT2R levels, and up-regulated MasR expression levels [7,23], which ultimately prevented DCM progression, whereas valsartan increased Ang(1-7) levels much lower than C/EBPb. Overall, C/EBPb decreased intracellular Ang II level by up-regulating ACE2 and Ang (1-7) levels, indicating that C/EBPb may be more effective in treating DCM than ARBs.
In conclusion, we first investigated the role of C/EBPb as a transcription factor that promotes ACE2 expression and C/EBPb overexpression as a protective factor against fibrosis and apoptosis by upregulating ACE2 expression in DCM. Additionally, the underlying mechanisms involve the down-regulation of the ACE-Ang II-AT1R axis and the up-regulation of the ACE2-Ang(1-7)-MasR axis that directly suppressed collagen deposition and apoptosis. Furthermore, C/EBPb-induced ACE2 up-regulation is more efficacious in treating DCM than ARBs. Our study revealed a novel potential therapeutic target for the amelioration of cardiac dysfunction and remodelling in patients with DCM. | v3-fos-license |
2020-12-03T09:02:41.215Z | 2020-11-30T00:00:00.000 | 227261449 | {
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} | pes2o/s2orc | Synthesis, Biological, and Computational Evaluation of Antagonistic, Chiral Hydrobenzoin Esters of Arecaidine Targeting mAChR M1
Muscarinic acetylcholine receptors (mAChRs) are a pivotal constituent of the central and peripheral nervous system. Yet, therapeutic and diagnostic applications thereof are hampered by the lack of subtype selective ligands. Within this work, we synthesized and chemically characterized three different stereoisomers of hydrobenzoin esters of arecaidine by NMR, HR-MS, chiral chromatography, and HPLC-logP. All compounds are structurally eligible for carbon-11 labeling and show appropriate stability in Dulbecco’s phosphate-buffered saline (DPBS) and F12 cell culture medium. A competitive radioligand binding assay on Chinese hamster ovary cell membranes comprising the human mAChR subtypes M1-M5 showed the highest orthosteric binding affinity for subtype M1 and a strong influence of stereochemistry on binding affinity, which corresponds to in silico molecular docking experiments. Ki values toward M1 were determined as 99 ± 19 nM, 800 ± 200 nM, and 380 ± 90 nM for the (R,R)-, (S,S)-, and racemic (R,S)-stereoisomer, respectively, highlighting the importance of stereochemical variations in mAChR ligand development. All three stereoisomers were shown to act as antagonists toward mAChR M1 using a Fluo-4 calcium efflux assay. With respect to future positron emission tomography (PET) tracer development, the (R,R)-isomer appears especially promising as a lead structure due to its highest subtype selectivity and lowest Ki value.
Introduction
Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors, which are strongly involved in the parasympathetic nervous system's signal transduction. mAChRs appear as five subtypes, which differ in their expression pattern and downstream signaling [1]. However, their conserved orthosteric binding units hamper subtype selective molecular targeting [2]. Current anticholinergic drugs with market approval are mainly antagonists or inverse agonists [3]. Regarding therapeutic applications, mAChR agonists are in clinical use for the treatment of glaucoma [4], Sjögren's syndrome [4], and underactive bladder [5]. mAChR agonists have also been proposed as promising drugs for the treatment of Alzheimer's disease symptoms, hitherto, no compound passed clinical trial phase 3 [6]. Several mAChR antagonists targeting primarily the M1 subtype are used in Pharmaceuticals 2020, 13 medicine mainly for the treatment of Parkinson's disease [7] and peptic ulcers [8]. However, the clinical applicability of mAChR agonists and antagonists is often restrained by side effects related to the activation or inhibition of other mAChR subtypes [7]. Considering the insufficient penetration of the blood-brain barrier as well as unsuitable pharmacokinetics and -dynamics in treatment regimens of e.g., Parkinson's disease, these drugs often merely have peripheral effects on disease symptoms. Hence, as they do not act on the disease origin [9], they do not have the ability to alter disease progression. Therefore, clinical management is solely based on symptomatic treatment. The pivotal role of mAChRs in human physiology and their involvement in neuropathogenesis is well known, yet the underlying molecular mechanisms remain poorly understood [2]. A suitable positron emission tomography (PET) tracer targeting the mAChRs would allow for in vivo molecular imaging of mAChRs and hence strongly contribute to increase the knowledge in mAChR research. The basic understanding of the molecular mechanism is the only guide to establish personalized precision medicine based on image-guided therapy applications for central nervous system (CNS) diseases. Irrespective of numerous efforts, so far, no mAChR PET radiotracer has found widespread clinical application. Predominantly, this can be attributed to unsuccessful attempts to meet all the stringent requirements such as high affinity (ca. 3-50 nM) [10], subtype selectivity, metabolic stability, in vivo specific binding, and many more [11]. The requirements and parameters defining an effective and safe medication are highly dependent on the purpose (therapy or diagnosis) and the site of action (central or peripheral targets) and have to be considered already during the drug discovery process. In contrast to imaging agents, therapeutic CNS drugs allow less stringent attributes in terms of structure and binding affinity (µM to nM), but toxicology, functionality profile, and functional subtype selectivity are more in the spotlight [9,12].
Concluding, the repertoire of mAChR ligands for both therapy and diagnosis is characterized by a lack of high affinity and simultaneously subtype selective compounds. Diphenylmethyl esters of arecaidine (a and b, Figure 1) were recently discovered as orthosteric mAChR ligands with low nanomolar affinity and pronounced M1 selectivity. Unfortunately, strong nonspecific binding observed in the preclinical evaluation restrained their use as PET tracers [13]. Considering that nonspecific binding is often caused by hydrophobic interactions [14], this work aims to synthesize and evaluate similar compounds with reduced lipophilicity. In this study, we introduce the synthesis of chiral hydrobenzoin esters of arecaidine, which are fully characterized by nuclear magnetic resonance (NMR) spectroscopy and high resolution-mass spectrometry (HR-MS), and the purity was assessed by chiral and reversed phase-high performance liquid chromatography (HPLC). Physicochemical parameters as well as stability, affinity, and functionality toward mAChRs were assessed by in vitro methods for all synthesized compounds. In order to understand the difference in binding affinities, the binding poses of the novel compounds were assessed by in silico docking studies.
Pharmaceuticals 2020, 13, x FOR PEER REVIEW 2 of 12 phase 3 [6]. Several mAChR antagonists targeting primarily the M1 subtype are used in medicine mainly for the treatment of Parkinson's disease [7] and peptic ulcers [8]. However, the clinical applicability of mAChR agonists and antagonists is often restrained by side effects related to the activation or inhibition of other mAChR subtypes [7]. Considering the insufficient penetration of the blood-brain barrier as well as unsuitable pharmacokinetics and -dynamics in treatment regimens of e.g., Parkinson's disease, these drugs often merely have peripheral effects on disease symptoms. Hence, as they do not act on the disease origin [9], they do not have the ability to alter disease progression. Therefore, clinical management is solely based on symptomatic treatment. The pivotal role of mAChRs in human physiology and their involvement in neuropathogenesis is well known, yet the underlying molecular mechanisms remain poorly understood [2]. A suitable positron emission tomography (PET) tracer targeting the mAChRs would allow for in vivo molecular imaging of mAChRs and hence strongly contribute to increase the knowledge in mAChR research. The basic understanding of the molecular mechanism is the only guide to establish personalized precision medicine based on image-guided therapy applications for central nervous system (CNS) diseases. Irrespective of numerous efforts, so far, no mAChR PET radiotracer has found widespread clinical application. Predominantly, this can be attributed to unsuccessful attempts to meet all the stringent requirements such as high affinity (ca. 3-50 nM) [10], subtype selectivity, metabolic stability, in vivo specific binding, and many more [11]. The requirements and parameters defining an effective and safe medication are highly dependent on the purpose (therapy or diagnosis) and the site of action (central or peripheral targets) and have to be considered already during the drug discovery process. In contrast to imaging agents, therapeutic CNS drugs allow less stringent attributes in terms of structure and binding affinity (µM to nM), but toxicology, functionality profile, and functional subtype selectivity are more in the spotlight [9,12].
Concluding, the repertoire of mAChR ligands for both therapy and diagnosis is characterized by a lack of high affinity and simultaneously subtype selective compounds. Diphenylmethyl esters of arecaidine (a and b, Figure 1) were recently discovered as orthosteric mAChR ligands with low nanomolar affinity and pronounced M1 selectivity. Unfortunately, strong nonspecific binding observed in the preclinical evaluation restrained their use as PET tracers [13]. Considering that nonspecific binding is often caused by hydrophobic interactions [14], this work aims to synthesize and evaluate similar compounds with reduced lipophilicity. In this study, we introduce the synthesis of chiral hydrobenzoin esters of arecaidine, which are fully characterized by nuclear magnetic resonance (NMR) spectroscopy and high resolution-mass spectrometry (HR-MS), and the purity was assessed by chiral and reversed phase-high performance liquid chromatography (HPLC). Physicochemical parameters as well as stability, affinity, and functionality toward mAChRs were assessed by in vitro methods for all synthesized compounds. In order to understand the difference in binding affinities, the binding poses of the novel compounds were assessed by in silico docking studies.
Chemistry
Hydrobenzoin esters of arecaidine were prepared and characterized by NMR spectroscopy, HR-MS, logP, RP-HPLC, and chiral HPLC. The esterification of (R,R)-and (S,S)-hydrobenzoin (d, f) yielded the Pharmaceuticals 2020, 13, 437 3 of 12 enantiomerically pure esters 1 and 2, whereas esterification with meso-hydrobenzoin (g) yielded racemic 3 ( Figure 2). Previously, arecaidine esters were prepared in low yields via 1,1 -carbonyldiimidazole (CDI)-mediated esterification [13]. Within this work, we describe a transesterification procedure from the readily available arecaidine methyl ester (e, arecoline). Regarding the synthesis of hydrobenzoin esters, the transesterification (synthesis of 2) is characterized by a 70% increased yield and less experimental effort compared to the CDI-mediated esterification (synthesis of 1). The vicinal coupling constant of the hydrobenzoin part is the most notable difference in NMR spectra between 1 or 2 (7.2 Hz) to 3 (5.8 Hz). Chiral HPLC confirmed that no racemization occurred during both methods (Supporting Information).
Chemistry
Hydrobenzoin esters of arecaidine were prepared and characterized by NMR spectroscopy, HR-MS, logP, RP-HPLC, and chiral HPLC. The esterification of (R,R)-and (S,S)-hydrobenzoin (d, f) yielded the enantiomerically pure esters 1 and 2, whereas esterification with meso-hydrobenzoin (g) yielded racemic 3 ( Figure 2). Previously, arecaidine esters were prepared in low yields via 1,1′carbonyldiimidazole (CDI)-mediated esterification [13]. Within this work, we describe a transesterification procedure from the readily available arecaidine methyl ester (e, arecoline). Regarding the synthesis of hydrobenzoin esters, the transesterification (synthesis of 2) is characterized by a 70% increased yield and less experimental effort compared to the CDI-mediated esterification (synthesis of 1). The vicinal coupling constant of the hydrobenzoin part is the most notable difference in NMR spectra between 1 or 2 (7.2 Hz) to 3 (
Evaluation of Physico-Chemical Parameters and Stability
HPLC-logP ow pH7.4 values were found to be 2.24 ± 0.12 (1=2) and 2.39 ± 0.10 (3), which are much lower compared to arecaidine diphenylmethyl ester (3.32 ± 0.04) [13]. Consequently, 1, 2, and 3 are expected to be less prone to nonspecific binding. The predictive power of logP for blood-brain barrier permeability is controversially discussed, and several desirable logP ranges were postulated. However, the HPLC-logP ow pH7.4 values of 1, 2, and 3 comply with every single favorable logP range compared recently [15]. In addition, other chemical characteristics of 1, 2, and 3, such as molecular weight (337.42 g/mol), number of hydrogen bonds (3 acceptors, 1 donor), polar surface area (49.8 Å 2 ), rotatable bond count (6), and possession of a tertiary nitrogen with a positive charge at pH 7-8 (calculated pKa = 7.8) are in good accordance with desired properties for CNS active drugs [16]. Furthermore, analysis by SwissADME [17] indicates that 1, 2, and 3 are no P-glycoprotein substrates and no PAINS [18] or Brenk [19] alerts were caused. The stability of 1, 2, and 3 in buffer and cell culture medium was investigated because their disintegration could impair biological testing. 1, 2, and 3 show a comparable rate of decomposition in Dulbecco's phosphate-buffered saline (DPBS) and Ham's F12 cell culture medium at 37 • C over a time period of 60 h (Figure 3), which is slow enough to allow for biological testing and drug development. For 1, 2, and 3, the decomposition is faster in Ham's F12 cell culture medium compared to DPBS. After 60 h in DPBS 1, 2, and 3 were decomposed to the same extent (not significantly different in Sidak's multiple comparisons test, α = 0.05). However, the decomposition of 1 in Ham's F12 cell culture medium was significantly stronger compared to 2 (p ≤ 0.001) and 3 (p ≤ 0.01). The different stability of the enantiomers 1 and 2 in Ham's F12 cell culture medium can be attributed to its numerous chiral constituents. Enantioselectivity in the presence of chiral amino acids is frequently observed in organic reactions; still, an unequivocal mechanism often remains obscure [20]. For the potential application as a carbon-11 PET tracer, compounds should exhibit a reasonable stability for at least six half-lifes (i.e., 2 h). The determined stabilities of 1, 2, and 3 support further development as a carbon-11 PET tracer, as a linear interpolation between the 0 h and 10 h indicates that only 0.8% and 2.5% are decomposed after 2 h in DPBS and Ham's F12 cell culture medium, respectively.
The stability of 1, 2, and 3 in buffer and cell culture medium was investigated because their disintegration could impair biological testing. 1, 2, and 3 show a comparable rate of decomposition in Dulbecco's phosphate-buffered saline (DPBS) and Ham's F12 cell culture medium at 37 °C over a time period of 60 h (Figure 3), which is slow enough to allow for biological testing and drug development. For 1, 2, and 3, the decomposition is faster in Ham's F12 cell culture medium compared to DPBS. After 60 h in DPBS 1, 2, and 3 were decomposed to the same extent (not significantly different in Sidak's multiple comparisons test, α = 0.05). However, the decomposition of 1 in Ham's F12 cell culture medium was significantly stronger compared to 2 (p ≤ 0.001) and 3 (p ≤ 0.01). The different stability of the enantiomers 1 and 2 in Ham's F12 cell culture medium can be attributed to its numerous chiral constituents. Enantioselectivity in the presence of chiral amino acids is frequently observed in organic reactions; still, an unequivocal mechanism often remains obscure [20]. For the potential application as a carbon-11 PET tracer, compounds should exhibit a reasonable stability for at least six half-lifes (i.e., 2 h). The determined stabilities of 1, 2, and 3 support further development as a carbon-11 PET tracer, as a linear interpolation between the 0 h and 10 h indicates that only 0.8% and 2.5% are decomposed after 2 h in DPBS and Ham's F12 cell culture medium, respectively.
In Silico Docking Experiments
Based on the in silico docking experiments, the enantiomers 1 and 2 feature substantially different binding poses ( Figure 4). In addition to the hydrophobic interactions and the ionic Asp105 interaction, both enantiomers show hydrogen bonds to two amino acid side chains. However, in the predicted binding pose of 1, the position of the hydroxyl group enables it to act as a hydrogen bond donor and acceptor to Asn382, which is an amino acid that is known to be strongly involved in the binding of high affinity antagonists [21]. Conclusively, 1 exhibits an additional hydrogen bond interaction compared to 2, which can be seen as an indicator for higher affinity.
In Silico Docking Experiments
Based on the in silico docking experiments, the enantiomers 1 and 2 feature substantially different binding poses (Figure 4). In addition to the hydrophobic interactions and the ionic Asp105 interaction, both enantiomers show hydrogen bonds to two amino acid side chains. However, in the predicted binding pose of 1, the position of the hydroxyl group enables it to act as a hydrogen bond donor and acceptor to Asn382, which is an amino acid that is known to be strongly involved in the binding of high affinity antagonists [21]. Conclusively, 1 exhibits an additional hydrogen bond interaction compared to 2, which can be seen as an indicator for higher affinity.
Biological Evaluation
The expected orthosteric binding of 1, 2, and 3 could be confirmed using [N-methyl-3 H]scopolamine methyl chloride ([ 3 H]NMS) in a competitive radioligand binding assay toward all mAChR subtypes. All competitive binding curves show a monophasic logistic behavior and justify the use of a one-site model (supporting information, Figure S1) [22]. Comparing the subtypes, all tested compounds show the highest affinity toward M1 followed by M5 and the lowest affinity to M2 (Table 1). However, the methyl ester of the free acid of 1, 2, and 3 (e, arecoline) shows pronounced mAChR M2 affinity [13], which illustrates the influence of chemical derivatization on subtype selectivity. 1 is considered most promising because it displays the highest affinity to the preferred M1
Biological Evaluation
The expected orthosteric binding of 1, 2, and 3 could be confirmed using [N-methyl-3 H]scopolamine methyl chloride ([ 3 H]NMS) in a competitive radioligand binding assay toward all mAChR subtypes. All competitive binding curves show a monophasic logistic behavior and justify the use of a one-site model (supporting information, Figure S1) [22]. Comparing the subtypes, all tested compounds show Pharmaceuticals 2020, 13, 437 5 of 12 the highest affinity toward M1 followed by M5 and the lowest affinity to M2 (Table 1). However, the methyl ester of the free acid of 1, 2, and 3 (e, arecoline) shows pronounced mAChR M2 affinity [13], which illustrates the influence of chemical derivatization on subtype selectivity. 1 is considered most promising because it displays the highest affinity to the preferred M1 subtype. Furthermore, 1 shows the strongest subtype selectivity against all subtypes (M1 over M2, M3, M4, and M5) and the highest overall selectivity between M1 and M2 (19-fold) within the tested compounds. Especially, the 7-fold M1 over M4 subtype selectivity has to be mentioned. Although it is the second lowest selectivity compared to the other receptor subtypes, it is even higher than found in pirenzepine (3.5-fold) and trihexyphenidyl (1.6-fold) [23]. The superior mAChR M1 affinity of the (R,R)-isomer 1 over the (S,S)-isomer 2 corresponds well to the in silico docking experiments. In terms of mAChR M1 minimum subtype selectivity, 1 performs better than the clinically established mAChR M1 antagonists pirenzepine and trihexyphenidyl ( Figure 5) and also better than our recent top candidates for mAChR M1 PET imaging [13]. Given the lower affinity of 1 compared to pirenzepine, antagonistic mAChR M1 treatment with 1 would require a higher dose, which might be well tolerated due to the broader M1 subtype selectivity. Compounds featuring two aromatic rings are very prominent in mAChR drug development. However, to the best of our knowledge, the hydrobenzoin scaffold has only been utilized in a single publication on muscarinic ligand development [24]. In that publication, compounds 18a-c contain the hydrobenzoin scaffold integrated in a 1,4-dioxane ring and also show the highest affinity toward subtype M1. Still, subtype selectivity is narrower compared to the compounds presented herein.
Pharmaceuticals 2020, 13, x FOR PEER REVIEW 6 of 12 integrated in a 1,4-dioxane ring and also show the highest affinity toward subtype M1. Still, subtype selectivity is narrower compared to the compounds presented herein. The significantly different binding affinities observed between the three stereoisomers underline the general possibility to increase mAChR affinity and subtype selectivity of arecaidine esters via stereochemical variations. In view of the vast majority of achiral small molecules, which have been studied as potential mAChR PET tracers [10], we believe that stereochemistry is a useful yet underappreciated tool to optimize compound properties. In addition to the assessment of the affinity by means of a radiometric competition binding assay [25] on cellular membranes, the functionality of the synthesized compounds was further evaluated in a cell-based fluorescence assay using Fluo-4 [26]. In contrast to the mAChR agonist carbachol, no increase in calcium release could be induced by incubation with 1, 2, and 3 using commercially Figure 5. Subtype selectivities related to M1 visualized as radar charts. For each substance, the logarithmic axis represents K i toward the respective subtype divide by the K i of M1. Compared to the clinically established M1 antagonists pirenzepine and trihexyphenidyl, 1 shows a pronounced subtype selectivity toward M4, which results in overall broader subtype selectivity. Selectivities of pirenzepine and trihexyphenidyl were calculated based on their literature reported K i values [23]. The significantly different binding affinities observed between the three stereoisomers underline the general possibility to increase mAChR affinity and subtype selectivity of arecaidine esters via stereochemical variations. In view of the vast majority of achiral small molecules, which have been studied as potential mAChR PET tracers [10], we believe that stereochemistry is a useful yet underappreciated tool to optimize compound properties.
In addition to the assessment of the affinity by means of a radiometric competition binding assay [25] on cellular membranes, the functionality of the synthesized compounds was further evaluated in a cell-based fluorescence assay using Fluo-4 [26]. In contrast to the mAChR agonist carbachol, no increase in calcium release could be induced by incubation with 1, 2, and 3 using commercially available Fluo-4 direct calcium flux assay on stably transfected CHO-M1 cells (Figure 6, Panel A). Applying compounds 1, 2, 3, and scopolamine as positive control prior to the treatment with a constant concentration of the signal inducing carbachol clearly illustrates the antagonistic binding of the tested compounds ( Figure 6, Panel B). The progression of the effect-concentration curve of both positive controls, carbachol for the agonistic assay and scopolamine for the antagonistic assay, are in good accordance with the results depicted by the manufacturer. Figure 5. Subtype selectivities related to M1 visualized as radar charts. For each substance, the logarithmic axis represents Ki toward the respective subtype divide by the Ki of M1. Compared to the clinically established M1 antagonists pirenzepine and trihexyphenidyl, 1 shows a pronounced subtype selectivity toward M4, which results in overall broader subtype selectivity. Selectivities of pirenzepine and trihexyphenidyl were calculated based on their literature reported Ki values [23].
In addition to the assessment of the affinity by means of a radiometric competition binding assay [25] on cellular membranes, the functionality of the synthesized compounds was further evaluated in a cell-based fluorescence assay using Fluo-4 [26]. In contrast to the mAChR agonist carbachol, no increase in calcium release could be induced by incubation with 1, 2, and 3 using commercially available Fluo-4 direct calcium flux assay on stably transfected CHO-M1 cells (Figure 6, Panel A). Applying compounds 1, 2, 3, and scopolamine as positive control prior to the treatment with a constant concentration of the signal inducing carbachol clearly illustrates the antagonistic binding of the tested compounds ( Figure 6, Panel B). The progression of the effect-concentration curve of both positive controls, carbachol for the agonistic assay and scopolamine for the antagonistic assay, are in good accordance with the results depicted by the manufacturer. The following day, cells were assayed for a calcium response to carbachol (positive control), 1, 2, and 3 using the Fluo-4 Direct™ Calcium Assay Kit. Cells were stimulated for potential agonistic activity (panel A) [27] or cells were treated for potential antagonistic activity, scopolamine (positive control), 1, 2, and 3 to block the calcium response elicited by 20 µM carbachol (panel B). Measurements are given in Figure 6. Dose-dependent calcium response to muscarinic 1 (M1) receptor agonists and antagonists. CHO-M1 cells were plated in black clear bottom 96-well plate and incubated overnight. The following day, cells were assayed for a calcium response to carbachol (positive control), 1, 2, and 3 using the Fluo-4 Direct™ Calcium Assay Kit. Cells were stimulated for potential agonistic activity (panel A) [27] or cells were treated for potential antagonistic activity, scopolamine (positive control), 1, 2, and 3 to block the calcium response elicited by 20 µM carbachol (panel B). Measurements are given in relative fluorescent units (RFU) as the maximum response minus the minimum response divided by the minimum response.
1 has a high binding affinity (K i -value) to the M1 receptor of 99 ± 19 nM, as assessed by means of the radioligand binding assay, being 2-fold out of the postulated ideal range [10]. As numerous mAChR ligands failed in PET radiotracer development due to too high binding affinity, being a disadvantage in radiotracer kinetics causing flow dependence of tracer accumulation [10], this "borderline" binding affinity should not be a common ground for excluding 1 from further development toward diagnostic application, especially when considering that the targeted mAChR subtype M1 is the most abundant subtype in the cortex [28]. Furthermore, 1 may serve as a lead structure for the development of derivatives with even higher affinity, retaining its suitable selectivity profile and moderate lipophilicity values.
It is an ongoing debate as to whether PET radiotracer targeting GPCRs are preferably agonist or antagonists [29] Agonists per se complicate the PET image analysis as they favorably bind to the activated form of GPCRs. The global picture shows a prevalence for antagonists carried by the easier access and unlikeliness of side effects. Considering the physico-chemical parameters, binding affinities, subtype selectivity, and effective functionality of the here presented subset of compounds, the potential application points into the direction as diagnostic imaging probes over a potential therapeutic application. Potential application as an imaging probe is further supported by the expected ease of carbon-11 radiolabeling of 1, considering that structurally related arecaidine esters were previously labeled with carbon-11 straightforwardly via reaction of [ 11 C]CH 3 I with N-desmethyl Pharmaceuticals 2020, 13, 437 7 of 12 precursors [13]. The observed antagonism is advantageous for PET radiotracers, as potential side effects are unlikely. Especially, compound 1 stands out with binding affinities in the nanomolar range and high subtype selectivity, proving the importance of stereochemical adjustments for successful mAChR ligand development.
Conclusions
Subtype selectivity represents an ambitious but necessary goal in the development of orthosteric mAChR ligands for therapeutics as well as for diagnostics. Within this work, we synthesized chiral hydrobenzoin esters of arecaidine and investigated their stability and binding properties toward mAChRs. Stereochemistry was shown to have a strong impact on in vitro mAChR M1 affinity, which was also anticipated by in silico molecular docking. Furthermore, the variable affinity toward the other subtypes highlights the relevance of chiral ligands in the quest for subtype selectivity. The antagonistic (R,R)-isomer 1 shows most promising characteristics to act as a suitable mAChR M1 PET tracer and especially stands out because of its broad subtype selectivity compared to clinically established mAChR M1 antagonists. These properties motivate us to radiolabel 1 with carbon-11 in the future to further evaluate its applicability as brain PET tracer.
Materials
Arecaidine was prepared according to the literature [30]
Instrumentation
NMR samples were measured in deuterated chloroform (CDCl 3 , ≥99.8%, stabilized with silver foil, Sigma Aldrich) at 25 • C on a Bruker Avance III 400 spectrometer (400 MHz for 1 H and 100 MHz for 13 C). An assignment of NMR signals was achieved by a combination of standard NMR techniques, such as COSY, NOESY, APT, HSQC, and HMBC experiments. The residual CHCl 3 signal was set to 7.26 ppm for 1 H spectra and 77.0 ppm for 13 C spectra. Coupling constants are given in Hz. Mass spectra were obtained on a Bruker maXis 4G instrument (ESI-TOF, HR-MS). Optical rotation was determined in CHCl 3 using a Schmidt + Haensch UniPol L2000 polarimeter with a 1dm cell. An Agilent HPLC system consisting of an autosampler (series 1100), pump (series 1200) and diode array detector (series 1100) was used for the determination of logP using an apHERA (5 µm, 10 × 6 mm) stationary phase [15]. Compounds 1-3 were shown to exceed a purity of 95% by gradient run using the logP HPLC method without the addition of toluene and triphenylene and by isocratic runs on an XSelect TM (HSS T3, 3.5 µm, 100 × 4.6 mm) stationary phase at a flow of 1 mL/min (supporting information, Figure S2). Fluorescence measurements were performed on a BioTek Synergy HTX multi-mode reader. A M-36 tygon tubed Cell Harvester (Brandel ® ) and Whatman™ GF/B filters were used for the filtration of radioligand binding assays. Filter disks were counted with 2 mL Ultima Gold™ (high flashpoint LSC cocktail, PerkinElmer) using a 300 SL Automatic TDCR liquid Scintillation Counter (HIDEX). stationary phase and 40% acetonitrile in 25 mM (NH 4 )H 2 PO 4 buffer (pH 9.3) at a flow of 1 mL/min (k 1,2 = 5.6, k 3 = 5.9). The fraction of stable compounds was calculated as the area under the curve of the compound peak for each combination of compound and matrix related to the 0 h time point with 100%. Three non-consecutive HPLC analyses were performed for each time point, combination of compounds, and matrix.
Molecular Docking Studies
Molecular docking was performed on a crystal structure of human mAChR M1 [31] using AutoDock 4.2 with default settings in LigandScout 4.4_RC6. 1 and 2 were docked as protonated tertiary amines to consider the expected species under physiological conditions. A pharmacophore of the binding poses exhibiting the expected ionic interaction to Asp105 was created to visualize proposed interactions of 1 and 2 to amino acid side chains.
Affinity Assay Toward mAChRs M1-M5 on CHO Membranes
Cell membranes bearing human mAChRs M1-M5 were prepared as described previously [13]. In short, stably transfected CHO cells were grown to confluence in T175 flasks, washed with ice-cold DPBS, and suspended in 2 mL 10 mM Tris/HCl, 1 mM EDTA-buffer (pH 7.4), and 200 µL protease inhibitor using a cell scraper. A cell homogenate was prepared by passing the cell suspension through a G29 needle and subsequently centrifuged (10 min, 1000× g, 4 • C). Ultracentrifugation of the supernatant (30 min, 100,000× g, 4 • C) yielded a membrane pellet, which was suspended in 125 µL 50 mM Tris/HCl-buffer (pH 7.4) per T175 flask and stored at −80 • C. Ten confluent T175 flask were processed for one batch.
Inhibition constants (K i ) were determined with a competitive radioligand binding assay using 50 mM Tris/HCl, 10 mM MgCl 2 , and 1 mM EDTA (pH 7.4) as assay buffer. Then, 5 µL of test compound in DMSO, 50 µL of [N-methyl-3 H]scopolamine methyl chloride ([ 3 H]NMS) in assay buffer, and 445 µL of membrane suspension diluted in assay buffer were incubated for 90 min at 23 • C. The membrane-bound activity was recovered by filtration through GF/B filter paper pre-soaked in 0.1 % PEI and measured with a liquid scintillation counter. Maximum binding was measured by using 5 µL plain DMSO, and nonspecific binding was measured by using 5
Calcium Efflux Assay for Agonist/Antagonists Discrimination
For the Fluo-4 direct TM calcium assay kit (invitrogen), 100 µL of a 5 × 10 5 cells/mL suspension of CHO-M1 cells were seeded in black clear bottom 96-well plates (Corning). After settling of the cells for 24 h, the kit was performed according to the supplier's recommendation. In detail, the media was Pharmaceuticals 2020, 13, 437 9 of 12 removed, and 50 µL of Hanks-balanced salt solution (HBSS; Sigma Aldrich) were added, followed by 50 µL of the Fluo-4 buffer solution (including probenecid). The 96-well plates were incubated for 60 min at 37 • C in the dark. For the agonist assay, 100 µL of a double-concentrated dilution series of carbachol (positive control), 1, 2, and 3 were added with the end concentration of 100, 10, 1, 0.1, 0.01 and 0.001 µM in duplicates. The relative fluorescence was measured with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. For the antagonist assay, 50 µL of a 4-fold concentrated dilution series of scopolamine hydrochloride (positive control), 1, 2, and 3 were added in duplicates. Subsequently a 40 µM stock solution of carbachol was added to all wells, and the relative fluorescence was measured with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. Stock solution of 1, 2, and 3 were in DMSO with a final concentration of at most 1% of DMSO. All experiments were performed at least in three repetitions.
Calculations and Statistics
If not indicated otherwise, all values are depicted as mean ± standard deviation. All quantitative results were analysed by means of MS Excel ® 2013. Significance was tested with GraphPad Prims 6 (version 6.07, San Diego, CA, USA). of activated 5 Å molecular sieves were added subsequently. The resulting mixture was refluxed for 16 h. Then, it was filtered through a pad of celite, and volatiles were removed under reduced pressure. The crude oil was purified via preparative TLC (EtOAc/n-heptane, 70:30 with 3% Et 3 N) to give the desired product in 16% yield (46 mg). 1 Figure S2: Isocratic HPLC chromatograms of compounds 1-3. 35-40% ACN in 25 mM NH 4 H 2 PO 4 buffer pH 9.3 at a flow of 1 mL/min, Figure S3: Chiral chromatography using a an AGP 0.3 cmØ × 5cm 5 µM column operated with 2% IPA in 10 mM NH 4 Ac pH 5.8 at a flow of 0.5 mL/min, Figure S4: 1 H-and 13 C-NMR spectrum of 1, Figure S5: 1 H-and 13 C-NMR spectrum of 2. Figure S6 | v3-fos-license |
2020-01-20T15:31:37.831Z | 2020-01-20T00:00:00.000 | 210717175 | {
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} | pes2o/s2orc | Nickel-catalyzed allylic carbonylative coupling of alkyl zinc reagents with tert-butyl isocyanide
Transition metal-catalyzed carbonylation with carbon nucleophiles is one of the most prominent methods to construct ketones, which are highly versatile motifs prevalent in a variety of organic compounds. In comparison to the well-established palladium catalytic system, the nickel-catalyzed carbonylative coupling is much underdeveloped due to the strong binding affinity of CO to nickel. By leveraging easily accessible tert-butyl isocyanide as the CO surrogate, we present a nickel-catalyzed allylic carbonylative coupling with alkyl zinc reagent, allowing for the practical and straightforward preparation of synthetically important β,γ-unsaturated ketones in a linear-selective fashion with excellent trans-selectivity under mild conditions. Moreover, the undesired polycarbonylation process which is often encountered in palladium chemistry could be completely suppressed. This nickel-based method features excellent functional group tolerance, even including the active aryl iodide functionality to allow the orthogonal derivatization of β,γ-unsaturated ketones. Preliminary mechanistic studies suggest that the reaction proceeds via a π-allylnickel intermediate.
P ioneering work developed by Heck in the 1970s catapulted palladium (Pd)-catalyzed three component reactions with carbon monoxide (CO) as a powerful strategy for introduction of carbonyl group 1-8 . Among these processes, the use of carbon-based nucleophiles allows for the convenient synthesis of ketones with broad applications 9 . Pd-catalyzed allylic reaction represents one of the most prominent carbon-carbon bondforming reactions with wide synthetic applications in organic chemistry, including synthesis of various biologically active natural products, pharmaceuticals, and agrochemicals [10][11][12][13] . Thus, the allylic carbonylation would be an important strategy for incorporation of both alkene and carbonyl functionality in one synthetic protocol, enabling the expedient synthesis of the versatile β,γ-unsaturated ketones, which are ubiquitous motifs in bioactive compounds and utilized as valuable synthetic building blocks [14][15][16][17][18][19][20] . The Stille group has realized the Pd-catalyzed allylic carbonylative Stille coupling, while the organotin reagents largely limited on aryl, vinyl, and allyl stannanes 21,22 . The Tamaru group has previously developed a Pd-catalyzed allylic carbonylative Negishi coupling with CO to access β,γ-unsaturated ketones 23,24 . However, the site selectivity of this process is highly dependent on the electronics of the organozinc coupling partner, and a mixture of the linear and branched coupling products were usually obtained when simple alkyl Negishi reagents were used (Fig. 1a). Despite the progress in Pd-catalyzed three component reactions with CO gas, it is still highly imperative to develop practical carbonylation utilizing earth abundant transition metal 25,26 for functionalized ketone synthesis, especially to circumvent the long-term limitations.
At the outset of our investigation, we recognized several issues that needed to be addressed in order to develop an effective nickel-catalyzed allylic carbonylation. First, the use of nickel catalysts in carbonylation reactions has been less explored likely due to the strong binding affinity of CO towards nickel [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] . As a rare example, the Skrydstrup group elegantly succeeded the catalytic carbonylation of primary benzylic electrophile using a nickel pincer complex with a CO-gen precursor via the slow addition of Negishi reagent to circumvent the direct Negishi coupling, although the use of allyl electrophiles received limited success with 8% ketone formation 51 . Additionally, the preference of imidoylnickel intermediates to undergo further migratory insertion with isocyanides to furnish poly(iminomethylene)s is a well-known process in polymer chemistry [54][55][56][57] , presenting a major hurdle to access monocarbonylated products. Furthermore, Zhu and co-workers 58 illustrate that the allyl imidoylpalladium intermediate is extremely prone to undergo β-H elimination to provide the ketenimine intermediate, which could further hydrolyze to β,γ-unsaturated amide 59 . To overcome these challenges, we envisioned that the use of functional group compatible organozinc reagents that are highly effective for transmetallation might disfavor the overcarbonylation process. Rapid C−C bond formation via reductive elimination would provide the monocarbonylated coupling product 60,61 . Moreover, the β,γ-unsaturated ketone easily undergoes the undesired isomerization step to afford the thermodynamically stable α,β-unsaturated ketone in the presence of transition metal catalyst 62 . Herein, we report the highly regio-and chemoselective nickel-catalyzed allylic carbonylative Negishi reaction with tert-butyl isocyanide 63 , which allows the expedient synthesis of β,γ-unsaturated ketones with broad substrate scope under mild conditions (Fig. 1c).
Results
Optimization of reaction conditions. We started our investigation by studying the nickel-catalyzed reaction of phenyl allyl NiCl 2 ·DME dppf 5 ND ND 4 NiCl 2 ·DME Phen 82 3 3 5 NiCl 2 ·DME SIPr·HCl 87 acetate 1a and n C 8 H 17 ZnBr (1.5 equiv). To our delight, when the commercially available tert-butyl isocyanide (1.5 equiv) was used as the CO equivalent, the reaction proceeded smoothly with 10 mol% bench-stable and easily accessible NiCl 2 ·DME in dimethylacetamide (DMA) at 25°C. The starting material was consumed in 30 min; affording the desired trans β,γ-unsaturated ketone 4a in 86% isolated yield after the simple acidic work up procedure, only small amount of direct coupling byproduct 6 (1%) could be observed at gas chromatography (GC) ( Table 1, entry 1). This nickel-isocyanide system features excellent linear selectivity. Neither the branched nor the secondary allylic products as observed in previous work 23 were detected. Ligand-free nickel species was found to be the most effective for this coupling process. The use of phosphine ligands including PPh 3 and other bidentate phosphines such as dppf (1,1′-ferrocenediyl-bis(diphenylphosphine)) was detrimental to this reaction, β,γ-unsaturated amide byproduct 5 was detected by GC (Table 1, entries 2 and 3). The use of 1,10-phenathorine also lowered the yield (Table 1, entry 4), and the inclusion of N-heterocyclic carbene ligand was also not beneficial ( Substrate scope of alkyl zinc reagents. With the optimized conditions in hand, we next explored the substrate scope of alkylated zinc nucleophiles (Fig. 2). The reaction tolerates a wide range of organozinc reagents, affording β,γ-unsaturated ketones with complete trans-selectivity. Furthermore, the undesired isomerization of alkene moiety was not detected. Diethyl zinc could be successfully applied in this carbonylation process with 84% isolated yield (4b); however, requiring sacrifice one equivalent alkyl source. Simple alkyl groups including methyl (4c), benzyl (4d), and 2-phenylethyl (4e) could be incorporated into the products in excellent yield. The alkene moiety was compatible in this carbonylative process, and homoallyl (4f) and prenyl (4g) substituted β,γ-unsaturated ketones could be formed in good yield with excellent regio-and stereoselectivity. Additionally, various functionalized alkylzinc reagents, including those possessing a fluoride (4h), a chloride (4i), a nitrile (4j), an ester (4k), and an ether (4l, 4m), were successfully converted to the corresponding ketone product. Moreover, secondary cyclopentyl zinc bromide could also serve as the nucleophile to afford 4p in moderate yield. The utilization of nickel catalyst also tolerated the halogen-containing (hetero)benzyl nucleophiles (4n and 4q-4s), allowing further functionalization to be carried out. Unfortunately, the use of the tertiary alkylzinc and arylzinc reagent did Substrate scope of allylic electrophiles. The scope of allylic electrophiles was also investigated, the reaction proceeded well with both aryl and alkyl substituted alkene, affording the carbonylation product in 43-96% yield with excellent regio-and stereoselectivities (Fig. 3). Aryl substituents including 2-Br (4v), 4-I (4w), and 4-Bpin (4x) were tolerated, providing the feasibility for subsequent derivatization. The employment of heteroaromatic ring including thiophene (4y), furan (4z), and indole (4aa) also provided the product in high isolated yield. When a methyl group was introduced at the C-2 position of the allyl electrophile (4ae), the reaction also proceeded with excellent E/Z selectivity. The migratory insertion of allylic nickel intermediate with isocyanide mainly proceeded in the least sterically hindered position, furnishing the trans ketone 4af-4ah in good yield.
To illustrate potential utility of this nickel-catalyzed threecomponent carbonylative coupling, we performed late-stage modifications on complex and/or biologically active compounds (Fig. 4). For instance, several pharmaceutical derivatives, naproxen (4aj), ibuprofen (4ak), hyodeoxycholic acid (4am), and indometacin (4an) could be readily applied in this reaction, affording the desired product in good yield. To our delight, the olefin containing citronellal (4ai) and oleic acid (4al) both could be effectively transformed to relatively β,γ-unsaturated ketones in regioselective way, while no alkene isomerization could be observed in the standard condition. Notably, vitamin-E (4ao) derivative is also amenable and the desired product could be obtained in moderate yield. These results clearly demonstrate that this carbonylation has promising applications in late-stage modification of complex molecules.
Synthetic applications. To further showcase the synthetic potential for this nickel catalyzed carbonylative Negishi reaction using tert-butyl isocyanide as carbonyl source, the gram-scale synthesis of 4t was carried out with 90% isolated yield (Fig. 5a). The imine intermediate could be reduced using NaBH 4 to provide the useful building block homoallylic amine 7 (83% yield), further demonstrating the advantage of this isocyanide coupling technology (Fig. 5b). The tolerance of active aryl halide under current conditions allows the orthogonal cross-coupling strategy (Fig. 5c). Pd-catalyzed Suzuki coupling of (E)-3-(4-iodophenyl)allyl acetate 1w with phenylboronic acid afforded intermediate 1ap, which could further undergo the allylic carbonylation with benzyl zinc reagent to provide β,γ-unsaturated ketones 4ap in 58% overall yield. Meanwhile, β,γ-unsaturated ketones 4w could be obtained Mechanistic studies and proposed mechanism. To shed light on the reaction mechanism, several experiments were performed (Fig. 6). When 1-phenylallyl acetate 1a′ was used as the allylic electrophile, the linear β,γ-unsaturated ketone 4t was obtained as the sole product in 88% yield. Additionally, when allyl acetate containing a cyclopropyl group (1ar) was subjected to the standard conditions, ring-opening product of the cyclopropyl group could not be detected (Fig. 6a). Together, these results indicate that the reaction might proceed via the πallylnickel intermediate, and the radical intermediates is unlikely involved. The stereochemistry of this carbonylative coupling reaction was also examined by using (R)-1af as the starting material (Fig. 6b): the desired product 4af was obtained with 75% ee (enantiomeric excess) with the inversed configuration, which indicated that the initiate step most likely undergo SN2 type in the oxidative addition process similar to the Tsuji-Trost-type oxidation [64][65][66] . The erosion of ee may arise from the tautomerization between imine and enamine intermediate in the acidic work up procedure. The tert-butyl isocyanide possesses strong binding affinity to the nickel center, we prepared the tetrakis(tert-butyl isocyanide)nickel (II) perchlorate complex 8. Under standard condition, using 0.38 equiv 8 in the absence of additional t BuNC delivered the desired product in 57% yield. In contrast, using 10 mol% 8 with the addition of 1.5 equiv t BuNC increased the yield to 82%. These results suggest that the binding of multiple t BuNC as ligands to the nickel catalyst may proceed with ligand dissociation process in the catalytic cycles and slow down the coupling process (Fig. 6c). When we preform the [1-(tert-butylimino)-butyl]zinc chloride, which is prepared in situ via the reaction of t BuNC and n BuLi followed by transmetallation with ZnCl 2 at room temperature, the desired product 4t was not observed under our conditions, in contrast to the previous findings of Ito and coworkers 67 and Dechert-Schmitt et al. 33 , where high temperature is essential for carbonylative coupling (Fig. 6d). This result highlights the distinctive mechanism in the Ni-catalyzed carbonylative Negishi coupling reaction.
Based on the preliminary mechanistic studies, a plausible reaction pathway is proposed in Fig. 7. Oxidative addition of allyl acetate 1 with the nickel catalyst affords the π-allylnickel(II) intermediate A. Migratory insertion of tert-butyl isocyanide then provides allyl imidoylnickel intermediate B, from which transmetallation and reductive elimination lead to the β,γ-unsaturated imine D. The desired β,γ-unsaturated ketone 4 could be obtained via acidic hydrolysis of D.
Discussion
In summary, nickel-catalyzed allylic carbonylative coupling with alkyl zinc reagent has been developed for the synthesis of β,γunsaturated ketones from allylic acetate and alkyl zinc reagent using commercially available tert-butyl isocyanide as a CO source. In this coupling process, the allyl imidoylnickel intermediate undergoes rapid transmetallation with the zinc nucleophile, thus avoiding the undesired polycarbonylation. This reaction features broad substrate scope with excellent regio-and chemoselectivity. Preliminary mechanistic studies reveal the reaction proceeds with the π-allyl nickel intermediate. Further effort on detailed mechanism and exploration of other electrophiles is currently underway in our laboratory will be reported in future.
Methods
General procedure A for the allylic carbonylative reaction. An oven-dried Schlenk tube charged with NiCl 2 ·DME (10 mol%) was evacuated and backfilled with N 2 . (This process was repeated for three times.) DMA (0.1 M) was added into the reaction mixture. To this solution was subsequently added allylic acetate (1.0 equiv), t BuNC (1.5 equiv), and Negishi reagent (1.5 equiv). The tube was equipped with a balloon filled with N 2 at 25°C until complete consumption of the starting material. The mixture was added 1 M HCl aq. and stirred at room temperature for 0.25 h. The mixture was then extracted with EtOAc and separated organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to yield the crude product, which was purified by silica gel flash column chromatography.
Data availability
The authors declare that all the data supporting the findings of this work are available within the article and its Supplementary Information files, | v3-fos-license |
2019-04-02T13:07:44.265Z | 2017-05-27T00:00:00.000 | 90822502 | {
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} | pes2o/s2orc | Bio-enriched Pleurotus mushrooms for deficiency control and improved antioxidative protection of human platelets?
The study investigated effect of substrate supplementation with Se alone or in combination with Cu or/and Zn Se on (1) the growth of Pleurotus ostreatus and Pleurotus eryngii; (2) elements accumulation in mushrooms; (3) the antioxidant activities of bio-enriched mushroom extracts in human platelets. The accumulation of elements generally increased over concentration gradient reaching its maximum at 1.2 mM for P. ostreatus and P. eryngii: (1) over 100 and 80 mg kg−1 of Se, respectively (Se supplementation); (2) over 15 and 30 mg kg−1 of Cu, respectively (Se+Cu); (3) over 30 and 85 mg kg−1 of Zn, respectively. Se was predominantly accumulated as an organic fraction. Contrary to P. eryngii, the P. ostreatus biomass decreased with substrate elements concentration but was satisfactory up to 0.9 mM of Se, Se+Cu and Se+Zn. The Se+Cu+Zn model yielded low biomass and elements accumulation. Extracts from mushrooms bio-enriched with Se and Se+Zn (0.6–1.2 mM) revealed significant antioxidant activities in human platelets by ameliorating reactive oxygen species (ROS) and preventing lipid peroxidation. The study demonstrated the potential application of Pleurotus mushrooms as functional food products bio-enriched with essential elements. ROS inhibition by extracts of these mushrooms may be useful in control of platelets activation cascade.
Introduction
Mushrooms are increasingly popular as foodstuffs in different world locations owing to their nutritional value and potential medicinal use, extensively evidenced over recent decades. Mushrooms belonging to the genus Pleurotus (higher Basidiomycetes) are, next to Agaricus sp., one of the most important cultivated species worldwide [1] and account for nearly 27% of global production [2,3]. Considered as a delicacy, they are rich in B vitamins, polysaccharides and minerals, e.g. potassium, phosphorus, calcium, magnesium and iron [4]. Various pharmacological effects exerted by compounds of Pleurotus, particularly derived from P. ostreatus, have been reported and include antiviral antibacterial, antidiabetic, antihypercholesterolic and antiarthritic activities [5][6][7][8]. All in all, it appears that Pleurotus mushrooms have particularly high potential as functional foods. Economically-wise, this is also supported by their short growth time, the inexpensive and readily available materials required for their cultivation (e.g. wheat, rice straw, cotton waste or sawdust) and their documented resistance to pathogens and pests [9].
Bio-enrichment (bio-fortification) of food is an interesting strategy for delivering nutrients and preventing deficiencies [10,11], and it may be attractive for consumers as it applies products of natural origin, increasingly preferred over synthetic food supplements [12]. However, most attention in this regard is focused on plant-based products [13]. Mushrooms, on the other hand, also have great potential for such use if one considers their ability to uptake and accumulate various elements in their fruiting bodies efficiently [14,15], and their already established place in various national cuisines [16]. Some previous studies have already demonstrated that Agaricus bisporus and Ganoderma lucidum grown on substrates intentionally supplemented with trace elements may reveal significantly increased nutritional value [17,18].
Selenium (Se), zinc (Zn) and copper (Cu) are all known to play pivotal metabolic roles in various biological processes, and their altered status may exert numerous adverse effects in humans. A decreased intake of Se can lead, inter alia, to hair loss, blotchy nails, diarrhoea, garlic breath, gastrointestinal issues and decreased thyroid function [19,20]. Zn deficiency, on the other hand, has been related to immunosuppression, retardation of physical growth and cognitive function as well as various dermatological problems [21]. Epidemiologicallywise, the worldwide frequency of Se and Cu deficiency is not high but can be observed in regions characterized by extremely low content of these elements in the soil and subsequently plant products [22,23]. This is particularly the case in some parts of Asia, a continent with a long tradition of mushrooms use, both as food and natural medicines [24]. Zn deficiency is becoming a worldwide problem with approximately 17% of the human population being at risk [25]. It is therefore essential to maintain the optimal status of these trace metals in humans in order to avoid any manifestation of the above-mentioned harmful effects and to support normal human growth, development and function.
Mushrooms bio-fortified with certain elements may also exert novel properties, e.g. their enrichment with lithium may have a potential use as a medical nutrition therapy in mood modification [26] while enrichment with selenium (Se) enhances the antitumor activities of mushroom polysaccharides [27] and significant immunomodulatory properties of proteins [28]. It has also been shown that P. ostreatus and P. eryngii enriched simultaneously with Se and Zn reveal a greater content of phenolic compounds and that methanolic extracts of these mushrooms exhibited an increased scavenging of radicals in assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH) [29]. This would indicate that biofortified mushrooms may have greater antioxidative properties, yet it remained unclear whether they could have any use in the amelioration of oxidative stress in human cells.
The aim of the present study was to investigate whether two edible mushroom species of worldwide popularity, P. ostreatus and P. eryngii, can be used in bio-enrichment with Se, Cu, Zn in a single-, double-and triple-element cultivation model, and whether such fortified mushrooms may exert some beneficial antioxidative effects. Specifically, the preventive effect of hot water extracts of fruiting bodies on tert-butyl hydroperoxide (tBHP)-induced oxidative stress in human platelets isolated from healthy donors. It has been evidenced that reactive oxygen species (ROS) play a role in triggering the activation cascade in platelets that involves tethering to the endothelium, rolling adhesion, aggregation and eventually formation of thrombus [30]. Therefore, any antiplatelet effect of bio-enriched mushrooms could play a potentially positive role in the prevention of cardiovascular complications.
Experimental design
The mushrooms of two species P. ostreatus and P. eryngii were grown on substrates supplemented in four different configurations of trace elements: (1) Se; (2) Se+Cu; (3) Se+Zn and (4) Se+Cu+Zn, and final concentrations of 0.1, 0.3, 0.6, 0.9 and 1.2 mM. The control was constituted of mushrooms grown without the addition of any element. The yield included fruiting bodies harvested successively as they matured. Collected material was subjected to analytical determinations of Se, Cu and Zn content in the fruiting bodies. Additionally, speciation analyses were applied to distinguish between the inorganic and organic Se fraction due to distinctive differences in their biological activity. These experiments were designed to choose those cultivation models at which the most efficient bioaccumulation and growth of fruiting bodies was observed. The fruiting bodies from the chosen model were then extracted at high temperature, and extracts were later used to test as regards their ability to attenuate tBHP-induced generation of ROS and lipid peroxidation in human platelets isolated from healthy donors.
Mushroom cultivation and harvesting
All experimental cultivations were performed in three independent replicates. The substrate for P. ostreatus was prepared from wheat straw cut into chaff 4-5 cm long. The straw for the experiment was moistened to a moisture content of 60% using distilled water and then pasteurized at 60 °C for 24 h. The following salts were used in the experiment: sodium selenite [Na 2 SeO 3 (IV)], sodium selenate [Na 2 SeO 4 (VI)] by Acros Organics (USA), copper(II) selenate (CuSeO 4 ·2H 2 O) and zinc nitrate hexahydrate [Zn(NO 3 ) 2 ·6H 2 O] by Sigma-Aldrich. These salts were dissolved in such an amount of sterile distilled water sufficient to obtain their appropriate concentration (0.1, 0.3, 0.6, 0.9 and 1.2 mM for all elements) in the substrate after which they were added to the substrate. Following the addition of the salt solution, the substrate water content reached 70%. The substrate was mixed with grain spawn (on wheat grain) which constituted 3% in relation to the wet weight of the substrate and placed in bags of perforated foil. Each bag contained 1 kg of the substrate. Mycelium incubation was conducted at a temperature of 25 °C and relative humidity of air at 85-90%. Once the substrate was totally covered with the mycelium, it was transferred to the cultivation chamber in which the temperature was maintained at 15-16 °C and relative humidity of air at 85-90%. The cultivation was additionally irradiated with fluorescent light of 500 lx intensity for 10 h a day. The growth facility was aerated in such a way as to maintain CO 2 concentration below 1000 ppm.
The substrate for P. eryngii was prepared from a mixture of beech sawdust and flax shives (3:1 vol.) which was additionally supplemented with wheat bran to the amount of 20%, corn flour 5% and gypsum 1% in relation to the substrate dry matter. The mixture was moistened with distilled water to a moisture content of 45%. The substrate prepared as described above was placed in PP bags and sterilized at a temperature of 121 °C for 1 h and was then cooled down to a temperature of 25 °C. Cu, Se and Zn salts were dissolved in sterile water, and the solution was added to the substrate with intensive blending using a POLYMIX PX-SR 90 D stirrer (Kinematica AG, Switzerland) in an amount that allowed their appropriate concentration in the substrate and, at the same time, making sure that the substrate water content reached 60%. The same concentrations of Se and Zn salts were applied in the substrate as for P. ostreatus. The substrates with Se and Zn addition were mixed with grain spawn (on wheat grain) of the examined mushroom species (5% of substrate weight) and placed in polypropylene bottles of 1 L volume. Each bottle was filled with 350 g of the substrate and closed with a cover consisting of a cellulose filter type 338 with a typical retention of 12-15 µm and a basic weight of 84 g m −2 (Munktell, Germany). The incubation was conducted at a temperature of 25 °C and air relative humidity of 80-85% until the substrate became completely covered with mycelium. Next, the covers were removed and placed in the cultivation chamber. For fructification, air relative humidity was maintained at 85-90% and temperature at 14 ± 1 °C. The cultivation was additionally irradiated with fluorescent light of 500 lx intensity for 12 h a day. The growth facility was aerated in such a way as to maintain CO 2 concentration below 1000 ppm.
The mushrooms were collected after their biomass no longer demonstrated further increase. The fruiting bodies were then weighed, dried using an electric drier SLW 53 STD (Pol-Eko, Poland) at 50 ± 2 °C for 48 h, weighed again for analysis of dry weight and ground in a Cutting Boll Mill 200 (Retsch GmbH, Germany) for 1 min.
Determination of trace elements
The powdered mushroom samples were sieved through a 0.02 mm sieve. 1.000 ± 0.001 g of sample was extracted by 1 mol L −1 phosphoric acid in an ultrasonic bath at an ambient temperature (30 min). Samples were then filtered through a paper filter (washed by 200 mL of water and 20 mL of phosphoric buffer). Sample pH was adjusted at 6.0-6.5 by the addition of 10 mol L −1 sodium hydroxide solution, and finally the samples were diluted to 20.0 mL by a phosphate buffer.
For Cu and Zn determination, flame atomic absorption spectrometry (FAAS) SpectrAA 22FS (Varian, Australia) was applied with stoichiometric flame acetylene (2.0 L min −1 ) and air (13.5 L min −1 ). Hollow cathode lamps (Varian) were used with the following parameters for Cu: wavelength 324.8 nm, slit 0.5, lamp current 10 mA and for Zn: wavelength 213.9 nm, slit 1.0, lamp current 5 mA, both with background correction with a deuterium lamp. The determination limits were 0.1 mg kg −1 with uncertainty about 5.0% (measured as RSD) for both elements determined.
For Se determination, electrothermal atomic absorption spectrometry (ETAAS) SpectrAA 280Z (Agilent, USA) with Zeeman background correction was applied. A selenium hollow cathode lamp (wavelength 196.0 nm, slit 1.0 nm, current 10 mA) was used, and a temperature programme was optimized as follows: drying 85-120 °C during 55 s; ashing 1000 °C during 8 s; atomization 2600 °C. Pyrolytic graphite tubes and palladium solution as a chemical modifier (10 µL of 500 mg L −1 for 20 µL of sample) were used. The limit of detection 0.01 mg kg −1 and the uncertainty of results (measured as RSD) at the level of 5.0% were obtained.
In the case of Se species analysis, hyphenated highperformance liquid chromatography with hydride generation atomic absorption spectrometry detection (HPLC-HG-AAS) systems was applied as described previously in detail [31]. The hyphenated analytical system consisted of a Shimadzu liquid chromatograph (LC-10A) equipped with an HPLC pump (LC-10AT), a vacuum degasser unit (GT-104), Rheodyne PEEK valve (IDEX, USA) and an anion-exchange column Supelco LC-SAX1 (250 mm, 4.6 mm i.d., resin particle size 5 µm) thermostatted by a column oven (CTO-10ASvp). The chromatographic run was isocratic at 3 mL min −1 with an injection volume of 200 µL. The measurements were performed with a Model SpectraAA 220FS spectrometer (Varian, Australia) equipped with an UltrAA selenium hollow cathode lamp.
3
The selenium species Se(VI) does not form volatile hydride, so in order to receive an analytical signal in the HG-AAS system it was necessary to perform a preliminary reduction of Se(VI) to Se(IV). The reduction was carried out on-line by heating the sample (90-100 °C) with the reducing agent: 0.5 mol L −1 thiourea solution in 10 mol L −1 hydrochloric acid (Kozak 2012). The eluate from the chromatographic column was joined to the stream of the reducing agent (flow rate 1 mL min −1 from the peristaltic pump) through a T-shape coupling and directed to the Tygon capillary loop (inner diameter 0.82 mm) heated in a water bath. The capillary loop outlet was connected to the hydride generation system. For both analytical systems, PEEK transfer tubing of the eluent from the LC column to the hydride generation unit was inserted into a Tygon sleeve. The continuous hydride generation system (VGA-77, Varian) consisted of a manually controlled, four-channel peristaltic pump with Tygon tubing (0.6 mm i.d.), one reaction coil (PTFE tubing 0.8 mm i.d., 75 cm length) and three-way connectors. The gas-liquid separator was made of glass, and the interior dead volume was 3 mL. For the atomization of the selenium hydrides (detected at 196.0 nm), a heating controller, an electrothermally heating mantle and a quartz tube (ETC-60, Varian) heated to 900 °C were used.
A number of validation parameters characterising the analytical method were determined. The limits of detection 0.01 mg kg −1 for both Se(IV) and Se(VI) and the uncertainty of results (measured as RSD) at the level of 10% for both selenium forms were obtained. As it was impossible to estimate the measurement traceability owing to the lack of any certified reference materials for determination of inorganic selenium species, the recovery of each selenium species was measured upon the addition of a standard to the sample. A recovery of 96-105% of each species was considered as satisfactory.
Mushroom extract preparation
Homogenized samples of fruiting bodies were extracted in distilled water at the ratio of 1:10 (w/v) at 95 °C for 30 min. The samples were then cooled to room temperature and centrifuged, and supernatants were sterilized by filtration on 0.22 μm syringeless filter devices (Roth). The extracts were kept at −20 °C until use.
Human platelet isolation
Human platelets were isolated from whole blood samples (6 mL) collected in acid citrate dextrose (ACD) from three healthy donors (screened by physical examination, medical history and initial blood tests), with normal platelet count (150,000-450,000 cells µL −1 ). The donors (aged 19-22 years old; three females) were non-smoking and normal weighted (BMI 18.5-24.9). Samples were collected at the Regional Centre of Blood and Blood Treatment in Poznan, Poland, according to accepted safeguard standards and legal requirements. Within 20 min of collection, platelet-rich plasma (PRP) was obtained by centrifugation at 200 g for 12 min. PRP was transferred into a polypropylene tube, and 1/10 volume ACD and 100 ng mL −1 prostaglandin E1 was added to prevent platelet activation during isolation. PRP was centrifuged at 900g for 15 min, and plasma was aspirated. Platelets were then suspended in 6 mL of Hepes-NaCl 2 buffer (10 mM Hepes, 0.85% NaCl, pH 7.4) and layered on a discontinuous 10-17% iodixanol gradient in Hepes/NaCl buffer. The gradient was centrifuged at 300g for 20 min, and the platelet fraction was collected. Platelets were centrifuged at 900g for 15 min, and the resulting pellet was washed and resuspended with Ca 2+ -free Tyrode-Hepes buffer (137 mM NaCl, 0.3 mM NaH 2 PO 4 , 3.5 mM Hepes, 5.5 mM [d]-glucose, pH 7.35).
Intracellular reactive oxygen species assay
Isolated platelets were loaded for 30 min at 37 °C in darkness with 20 μM of 2′,7′-dichlorofluorescin diacetate (DCFDA; Abcam, UK), a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. Cells were then washed, pretreated with 10 μL mushroom extracts for 30 min, washed again, seeded in black clear bottom 96-well plate and exposed to 10 µM tBHP (Abcam, UK) for another 60 min. The negative/positive controls consisted of DCFDA-loaded platelets pretreated with 10 μL of Tyrode-Hepes buffer and not exposed/exposed to 10 µM tBHP. Fluorescence of DCFDA in all samples was measured kinetically after 15, 30 and 60 min of incubation using a Synergy HTX multi-mode plate reader (BioTek, USA) at an excitation of 495 nm and emission of 528 nm. The final results were presented as a percentage of the negative control.
Lipid peroxidation assay
Lipid peroxidation was analysed using a Lipid Peroxidation (MDA) Colorimetric/Fluorometric Assay Kit (BioVision, UK) by means of malondialdehyde (MDA) equivalents. Isolated platelets were pretreated with 10 μL mushroom extracts for 30 min, washed and exposed to 10 µM tBHP for another 60 min. The negative/positive controls constituted of platelets pretreated with 10 μL of Tyrode-Hepes buffer and not exposed/ exposed to 10 µM tBHP. After the experiments, cells were harvested from each well and homogenized on ice in 300 μL of provided lysis buffer and centrifuged to remove insoluble material. The resulting 200 μL of supernatants was transferred to a microcentrifuge tube and supplemented with 600 μL of thiobarbituric acid (TBA) to generate an MDA-TBA adduct. To accelerate the reaction, samples were incubated at 95 °C for 60 min and the final product was measured colorimetrically at 532 nm. The calculated values were compared to a calibration curve prepared using MDA standard (BioVision, UK). The coefficient of variation (r 2 ) for the calibration curve was 0.99. The final results were presented as a percentage of the negative control.
Statistical analysis and calculations
The results were analysed using STATISTICA 10.0 software (StatSoft, USA). Because the element accumulation and mushroom biomass data met the assumption on Gaussian distribution (analysed with the Shapiro-Wilk test), comparison of element accumulation and yielded biomass between cultivation models was assessed with multivariate analyses of variance (MANOVA) with the Tukey HSD method as a post hoc test. Since the data of platelet ROS concentration and lipid peroxidation had no normal distribution, the Wilcoxon signed-rank test was employed to compare the studied samples with the positive control. p < 0.05 was considered as statistically significant.
Accumulation of trace elements in bio-enriched mushrooms
The addition of Se to the growth substrate increased its accumulation in the fruiting bodies of both investigated species in a concentration-dependent manner. Greater concentrations were, however, found for P. ostreatus. Nevertheless, Se concentrations were 40-fold higher compared to the control once the substrate overgrown by both species was supplemented with 1.2 mM of Se. It is worth noting that an increase in Se content was over 10-fold for P. ostreatus and 20-fold for P. eryngii when the lowest assayed concentration, 0.1 mM, was added (Fig. 1). For both species, organic Se had the greatest share for nearly all the studied substrate concentrations (Table 1). P. ostreatus, however, revealed higher concentrations, reaching maximally over 100 mg kg −1 after the substrate had been supplemented with 1.2 mM of Se (Table 1). These findings are in line with previous studies demonstrating the potential of various mushrooms from the Pleurotus genus in the production of food bio-fortified with Se [31,32].
Compared to mushrooms cultivated on substrates enriched only with Se, the simultaneous addition of Se and Cu resulted in decreased Se accumulation in both Pleurotus species although this effect was more evident for P. eryngii. For this species, Se concentrations did not exceed 20 mg kg −1 regardless of the initial substrate content of trace elements. To compare, for P. ostreatus the minimum Se level did not fall below 40 mg kg −1 once the initial concentrations were in the 0.6-1.2 mM range (Fig. 1). Moreover, greater organic Se levels were observed in P. ostreatus. In turn, P. eryngii was more effective in Cu bioenrichment, particularly at the 0.6-1.2 mM concentration range at which the Cu concentrations in the fruiting bodies exceeded at least 20 mg kg −1 (Fig. 2).
Compared to exclusive enrichment with Se, mushroom cultivation on substrates supplemented with Se in combination with Zn decreased the Se accumulation only 0.1 and 0.3 mM (P. ostreatus) or 1.2 mM (P. eryngii) concentration of elements had been applied. The greatest organic Se levels in fruiting bodies of both species were found in mushrooms growing on the substrate supplemented with 0.9 mM element concentration. In this combination, the organic Se content reached nearly 80 mg kg −1 for P. ostreatus and exceeded 30 mg kg −1 for P. eryngii (Table 1). The latter species was, in turn, more effective in Zn accumulation; the concentrations in the fruiting bodies exceeded at least 45 mg kg −1 after the substrate was supplemented in the 0.6-1.2 mM element concentration range (Fig. 2).
The observed concentrations of elements in Se, Se+Cu and Se+Zn cultivation combinations revealed relatively low variability (with some exceptions), demonstrated by low values of standard deviation (Figs. 1, 2), especially that our experiment was performed in three independent replicates. This, in turn, allows repetitive levels of trace elements to be obtained.
Simultaneous substrate supplementation with Se, Cu and Zn resulted only in a slight increase in Se levels in the fruiting bodies of both, P. ostreatus and P. eryngii, and usually significantly lower Cu and Zn levels compared to the control (Figs. 1, 2). This is most likely due to high competitive sorption of metals from the substrate, an effect observed in various organisms including mushrooms [33,34]. Due to unsatisfactory Se concentrations, speciation analysis was not performed for this cultivation model.
Considering that the recommended daily allowance (RDA) values were set at 55 µg for Se, 900 µg for Cu and 11 mg (male) or 8 mg (female) for Zn, for adult individuals [35,36], the fruiting bodies of P. ostreatus and P. eryngii bio-enriched with Se alone or in combination with Cu or Zn demonstrate a potentially high nutritional value. However, the partial loss of trace elements during mushroom processing (e.g. washing, drying, cooking) has to be taken into account [14]. The Se in Pleurotus mushrooms has been previously shown to be highly bioaccessible through in vitro studies, particularly in organic form [32]. In the present study, this form accounted for up to 89% for P. ostreatus and 74% for P. eryngii once the inorganic Se salts were added to the substrate at 1.2 mM concentration (Table 1). This is an important finding because the retention of organic forms of Se (selenocysteine and selenomethionine) is higher than that of inorganic Se (selenate-SeO 4 2and selenite-SeO 3 2− ) although both were shown to be absorbed in the gastrointestinal tract [37]. Further studies would be required to establish the bioaccessibility of Cu and Zn from mushrooms.
The results of the present study indicate that bioenriched Pleurotus mushrooms may help in deficiency prevention. It should, however, be highlighted that an excessive intake of Se, Zn or Cu may also bring about toxic effects in humans [38,39]. On the other hand, mushrooms are not usually consumed on a daily basis and, moreover, the bio-fortified fruiting bodies of Pleurotus species may be used in adequate amounts (e.g. in form of powder) as a natural ingredient of various food supplements.
Growth of bio-enriched mushrooms
The growth of the studied mushroom species revealed distinctively different patterns in response to the investigated combinations and concentrations of trace elements. In the case of P. ostreatus, the biomass generally decreased with the increasing initial element concentration in the substrate (Fig. 3). This effect was observed on substrates supplemented exclusively with Se but also in combination with Cu and Zn. Regardless of the combination of metals in the substrate, the lowest yield was found after supplementation of 1.2 mM. Nevertheless, with the exception of the Se+Cu+Zn model, the biomass of fruiting bodies did not fall below 100 g in samples with an initial substrate element concentration up to 0.9 mM (Fig. 3).
Pleurotus eryngii, in turn, exhibited a tendency to increase the biomass with initial concentrations of elements, with the exception of the Se+Cu+Zn model. This was particularly observed for the Se+Cu combination for which the greatest biomass production was found after the substrate had been supplemented with 1.2 mM of each element (Fig. 4). . Bars represent mean with standard deviation (n = 3). Identical superscript denotes no significant difference (p > 0.05) between cultivation models according to Tukey's HSD test (MANOVA) analysed separately for each studied concentration However, no sensory or instrumental measurement was applied; no change in colour or discolouration of the fruiting bodies of P. ostreatus and P. eryngii was noticeable after the mushrooms were grown on substrates supplemented with Se alone or in combination with Cu and/ or Zn (Figs. S1, S2). This finding is important from the consumer point of view as it has been shown that changes in the appearance of the food, e.g. the yellowish colour of maize enhanced with vitamin A [40], can be discouraging and lead to a decrease in the commercial value of such food products.
Antioxidant activity of bio-enriched mushrooms
Antioxidant activities were tested on hot water extracts obtained from fruiting bodies collected from substrates supplemented with 0.6, 0.9 and 1.2 mM concentration of Se, Se and Cu, and Se and Zn, as these cultivation models yielded the most satisfactory results of element bioaccumulation, organic selenium fraction and mushroom growth.
As observed, extracts of both species grown on substrates not supplemented with any element slightly decreased the intracellular ROS content (Figs. 5, 6) and level of lipid peroxidation (Fig. 6) generated by tBHP in human platelets (Fig. 7). It has been previously shown using in vivo rodent studies that both mushroom species possess antioxidant properties, a phenomenon associated with mushroom-derived polysaccharides [41,42].
The present study further demonstrates that extracts obtained from mushrooms bio-enriched with Se alone or in combination with Zn in the tested concentration range (0.6-1.2 mM) enhance their antioxidative effect exerted in platelets. The most significant amelioration of radicals was observed in cells pretreated with extracts from 1.2 mM of the Se and Se+Zn cultivation model with ROS levels decreased by 65.8 and 91.1% for P. ostreatus, respectively (Fig. 5) and by 54.3 and 79.6% for P. eryngii, respectively (Fig. 6). The counteraction of mushrooms extracts against tBHP-generated ROS resulted in significantly prevented lipid peroxidation in platelets, measured by means of MDA equivalents. The most beneficial effect was observed for extracts obtained from 1.2 mM Se+Zn supplementation-compared to the positive control the lipid peroxidation was decreased by 83.5% for P. ostreatus (Fig. 5) and 67.8% for P. eryngii (Fig. 6). Peroxidation of lipids is a chain reaction initiated by the hydrogen abstraction or addition of an oxygen radical, resulting in the oxidative damage of polyunsaturated fatty acids. If not terminated fast enough, a decrease in membrane fluidity and in the barrier functions of membranes is induced, final products of peroxidation (predominantly MDA and 4-hydroxy-2-nonenal) can induce genotoxicity, and cell death is promoted [43,44]. Its prevention is thus essential for cell survival and homeostasis.
The observed in vitro effects of mushroom extracts are not only beneficial in view of the prevention of oxidative stress and its potential consequences (e.g. DNA damage, Fig. 4 Biomass of P. eryngii cultivated on variously supplemented substrates (a) with the trend lines, linear equations and R 2 values (b). Bars represent mean with standard deviation (n = 3). Identical superscript denotes no significant difference (p > 0.05) between cultivation models according to Tukey's HSD test (MANOVA) analysed separately for each studied concentration Fig. 5 The intracellular ROS concentrations in human platelets exposed for 1 h to 10 µM tBHP and not pretreated with any mushroom extracts (tBHP; positive control), pretreated with extract of unenriched P. ostreatus (not enriched + tBHP) and pretreated with extracts of P. ostreatus bio-enriched with 0.6-1.2 mM of Se (Se + tBHP), Se and Cu (Se&Cu + tBHP), Se and Zn (Se&Zn + tBHP). Bars represent mean ± SD from three independent experiments corresponding to different donors. Asterisks represent statistically significant difference to the positive control (*p < 0.05; Wilcoxon signed-rank test) Fig. 6 The intracellular ROS concentrations in human platelets exposed for 1 h to 10 µM tBHP and not pretreated with any mushroom extracts (tBHP; positive control), pretreated with extract of unenriched P. eryngii (not enriched + tBHP) and pretreated with extracts of P. eryngii bio-enriched with 0.6-1.2 mM of Se (Se + tBHP), Se and Cu (Se&Cu + tBHP), Se and Zn (Se&Zn + tBHP). Bars represent mean ± SD from three independent experiments corresponding to different donors. Asterisks represent statistically significant difference to the positive control (*p < 0.05; Wilcoxon signed-rank test) Fig. 7 The level of lipid peroxidation (measured by means of MDA concentration) in human platelets exposed to 10 µM tBHP for 1 h and not pretreated with any mushroom extracts (tBHP; positive control), pretreated with extract of unenriched mushrooms (not enriched + tBHP) and pretreated with extracts of mushrooms bio-enriched with 0.6-1.2 mM of Se (Se + tBHP), Se and Cu (Se&Cu + tBHP), Se and Zn (Se&Zn + tBHP). a Data for P. ostreatus, b data for P. eryngii. Bars represent mean ± SD from three independent experiments corresponding to different donors. Asterisks represent statistically significant difference to the positive control (*p < 0.05; Wilcoxon signed-rank test) protein modification, lipid peroxidation) but also in terms of the potential inhibition of platelet activation. Although the present study did not investigate the effect of extracts on platelet aggregation, it is known that ROS may play, along with nitric oxide, adenosine and prostacyclin, a profound role in platelet activation by scavenging nitric oxide (which has a preventive effect on platelet aggregation), triggering tyrosine phosphorylation of β3 or supporting the metabolism of collagen and arachidonic acid [23]. Considering that primary mechanism of tBHP action is generation of oxidative stress [45] and that tBHP has been previously demonstrated to support platelet activation cascade [46], it is plausible that the antioxidant properties of mushroom extracts may also prevent platelets from aggregation. Moreover, a recent study demonstrated that an extract of P. eous inhibited human platelet activation in vitro, the effect linked to the high mushroom content of phenolic compounds and flavonoids [47]. Their concentrations in P. ostreatus and P. eryngii have been previously shown to be significantly increased by substrate supplementation with Se alone and particularly in combination with Zn [22]. Therefore, it might be interesting to investigate a potential application of bio-enriched mushrooms as antiplatelet agents. There is a clear evidence that dual antiplatelet treatment with aspirin and P2Y12 receptor inhibitors is essential in pharmacological treatment of acute coronary syndromes; it reduces the risk of ischaemic events and improves patients outcome [48,49]. Further studies, preferably employing assays such as VerifyNowP2Y12, Multiplate Aggregometry or Light Transmission Aggregometry, are required to evaluate their possible use in prevention of major adverse cardio-and cerebro-vascular ischaemic events such as myocardial infarction, stent thrombosis and stroke [50].
Importantly, extracts obtained from mushrooms cultivated on substrates supplemented with 0.6 and 0.9 mM of Se and Cu did not prevent generation of ROS (Figs. 5, 6) nor peroxidation of lipids (Fig. 7). This indicates that Cu can ameliorate the beneficial antioxidative properties of Se, as well as other compounds extractable from Pleurotus mushrooms. However, Cu (along with Zn) is a constituent of cytoplasmic superoxide dismutase (Cu, Zn-SOD), an important enzyme of the cellular anitoxidant system [51], and at higher concentrations it reveals significant pro-oxidant properties. It was shown that cultivation of Pleurotus sp. in a medium with increased Cu 2+ concentration results in enhanced activity of laccases [52]; enzymes performing monoelectronic oxidation of phenols and aromatic or aliphatic amines to corresponding reactive radicals [53]. In summary, the findings of the present study indicate that P. ostreatus and P. eryngii bioenriched with Se in combination with Cu do not reveal any promising antioxidative action, contrary to the other combinations tested.
Conclusions
The present study highlighted the potential of P. ostreatus and P. eryngii in the production of food bio-enriched with Se alone or in combination with Cu or Zn. Both species were able to grow on supplemented substrates and accumulate significant levels of elements (with a higher share of organic Se fraction) in their edible fruiting bodies. The only unsatisfactory results were obtained in the tripleelement cultivation model-the biomass and accumulated content of elements were low. The study further demonstrated that bio-enrichment with Se alone and in combination with Zn largely enhances the antioxidant properties of these mushrooms in human platelets as observed by ameliorated ROS generation and prevented lipid peroxidation. These findings indicate that bio-enrichment of Pleurotus mushrooms with the studied elements may not only be a promising strategy in deficiency control but could also have a potential biomedical application, particularly in antiplatelet therapy. | v3-fos-license |
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} | pes2o/s2orc | Flow Injection Analysis System for Screening Organophosphorus Pesticides by their Inhibitory Effect on the Enzyme Acethylcholinesterase
A flow-injection spectrophotometric procedure was developed for screening organophosphorus pesticides. The method is based on the inhibition of acetylcholinesterase immobilized on controlled porous glass beads with acetylcholine chloride as the substrate. Methyl parathion, chlorpyrifos, malathion and dichlorvos have been tested. The analytical peak height for a given acetylcholine chloride concentration correlates linearly with the logarithmic concentration of the pesticides between 1.0 × 10-3 mol L-1 and 1.0 × 10-6 mol L-1. If bromine water is added to the pesticide solution, a dramatic increase is observed in the analytical signal, and correlations with the logarithm of the concentrations are observed from 1.0 × 10-3 to 1.0 × 10-10 mol L-1.
Introduction
Pesticides (herbicides, fungicides, insecticides) are natural or manufactured chemical compounds used in modern agriculture to exterminate or control pests; these chemicals can damage crops and livestock and reduce farm productivity.Among the various pesticides, organophosphorus insecticides (OPs) are most often applied because they exhibit low environmental persistence and display high acute toxicity to their targets. 1,2rganophosphates comprise a group of chemical compounds that are used extensively in agriculture as insecticides.However, overuse of OPs results in pesticide residues in food, water and the environment, which poses a significant threat to human health.These compounds express acute lethality not only to insects but also to mammals because they are potent inhibitors of acetylcholinesterase (AChe), which is an enzyme that is vital to nerve function.AChe inhibition results in the buildup of the neurotransmitter acetylcholine, which interferes with muscular responses and, in vital organs, produces serious symptoms and eventually death.][5] Organophosphorus pesticides can be identified and quantified by classical analytical techniques, such as gas chromatography (GC), high performance liquid chromatography (HPLC) or HPLC coupled with mass spectrometry (MS).However, these methods require time-consuming sample preparation with application of different extraction and cleanup procedures using toxic and expensive organic solvents. 6In laboratories where a large number of samples must be processed rapidly, a method with sufficient sensitivity that can be used for preliminary screening is a good alternative.
Method
The method is based on the determination of the acetate formed by the enzymatic reaction of AChe immobilized on glass beads with the substrate acetylcholine at a fixed concentration.The enzymatic process is depicted in Scheme 1.The acetic acid that is formed after the reaction of the acetate with sulfuric acid permeates through a polytetrafluoroethylene (PTFE) membrane and is received by an aqueous solution of bromocresol purple (BCP; 5.0 × 10 -5 mol L -1 ) causing a color change from purple (l max = 590 nm) to yellow (l max = 400 nm).The variation in the absorbance of the solution is detected spectrophotometrically at 590 nm.1,1' trimethylene-bis(4formylpyridinium bromide) dioxime (TMB-4) was used to regenerate the enzyme. 22
BCP solutions (5.0 × 10 -5 mol L -1 ) were prepared by dissolving 0.27 g of BCP in 10 mL of ethanol.Further dilutions with water where performed to obtain the desired concentration.The pH was adjusted to 7.0 by dropwise addition of a dilute 0.1 mol L -1 NaOH solution.To avoid absorption of CO 2 from the air, the BCP solution was maintained in a bottle protected by a tube containing soda lime with an indicator.
The water used to prepare the solutions was first distilled in a glass distillatory, deionized in a Milli-Q Plus system and degassed prior to use.
Stock solutions of the pesticides (0.1 mol L -1 ) were prepared in acetone by dissolving adequate quantities in 5.0 mL of acetone in a volumetric flask.When necessary, an ultrasound bath was used to promote dissolution.More dilute solutions were obtained by successive dilutions with distilled water.For example, for methyl parathion, 0.1316 g was dissolved with acetone in a 5.0-mL volumetric flask, and the volume was completed with the same solvent (concentration: 0.1 mol L -1 ).From this solution, 500 mL was transferred to another 5.0-mL volumetric flask, and the volume was completed with distilled water solvent (concentration: 0.01 mol L -1 ).More dilute solutions were obtained by successive dilutions with distilled water using 500 mL of a solution and completing to 5.0 mL.The same procedure was used to obtain the solutions of the other pesticides.The initial quantities were as follows: dichlorvos, 0.1105 g; malathion, 0.1652 g and chlorpyrifos, 0.1753 g.For orange juice, distilled water was substituted by the juice in the final solution that was introduced in the flow system.
The silanization of the surface of the glass beads and the immobilization of the enzyme on the CPG were performed as previously described. 22heme 1.
Flow Injection Analysis System for Screening Organophosphorus Pesticides J. Braz.Chem.Soc.486
Solutions
Acetylcholine (5 × 10 -3 mol L -1 ) in an aqueous buffered solution was prepared by dissolving 0.0454 g in a 50.0-mL volumetric flask and filling to the mark with Sörensen pH 7.0 buffer (0.1 mol L -1 ).This solution was always freshly prepared prior to use.
A 5 × 10 -5 mol L -1 solution of TMB-4 in an aqueous buffer was prepared by dissolving 0.0223 g in a 10.0-mL volumetric flask and filling to the mark with Sörensen pH 7.0 buffer.This solution was always freshly prepared prior use.
Saturated bromine water was prepared from elemental bromine, and the final concentration of 20 mmol L -1 was obtained by dilution with water.A 20 mmol L -1 hypochlorite solution was prepared by adequate dilution of a 4-5% solution in water.20 mmol L -1 iodine was prepared by dissolving 1.27 g of solid I 2 in 250 mL of water in the presence of dissolved potassium iodide (4.0 g).
Flow-injection system
The scheme of the flow injection system is shown in Figure 1.Tygon® tubing is used at the pump to push or pull solutions, and the remaining tubes of the circuit are composed of polyethylene.The solution of acetylcholine chloride (Ac) is introduced by valve (V) into the carrier stream (F 1 ) consisting of 5.0 × 10 -3 mol L -1 Sörensen buffer (pH 7.0), which is pumped by the peristaltic pump (P) at a rate of 1.0 mL min -1 (rates: F 1 = F 2 = F 3 ).Then, the solution passes through the enzymatic reactor (ER) in a polyethylene tube (3.5 cm long and 3.0 mm in internal diameter) containing the immobilized enzyme.In sequence, the solution is mixed after the ER with a 0.5 mol L -1 sulfuric acid solution (F 2 ).The acetate formed in ER is mixed with the sulfuric acid solution in order to form acetic acid, which is the form that passes through the PTFE membrane in the permeation cell (PC) and is carried out by the BCP solution (F 3 ) to the spectrophotometer (SP).The formed acetic acid causes a color change from purple (l max = 590 nm) to yellow (l max = 400 nm).The signal, which is monitored at 590 nm, is registered on the chart recorder (R).The organophosphorus pesticide solutions (OPs) and the TMB-4 solution in a phosphate buffer are also introduced by the same valve (V) into flow F 1 .The sample loop consisting of 100 µL was composed of a polyethylene tube (1.0 mm i.d.).All of the waste was collected in (W).A Lauda Model RCS RC6 thermostated bath was used to study the influence of the temperature on the enzymatic column.
Results and Discussion
The pesticides studied in this work are methyl p a r a t h i o n ( O , O -d i m e t h y l -O -4 -n i t r o p h e n y l phosphorothioate), chlorpyrifos (O,O-diethyl O-3,5,6trichloropyridin-2-yl phosphorothio-ate), malathion (2-(dimethoxyphosphinothioylthio) butanedioic acid diethyl ester) and dichlorvos (2,2-dichlorovinyl dimethyl phosphate).Their molecular formulas are shown in Table 1.
The analytical signal (Figure 2) is calculated from the difference between the height of the signal corresponding to the acetic acid formed from the acetylcholine before the inhibition (peaks 1) of the enzyme and the signal from the formed acetic acid after the inhibition (peaks 3) of the enzyme by the organophosphorus pesticide for the same acetylcholine concentration.The difference between the peak heights corresponding to the uninhibited acetylcholinesterase and those related to the inhibited one is the analytical signal E 1 .Peaks 2 and 4 represent the introduction of the pesticide and the regenerator TMB-4, respectively.Their negative values are due to the presence of CO 2 in the BMP indicator that permeated through the membrane in the direction of the introduced solution, which A study of the influences of parameters, such as temperature, acetylcholine chloride concentration, flow rate, sample volume, pH of the reaction medium (phosphate buffer) and sulfuric acid concentration, on the flow injection analysis (FIA) system was performed to optimize the analytical signal.By selecting the best compromise between the signal intensity and the operational characteristics (i.e., cleaning time, temperature control of the reaction, analytical frequency, volume of the solutions, and concentration of the reagents) of the flow system, the conditions reported in Table 2 were established for the method.
The determination of the pesticide concentrations was made with and without oxidation using bromine water.The oxidation of the samples was performed by simply adding 1 mL of 20 mmol L -1 bromine water in a 5-mL volumetric flask and filling to the mark with the pesticide solution, without any other pretreatment.The introduction into the analytical flow system was performed after 15 min.
The use of TMB-4 to reactivate the enzyme always exhibited excellent results, which allowed for normal operation of the analytical procedure.
The experiments were performed with 20 mmol L -1 hypochlorite and iodine (I 3 -) solutions as substitutes for bromine water.However, in contrast to bromine, these reagents irreversibly and cumulatively damage the enzyme during pesticides injection into the flow system, and therefore, the complete recovery of the enzymatic activity does not occur with the TMB-4 regenerator.
The possible direct effect of the bromine water on the enzyme was studied, and no influence of this reagent on the enzyme activity was observed.
Table 3 shows the relationship between the percentage inhibition of the enzyme and the pesticide concentration.The limits of detection were drastically lowered when some drops of bromine water were directly added to the pesticide solution.The resulting compounds are much more vigorous inhibitors of acetylcholinesterase, and therefore, the analytical signal significantly increased.Morita and Kumaran 29 reported that organophosphorus pesticide oxidation with bromine water changes the P=S group to the corresponding P=O group, and these compounds typically exhibit increased enzyme inhibition.However, this explanation does not clarify the results for dichlorvos, which contains a P=O group in the original molecule.Sampling rate / h Duirk et al. 30 have elucidated the distribution of chlorpyrifos in the presence of aqueous hypochlorite solution in the pH range of 6.3 to 11.They observed the fast oxidation of chlorpyrifos (CP) by OCl -, which resulted in a more toxic compound (i.e., chlorpyrifosoxon (CPO)).Duirk et al. 30 also studied the hydrolysis of dimethylformamide (DMF) for CP and 3,5,6-trichloro-2pyridinol (TCP).In a study of the degradation products of organophosphorus pesticides, Kralj et al. 31 observed that, for the pesticide chlorpyrifos, a product analogous to chlorpyrifos-oxon was detected.Duirk et al. 30 also reported that one of the degradation products is chlorpyrifos-oxon.
One possible suggestion for dichlorvos is that bromine addition occurs at the carbon-carbon double bond.Certainly, a more detailed study must be performed to explain the increase in the toxicity of these types of pesticides after oxidation with bromine water, but this work is beyond the scope of the current work.
Table 3 shows the relative percent inhibition of the aqueous solutions for the four pesticides on the acetylcholinesterase.The solutions in fresh orange juice were also studied.The inhibition was studied in the presence and absence of bromine water in all of the cases.The percent inhibition was calculated for a pesticide concentration according to %(inhibition) = ((E 1 /E 2 ) × 100), where E 1 is the difference between the two signals before and after inhibition and E 0 is the signal before inhibition.
The higher toxicity of the pesticides oxidized by the addition of bromine is evident, which is reflected in the increase in the inhibition of the enzyme.In the more dramatic case of dichlorvos, concentrations with a magnitude of 10 -10 mol L -1 can be detected.
To test the method in a real matrix, orange juice was separately spiked with known quantities of methyl parathion, chlorpyrifos, malathion and dichlorvos.The results are shown in Table 3.No similar equations correlating the inhibition of the oxidized forms of the pesticides and concentration were observed for the four organophosphorus compounds.Therefore, for comparison, dichlorvos was used as the reference because it exhibits inhibitory action over a wider range of concentrations.For this comparison, the parameter dichlorvos proportion (dp), which is the rate between the inhibition of the enzyme by a pesticide in a given concentration and the inhibition caused by 1.0 × 10 -3 mol L -1 dichlorvos, is introduced.The calculated values are also shown in Table 3.
This method provides a good indication of the level of toxicity of a sample containing an organophosphorus pesticide even without specifically knowing which compound is present, allowing for rapid medical decisions due to intoxication.In this type of analysis, a standard sample of 1.0 × 10 -3 mol L -1 dichlorvos must be analyzed for comparison.
Conclusion
Based on the observed results, the proposed method is reliable, low cost, rapid and easily performed.This method also exhibits very good detection because very low concentrations of organophosphorus pesticides, for which acetylcholinesterase is a specific sensor, were detected.Therefore, the method can be applied for the detection of these types of substances.
Table 1 .Figure 2 .Table 2 .
Figure 2. Example of the analytical signal.The group of peaks labeled 1 is related to signal 1 of the acetic acid produced from 5.0 × 10 -5 mol L -1 acetylcholine prior to the inhibition of the enzyme by the pesticide.Signal 2 corresponds to the introduction of the pesticide solution in the FIA system.Group 3 is related to the signal after the inhibition of the enzyme by the pesticide.Peak 4 is related to the introduction of the TMB-4 solution into the FIA system.Group 5 is related to the signals for the acetylcholine after the regeneration of the enzyme.
Table 3 .
Inhibition of the enzyme acetylcholinesterase as functions of the concentration of the pesticides methyl parathion, chlorpyrifos, malathion and dichlorvos.Comparison of the inhibition power of the pesticides with respect to dichlorvos arbitrarily taken as reference.Determination of these pesticides in fresh orange juice purposely contaminated | v3-fos-license |
2016-05-07T11:54:55.185Z | 2014-03-05T00:00:00.000 | 17256727 | {
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} | pes2o/s2orc | Mapping the force field of a hydrogen-bonded assembly
Hydrogen bonding underpins the properties of a vast array of systems spanning a wide variety of scientific fields. From the elegance of base pair interactions in DNA to the symmetry of extended supramolecular assemblies, hydrogen bonds play an essential role in directing intermolecular forces. Yet fundamental aspects of the hydrogen bond continue to be vigorously debated. Here we use dynamic force microscopy (DFM) to quantitatively map the tip-sample force field for naphthalene tetracarboxylic diimide molecules hydrogen-bonded in two-dimensional assemblies. A comparison of experimental images and force spectra with their simulated counterparts shows that intermolecular contrast arises from repulsive tip-sample interactions whose interpretation can be aided via an examination of charge density depletion across the molecular system. Interpreting DFM images of hydrogen-bonded systems therefore necessitates detailed consideration of the coupled tip-molecule system: analyses based on intermolecular charge density in the absence of the tip fail to capture the essential physical chemistry underpinning the imaging mechanism.
z=2.6A x Hydrogen bond Figure 4E of the main text.
Experimental methods
All experiments were carried out in ultrahigh vacuum (UHV) using an Omicron Nanotechnology GmbH LT STM/AFM in the qPlus configuration, using MATRIX electronics as the control system. Clean Si(111) 7 x 7 surfaces were prepared by the standard method of Experiments were performed at either ∼ 5 K (using liquid helium) or ∼ 78 K (when using liquid nitrogen). Although operating at 5 K offered significant advantages in terms of reducing drift and creep as well as improving the overall noise level, we did not otherwise observe any qualitative differences in the measurements performed at different temperatures.
We used commercial qPlus sensors supplied by Omicron Nanotechnology, with an electrochemically etched tungsten wire attached to one tine of the tuning fork as a tip. These were introduced into the scan head without any further conditioning. The tips were then prepared on clean silicon surfaces by standard STM tip preparation (tip pulses and controlled crashes into the surface) until good STM and DFM resolution was obtained. Consequently, before we start imaging the NTCDI molecules, we expect that our tips are silicon, rather than tungsten, terminated.
Experimental techniques
When imaging the islands of NTCDI, typically the region of interest was first imaged in STM, and left to scan in order to minimise drift and creep, before subsequently attempting to image the same region in constant height DFM. When imaging in constant height DFM, we ensured the gap voltage was set to 0V (i.e. no detectable tunnel current) in order to eliminate any effect of either electronic crosstalk [2], or the so-called phantom force [3]. For STM measurements the sample was biased and the tip held at ground.
When imaging the molecular islands on the silver-terminated surface in STM we observed a large number of spontaneous changes in resolution, suggesting that the tip was picking up or depositing material from the substrate. It is partly on this basis that we suggest that our tips become NTCDI-terminated during the initial STM imaging.
Atom tracking and 3-D forcefield measurement
At both 5 K and 77 K we used a home-made atom tracking system developed by Rahe et al. at. [4], interfaced with the Omicron MATRIX electronics, to both measure the relative tip-sample drift/creep and subsequently apply feedforward correction. At liquid helium temperatures this was not necessary in the case of taking a single constant height image, but was required over the long periods of time needed to acquire the 3-D datasets presented (see below for more details). At liquid nitrogen temperatures we found the use of feedforward correction was essential in order to allow reliable constant height imaging of the molecules and surface, even in the case of single scans.
In order to acquire the 3-D force fields we used a grid spectroscopy protocol combined with the atom tracking previously described by Rahe et al. [4]. In brief, once the tracking parameters had been determined, the tip was left for some time tracking a single molecule.
To perform a measurement the tracking and dither would be disengaged and the tip would move to a predefined position and perform a single ∆f (z) spectroscopy measurement, the tip would then move back to the original location, reengage the dither, and re-enter tracking.
We note additional delays were incorporated during repositioning steps to reduce the effect of piezo creep. This procedure was repeated for every pixel of the 3-D grid. The x-y position of the tip was recorded each time the tip re-entered tracking, and the feed-forward vector updated every 10-30 minutes based on the recorded motion in that time. A typical 3-D grid would entail between 1000 and 2000 ∆f (z) measurements depending on the size and pixel density. We prefer to construct the 3-D force fields using this protocol rather than the alternative x-y "slice" methods as we believe that there is less potential for introducing artefacts either from drift correction, or errors in the amplitude/phase signals due to the high ∆f corrugation present when imaging in constant height. During ∆f (z) measurements integration times and PLL/AGC settings were selected such that the phase and amplitude signals of the PLL remained constant within the instrumental noise. The use of the atom tracking to correct for long-term drift, and to continuously relocate the tip over the molecule, was necessary due to the very long acquisition times required in order to record these datasets (approximately 29 hours per 3D grid). For constant height images taken using atom tracking all ∆z values quoted are relative to the height of the tip in ∆f feedback during tracking.
F(z) curves were extracted from the ∆f (z) spectroscopy by subtracting ∆f (z) measurements taken off the island from ∆f (z) measurements on top of the molecules. This "on minus off" method (used for example by both Lantz et al, and Ternes et al [5,6]) produced a "short-range" ∆f that was then inverted to force by the Sader-Jarvis method [7].
Although all measurements were acquired in constant height we note it was necessary to process the 3D data to remove variation in ∆f due to drift in the frequency of the PLL (of order 40-100mHz over a 30 hour period). After this processing step we ensured the long range part (i.e. those parts of the curve not showing site specific contrast, usually over +1 to +1.5nm from the feedback position) of the ∆f (z) curves were aligned using a least mean squares fitting method in order to check the drift in ∆f had not significantly altered the feedback position during atom tracking. The alignment checked by this method was generally better than 5pm, below our vertical noise limit in ∆f feedback. Consequently, the grid datasets could be processed as constant height datasets within our experimental noise.
As a result of the "on minus off" method the calculated force values only include sitespecific interactions, as long-range van der Waals and electrostatic interactions are subtracted out. We explicitly note, however, that any site specific van der Waals interactions (that is to say, the short range dispersion attraction between the apex of the tip and the molecule on the surface) are still included (and, as described below, are also accounted for in our density functional theory calculations). The "on" and "off" measurements are both taken over the same range of z, but only the "on" measurement contains contributions from the short range (site specific) force. Following subtraction of the "off" curve, we therefore obtain the short range signal, which is directly comparable to single DFT F(z) calculations.
Simulation methods
Calculations were performed using an ab initio density functional theory (DFT) method and the CP2K code [8,9]. The algorithm implemented in the code is based primarily on using a Gaussian basis set for evaluating most terms in the Hamiltonian. However, an on-the-fly conversion is made to a plane wave basis set to calculate the electrostatic energy. Goedecker, Teter, and Hutter (GTH) pseudopotentials and the PerdewBurkeErnzerhof (PBE) generalized gradient approximation method [10] were used. In all calculations dispersion interactions were approximately taken into account using the DFT-D3 method due to Grimme et. al. [11]. Geometry relaxation in most calculations was run until the forces on atoms that were allowed to relax were no more than 0.01 eV/A.
Several tips were used based on two models: a Si pyramidal tip terminated with either
Imaging low coverages of NTCDI on silver-terminated silicon
As previously shown, at larger coverages NTCDI readily forms large islands on the silver terminated silicon surface. At low coverages the molecules are mostly found absorbed either individually or in small clusters at defects on the surface. These pinning sites may include unpassivated silicon atoms or disordered silicon -silver clusters, and, consequently, the exact nature of the binding of the molecules to the substrate is not well defined in these instances. However, it is almost certainly much stronger than the weak van der Waals forces underpinning the molecule-substrate interaction on the clean, passivated Ag:Si(111)- likely perturbs the electronic structure of the molecules more strongly than those absorbed on the silver. The constant height DFM imaging of the same molecules reveals the same carbon backbone structure as observed in the images of the large islands. The pinning of the molecules in this case means that the molecules are arranged in an unfavourable "endon" configuration. In this instance we also observe intramolecular contrast between the molecules. Here there are two possible explanations of the contrast. The first is that we are observing bonding between the NTCDI, and one of its tautomers (see Supplementary Figure 3). The other is that both molecules have the same tautomerisation and that the close spacing of the molecules in this instance produces contrast between the molecules similar to that observed in the hydrogen bonding configuration, although we note explicitly that in the configuration shown no hydrogen bonding would occur between the aligned groups.
Consequently, we suggest a degree of caution must be applied to the interpretation of intermolecular contrast as it is not trivial to see how repulsive effects purely due to the close proximity of the molecules could be distinguished from hydrogen bonding effects.
Supplementary Figure 4 shows another two molecules obtained at low coverage, in a similar bonding configuration to Supplementary Figure 2, the same sub-molecular resolution is obtained. The right-hand molecule appears slightly distorted at the far end, possibly due to its bonding to the substrate.
Mapping the potential field
In addition to the force-field presented in the main paper we also calculated the potential energy of interaction between the tip and molecule (Supplementary Figure 5). Qualitatively we observe the same contrast in the x-y maps as for the force, and we observe reasonable quantitative agreement between the experimental potentials and the energies calculated in our DFT simulations. However, on a cautionary note, we find that both the magnitude, and shape, of the potential curves are much more sensitive to any variation in the alignment of the curves, or variations in experimental noise, than the corresponding force curves. This can be readily understood from the relationship between force and potential, F (z) = −dU/dz, and thus although the force measurements are therefore 'noisier' "point by point", they are much less sensitive to any 'integrated' variation in the tail of the potential. Conversely, it is extremely instructive to examine the potential curves during data processing, even if the quantity of interest is the force, as any variation is often much clearer in the potential than the force. Similarly, STM images of NTCDI islands are complicated to interpret and cannot be used to determine whether the tip has become sharper through pickup of the molecule -as is the case for other tip terminations such as Br and Xe [12]. Consequently, the primary method used to check for tips capable of submolecular resolution was to directly attempt imaging in the constant height AFM mode. We therefore rely on corroborating information and comparisons with DFT simulations described elsewhere in the paper to determine our tip state.
Bond length measurements
In their recent work, Gross et al. [13] however, we stress again that accurate determination of the hydrogen-bond length from the images is problematic, and therefore should be treated with a degree of caution.
Atomic relaxations at very close approach
Due to the precision available at 5K temperatures, combined with the atom-tracking system, thermal drift was eliminated such that the variation in molecular resolution with scan height could be carefully examined. Particularly noticeable in Figure 2 of the main paper (see also movie file 3D grid Force 01) is a slight distortion of the NTCDI molecule when the tip height is reduced below -0.15nm, shown as point 1 in Figure 2. As all of our 3D data was collected using a grid spectroscopy protocol (see Section II.1), we can be confident that the distortions of the molecule are in fact real, likely due to the tip tilting under compressive strain at very close approach. It is important to note that we clearly observe both intramolecular and intermolecular features as early as the force turnaround suggesting that only a small amount of repulsion is required to distinguish these features.
To further investigate the effect of tip distortions we extracted the atomic displacements from our DFT calculated spectra. Shown in Supplementary Figure 14 is the calculated force spectra and atomic displacement for the NTCDI tip in the oxygen-down orientation at the primary locations investigated. The atomic displacement characterises the x-y-z movement of the lowest oxygen atom of the NTCDI-tip. Significant deformations of the NTCDI molecule are only observed when the tip-sample separation is <0.3nm, which corresponds to the same region of the experimental 3D force data where distortions are observed for the NTCDI molecule described above. As such our DFT calculations support the proposal that the distortions observed at very close approach for our data in Figure 2a of the main paper is a consequence of tip (and NTCDI island) relaxations. However, it is useful to note that the regime of very close approach corresponds to a significantly smaller scan height, around 50-100pm less, than what is required to observe the intramolecular and intermolecular resolution shown in this paper.
Movies
Movies of the 3D datasets presented in the main paper and supporting information are available as separate files to show the full evolution of the force contrast through the dataset.
Total electronic density calculations
To interpret the experimental images the total electron density (TED) was calculated for the simulated NTCDI island adsorbed onto the Ag:Si(111)-( Whilst reasonable agreement was found for the intramolecular resolution observed within each NTCDI molecule, the inter molecular resolution in the regions of H-bonding was not reproduced. The TED is plotted on a linear scale in Supplementary Figure 11a and b which shows that the density in the region of H-bonding is over an order of magnitude (∼0.08 versus ∼1.5 electrons/Bohr 3 ) smaller than that over the C-C bond regions. Supplementary Figure 11 highlights this disparity by plotting the minimum density isovalue at which charge is observed in the H-bond locations. As such, consideration of the TED is not sufficient to explain our inter molecular DFM observations. We note that in previous observations of intermolecular bonding [14] the calculated TED images required significant saturation of the scale bar for any intermolecular contrast to be visible, a process that is not required for the experimental DFM images.
Electron density difference
As discussed in the main paper, Supplementary Figure 12 shows calculated TED and electron density difference(EDD) plotted as an x-y slice positioned 100pm above the molecular plane. The electron density difference was obtained by first calculating the TED for the isolated surface (Ag-√ 3 + NTCDI molecular island) and also the isolated NTCDI tip at each step of the F(z) calculation. These two densities were then summed together and subtracted from the total density for the full relaxed system. Therefore the remaining subtracted electron density difference will describe the interaction caused by the presence of the tip, allowing us to examine its effect on the simulated system.
At this position the difference in electron depletion between the C-C and H-bond positions appears to be greater than in the main text. This is because for the H-bond location the electron depletion is centred along the axis of the H-bond, in plane with the NTCDI, whereas for the C-C bond the depletion is stronger above the plane of the molecule (at 50pm as shown in Figure 4 of the main paper). It is interesting to note that in Supplementary Figure 12c the EDD above the C-C bond takes on a sharp appearance, similar to that observed in experimental images. We also note that in Supplementary Figure 12 f there appears to be enhancement of the TED in the H-bond location, however, this is a misleading observation.
In Supplementary Figure 12 the x-y slices are plotted 100pm above the molecular plane, 50pm higher than that shown in Figure 4 of the main paper, whereas the DFM tip position (defined by the location of the lowest atomic core) is 250pm above the molecular plane.
Therefore the position of the x-y slice at 100pm is high enough to intersect the electron density originating from the atoms within the DFM tip, which is unrelated to the tipsurface interaction. The enhancement is thus purely apparent and bears no relation to interpretation of our DFM images.
Supplementary Figure 13 shows the calculated EDD for (a) the carbon ring and (b) void positions marked in Figure 2 and Figure 3 of the main text, plotted with the same position and scale as that in Figure 4 and overlaid with the ball-and-stick model. Compared to the other positions the void location shows a dramatic reduction in the amount of electronic density redistribution from the presence of the AFM tip due to the absence of any NTCDI molecules or intermolecular bonding in this location. Furthermore, the observed density depletion occurs in locations other than below the AFM tip, and is in fact centred on the atomic cores of the surface NTCDI molecules. This supports our observation that the density observed for the H-bond location is in fact due to real interaction between the surface network hydrogen bonds and the AFM tip. For completeness, the Carbon Ring location is also shown. Here we observe that the density depletion is strongest over the individual carbon atoms, with an intensity between that of the C-C and H-bond locations, possibly reflecting the same ordering observed in the calculated F(z) curves at negative gradients.
Carbon monoxide tip calculations
We have shown that to achieve intra-and inter-molecular resolution in our experiments our tip is most likely terminated with a single NTCDI molecule oriented with its C=O bond pointing towards the surface molecular island. However, although the technique of controlled carbon monoxide pickup was not used in our study it is instructive to make a comparison between the results of the more commonly used CO tip and an oxygen-down NTCDI. Supplementary Figure 15 shows the calculated force profile for a CO and O:NTCDI tip positioned over the four main sites considered in our study (as shown in Figure 3 of the main text). The initial position of each molecule was set such that each oxygen tip atom is located 0.6nm above the plane of the molecular island. As can be seen from Supplementary Figure 15 the behaviour of the CO and O:NTCDI tip is very similar, supporting our claim that an O:NTCDI tip is capable of comparable resolution to previous studies using the CO method. A key difference observed in our calculations, however, is that the CO tip is significantly more flexible than the NTCDI molecule, evidenced by the behaviour of the force curves at very close approach. This is particularly relevant to the discussion in section V.
Calculated spectra with fixed density
To examine whether atomic and electronic relaxation plays a significant role in determining F(z) we repeated the simulations shown in Figure 3A of the main paper using a simplified approach. In principle the total density is the sum of two terms. 1) the densities of the isolated surface and molecules, and the isolated tip, which can both be obtained by relaxing each of the two systems separately.
2) The density change induced when the sur-face+molecules and the tip are brought together, which is calculated with the full electronic self-consistency and geometry relaxation: The total energy is a function (in DFT) of the density, so contains terms due to the first and the second terms of the density. Both terms in the density depend on the position of the tip, and hence both contribute to the tip force and eventually to the contrast. However, if the energy due to the first term is more important than that due to the second, then a possible explanation would be that the total density is responsible and its fairly small change due to interaction of the tip with the surface contributes only marginally to contrast. If, however, the energy due to the second term in the density appears to be more important, then the EDD is a better predictor of the contrast in the dynamic force microscopy image.
We therefore ran a series of calculations to test this hypothesis. Specifically, we calculated the isolated densities of surface + molecule and the tip, and then at each tip height in an F(z) spectrum we calculated only the total energy/force, without allowing the density to further relax. This calculation provided the energy contribution to the first term(isolated density). We then compared the results of this fixed density approach to the fully relaxed case. The results from these calculations are shown in Supplementary Figure 16. From a comparison of the force curves it is clear that there remains a clear separation between the force curves in the repulsive region. Therefore in each case both intermolecular and intramolecular resolution will be observable. In particular we note that the intermolecular features are still visible even when the NTCDI tip is rigidly held in place. The effect of the relaxation of the density, however, is to increase the separation of the force curves in the repulsive region, leading to an improvement in the contrast of the bonds. From these results it appears that the tip-sample force interaction is primarily dominated by the TED, with a small direct contribution from the EDD. As described in the main paper, however, only a percentage of the TED is affected by the repulsive forces that varies with bond type. We believe this observation is crucial to understanding why intra and intermolecular features appear with a similar brightness in experimental measurements.
Simulated images and line profiles
Due to the significantly reduced computational cost of running fixed density and geometry calculations we simulated the full three dimensional force field above a hydrogen bond within the NTCDI island, as shown in Figure 4 of the main paper. The simulated grid was 6×6×2 A in size using 30×30×10 points corresponding to a grid spacing of 0.2Å. Two dimensional line profiles positioned above single hydrogen and carbon bonds were also calculated using a smaller lateral spacing of 0.1Å (as shown in Figure 4D of the main text). Spectra were calculated over a tip-sample distance range of 5Å to 2Å for the line profiles and 4.5Å to 2.5Å for the full simulated image. Supplementary Figure 17A shows a series of 'slices' taken from the simulated 3D force data. At larger tip-sample distances the image contrast is dominated by short range dispersion interactions which appear as dark depressions, similar to the experimental image in Figure 1D of the main paper. At smaller tip-sample distances the repulsive contribution to the force increases and inter and intra molecular features become apparent as bright protrusions. We note in particular the observed intermolecular contrast in the hydrogen bonding regions. Additional line profiles calculated at multiple tip-sample separations are shown in Supplementary Figure 17B. Despite the approximations made using a fixed density and geometry the line profile data agrees well with experimental profiles, even though the simulated tip structure is completely fixed in place. The observed oscillations in the simulated images (particularly noticeable at z≥2.8Å) are due to a numerical error associated with the finite grid used in the DFT calculations of the density. A finer SCF grid was tested for a number of points in the simulated force-field and the amplitude of the oscillations reduced significantly. However, this greatly increased the computational cost.
Regardless of the grid size used to calculate the density, however, the same overall conclusion is reached: the intermolecular features at the hydrogen-bond positions are observed even when the atomic coordinates and electron density are fixed. | v3-fos-license |
2021-09-28T01:09:06.116Z | 2021-07-13T00:00:00.000 | 237746384 | {
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} | pes2o/s2orc | Upregulation of MiR-340-5p Reverses Cisplatin Sensitivity by Inhibiting the Expression of CDK6 in HepG2 Cells
Cisplatin (CDDP) has been successfully used in chemotherapy for liver cancer. However, the development of CDDP resistance in HepG2 cells usually leads to relapse and a worsening prognosis. MiR-340-5p has attracted much attention because of its ability to affect cell resistance. This project is intended to explore the role of miR-340-5p and CDK6 in CDDP-R HepG2 cells and provide new ideas for the treatment of liver cancer. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-340-5p and CDK6. We constructed a CDDP-resistant model of HepG2 cells to examine the effect of miR-340-5p on the drug sensitivity of HepG2 cells. CDDP-R HepG2 cells were transfected with miR-340-5p overexpression plasmid and CDK6 silencing plasmid. QRT-PCR was used to detect the expression of miR-340-5p and CDK6. A western blot was performed to determine the expression of CDK6, CyclinD1, and CyclinD2. CCK-8, flow cytometry, TUNEL and Clonogenic assays were also carried out to detect CDDP-R HepG2 cells. There is a targeting relationship between miR-340-5p and CDK6. The drug resistance of CDDP-R HepG2 cells was significantly higher than that of CDDP-S HepG2 cells. CDDP-R HepG2 cells transfected with both miR-340-5p overexpressing plasmid and CDK6 silencing plasmid showed a lower proliferation ability, cell cycle arrest in the G0/G1 phase, and lower drug resistance compared with single CDDP-R HepG2 cells. Overexpression of miR-340-5p aggravated CDDP-R HepG2 cells’ apoptosis and inhibited cell viability. Overexpression of miR-340-5p could reverse the sensitivity of HepG2 cells to CDDP by inhibiting the expression of CDK6 in HepG2 cells.
Liver cancer is one of the most common malignant tumors in the world and the second leading cause of cancer death (ZHAI et al. 2018). Especially in the developing countries of Asia, the incidence of liver cancer has increased significantly in recent years (ARGYROU et al. 2017). Globally, about 630,000 new cases of liver cancer occur every year, and more than half of these new cases occur in China . Currently, chemotherapy is the main method for treating advanced liver cancer and treating patients with poor liver function. Cisplatin (CDDP) is one of the most commonly used chemotherapy drugs for advanced liver cancer and postoperative patients, combined with arterial chemotherapy (GENG et al. 2019). CDDP chemotherapy drugs will circulate throughout most of the organs and tissues of the body along with blood circulation, and is a means of systemic treatment. It is very effective in the treatment of cancer, but the subsequent side effects of chemotherapy and the adverse reactions of chemotherapy have brought intense pain to patients (CHU et al. 2016). Due to the existence of self or acquired chemotherapy resistance, its efficacy may be limited (CUI et al. 2018;SHAH 2015). Therefore, we urgently need to explore a combination therapy that could reduce the drug resistance of liver cancer cells and restore the sensitivity of HepG2 cells to CDDP.
The side effects and drug resistance of CDDP limit its application and effectiveness. Side effects are mainly reflected in nephrotoxicity, ototoxicity, and neurotoxicity (SONG et al. 2016). Previous studies have shown that drug resistance is a complex process involving many genes, including microRNA (miRNA) (CHENG et al. 2015). Recent studies have shown that miRNAs are key regulators of tumorigenesis and development (POWERS et al. 2016). They usually regulate tumor cell proliferation, migration, invasion, and drug resistance by targeting oncogenes, tumor suppressor genes, transcription factors, and other regulatory factors involved in cell death and survival (DVINGE et al. 2013;SHI et al. 2016;SHINDO et al. 2018). miR-340-5p inhibits cell proliferation and drug resistance in breast cancer cells by downregulating the LGR5 expression of the Wnt/â-catenin pathway . miR-340-5p reduces cell chemoresistance by targeting ZEB1 in osteosarcoma . miR-340-5p exerts its tumor suppressor function by directly targeting ANXA3 in liver cancer, and may be a new prognostic biomarker and therapeutic target . Our previous work confirmed that miR-340-5p is abnormally expressed in CDDP-R HepG2 cells, and found that most HepG2 cells have a low G1/S ratio. However, we are not certain about the role and mechanism of miR-340-5p involved in CDDP-R HepG2 cells. We speculate that miR-340-5p may interfere with cell division by regulating cell cycle factors.
Cyclin-dependent kinase 6 (CDK6) is a protein kinase that binds to cyclin and has kinase activity (BELLUTTI et al. 2018). CDK6 is indispensable in the normal progress of the G1/S process (ROMERO-POZUELO et al. 2020). There is evidence that cell cycle arrest is related to CDK6 and D-type cyclin. D-type cyclin competes with CDK inhibitor family 2 to bind to monomeric CDK4/6 to form an active cyclin D-CDK4/6 complex (ÁLVAREZ-FERNÁNDEZ & MALUMBRES 2020). A cyclin D-CDK4/6 complex could directly phosphorylate the transcription factor forkhead box protein M1 (FOXM1), and then induce the transcription of other genes involved in the G2/M phase of the cell cycle (ANDERS et al. 2011). There are three D-type cyclins in mammals (CyclinD1, CyclinD2, and CyclinD3) (LIU et al. 2016). The cyclinD-CDK4 complex is a regulator of the stability of CDC25A in the G1 phase, which generates a negative feedback loop to control the G1/S transition (DOZIER et al. 2017). CDK6 may mediate the cell cycle progression of HepG2 cells through the cyclin pathway, thereby changing cell proliferation, drug resistance, and apoptosis.
Although the results of previous studies suggest that miR-340-5p may affect the drug resistance of HepG2 cells, there is no relevant research on whether miR-340-5p could reverse the sensitivity of HepG2 cells to CDDP through CDK6. Through the research of this subject, we clarified the regulation mechanism of miR-340-5p on the HepG2 cell cycle and it's drug resistance through CDK6 so as to provide new ideas and a new basis for a CDDP combined treatment of liver cancer.
Cell Counting Kit-8 (CCK-8) assay
Cells in different groups were inoculated on 96-well plates (NU679, Dojindo, Japan) with 1×10 4 cells/well density, and 100 ìl per well. After adherent culture, 10 ìl/well of CCK-8 was added to each well for the corresponding time as described above. The CCK-8 solution was prepared in a complete medium. The Optical Density (OD) value of the absorbance of all samples at 450 nm was measured with a microplate reader (MB-530, China) after incubation at 37 o C and 5% CO 2 for 4 h. Cell viability = (OD Drug -OD Blank )/(OD Control -OD Blank ) ×100%. The IC50 of cells was calculated by the SPSS18.0 software profit regression model.
Clonogenic assay
The cells in the exponential growth stage were inoculated in a 6-well plate with 200 cells per well and cultured in an incubator at 37°C with 5% CO 2 and 95% humidity. The culture medium was replaced once every 5-7 days. Cell culture was terminated when macroscopic clones were observed. Then, the culture medium was discarded and the cells were carefully soaked in PBS twice. 1 ml of 4% paraformaldehyde was added to each well to fix the cells for 15 min. The fixation solution was removed and 1 ml of dye solution was added for dyeing at room temperature for 30 min. After decolorization, the OD value at 550 nm was determined by a microplate reader.
Quantitative real-time PCR (qRT-PCR)
The total RNA of the HepG2 cells (500 ìl) was extracted after 3 min of cell lysis with Trizol. In the next step, the total extracted RNA was reversely transcribed into cDNA in accordance with the instruction of a Reverse transcription kit (CW2569, CWBIO, China). Subsequently, real-time PCR was performed on a Fluorescence quantitative RCP instrument (QuantStudio1, Thermo, USA) with reference to the instructions of the UltraSYBR Mixture kit (CW2601, CWBIO, China). Reaction conditions were: predenaturation at 95 o C for 10 min, denaturation at 94 o C for 15 s, annealing at 60 o C for 30 s and 40 cycles. The internal references of the primer were U6 and â-actin, and the primer sequences are listed in Table 1. The relative transcription level of the target gene was calculated using 2 ìg total cDNA as a template by the relative quantitative method (2 !))Ct method): ))Ct = ) experimental group !) Control group, )Ct = Ct(target gene) ! Ct(â-actin). The experiment was repeated three times.
Dual-luciferase reporter assay
293A cells were inoculated in 24-well plates and grew for 10-24 hrs (80% confluence). PHG-miRTarget CDK6-3U plasmid (1 ìg/ìl) was co-transfected with miRNA NC (NC+CDK6 group) and hsa-miR-340-5p mimics (hsa-miR-340-5p+CDK6 group), respectively, into the 293A cells. 20 ìl of cell lysate was added into each plate, followed by 100 ìl of Luciferase Assay Reagent II (LAR II) working fluid. After mixing, the mixture was put into the luminescent detector to detect the Firefly luminescence value. Then, we added 100 ìl of Stop&Glo detection solution to stop the first light emission and detected the Renilla luciferase value.
Cell cycle assay
Cell suspension was collected and centrifuged at 800 rpm for 5 min to obtain cell precipitation. PBS was washed 2-3 times to prepare a single cell suspension with a cell concentration adjusted to 1 × 10 6 cells/ml, which was fixed with 70% ethanol. After the residual ethanol was removed, 150 ìl of propidium iodide (PI) solution was added and dyed in the dark at 4 o C for 30 min. Finally, the cell cycle was measured by flow cytometry (A00-1-1102, Beckman, USA) at 488 nm. The adherent cells and debris were excluded by the gating technique, and the percentage of each cell cycle of the PI fluorescence histogram was analyzed.
Apoptosis assay
The cells were digested and collected by trypsin without EDTA. About 5 × 10 5 cells were collected after washing with PBS twice and centrifuged at 2000 rpm for 5 min. 500 ìl of binding buffer was added to resuspend the cells. 5 ìl of annexin V-FITC (KGA108, KeyGen, China) and 5 ìl of propidium iodide were added to the cell suspension and mixed. Then, the reaction was kept at room temperature and kept away from light for 15 min. Within 1 hr, flow cytometry (A00-1-1102, Beckman, USA) was utilized to detect cell apoptosis.
TUNEL assay
Cells were inoculated in 24-well plates to make cell slides. The slide was fixed with 4% paraformaldehyde for 30 min and washed with PBS for 5 min x 3 times. TUNEL staining was performed using the Annexin V-EGFP Apoptosis Kit (KGA702, KeyGen, China) according to the manufacturer's instructions. DAPI (Wellbio, China) solution was used to restain the nuclei at 37 o C for 10 min, and PBS was used to wash the nuclei for 5 min x 3 times. Cell images were obtained under fluorescence microscope (BA410T, Motic, China) to count TUNEL positive cells.
Statistical analysis
All data were analyzed using Graphpad Prism 8.0 software (GraphPad Software, San Diego, California, USA). The unpaired T test was used between the two groups conforming to the normal distribution. Comparisons among multiple groups were conducted by a one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. A value of p<0.05 was considered to be statistically significant.
IC50 value of HepG2 in CDDP
To investigate the drug resistance of liver cancer cell lines with cisplatin, we compared the CDDP resistance of four different HCC cell lines. We found that the drug resistance of HepG2 was significantly lower than other liver cancer cell lines, including SMMC-7721, Huh-7, and Hep3B (Fig. 1A). Then, we constructed CDDP-R HepG2 cells to investigate whether there is a substance to reverse CDDP-resistance. CDDP-R HepG2 cells were selected by gradually increasing the concentration of CDDP in the medium. After 6 months, we calculated the IC50 value of sensitive and resistant CDDP HepG2. Compared with the IC50 value of 5.8 ìM of CDDP-S HepG2 cells as shown in Figure 1B, the IC50 value of CDDP-R cells was significantly increased to 115 ìM (Fig. 1C). The results indicated that the CDDP-R HepG2 cells were successfully constructed.
Expression of miR-340-5p and CDK6 in CDDP-S and CDDP-R HepG2 cells
In order to study whether miR-340-5p and CDK6 are related to drug resistance of hepatocellular carcinoma cells, we did qRT-PCR and WB experiments. We first detected the expression of miR-340-5p and CDK6 in CDDP-R HepG2 cells by qRT-PCR. The data showed that compared with CDDP-S HepG2 cells, the expression of miR-340-5p in CDDP-R HepG2 cells was much lower, but the mRNA and protein expression levels of CDK6 increased. Figure 2 shows that miR-340-5p and CDK6 are abnormally expressed in CDDP-R HepG2 cells. This may suggest that the drug resistance of HepG2 cells is related to miR-340-5p and CDK6.
Overexpression of miR-340-5p could inhibit CDK6 expression in HepG2 cells
Biological information showed that miR-340-5p could specifically target the 3'UTR of CDK6. We verify through experiments whether miR-340-5p could regulate CDK6 expression. We used a dualluciferase report assay and the results showed that miR-340-5p could target CDK6. Figure 3A and 3B suggested that miR-340-5p may affect HepG2 cells by regulating the expression of CDK6. We constructed the CDDP-R HepG2 cell miR-340-5p overexpression group. The data indicated that miR-340-5p is highly expressed in the miR-340-5p mimic group. Figure 3C shows that the miR-340-5p overexpression group was successfully constructed. We further detected the expression of the cyclerelated genes CDK6, CyclinD1, and CyclinD2. The results demonstrated that compared with the Control and mimic-NC groups, the mRNA and protein expression of CDK6, CyclinD1, and CyclinD2 in the miR-340-5p mimic group were suppressed ( Fig. 3D and 3E). We finally used flow cytometry to detect the cell cycle. It can be seen from Figure 3F, compared with the Control and mimic-NC group, the miR-340-5p mimic group showed a higher cell cycle staying in the G0/G1 phase, and the S phase cell ratio was significantly reduced. The above results indicated that miR-340-5p could inhibit CDK6 expression, thus affecting the cycle of CDDP-R HepG2 cells.
Upregulation of miR-340-5p could reverse the sensitivity of HepG2 cells to CDDP In the above results, it was found that miR-340-5p was abnormally expressed in CDDP-R HepG2 cells. We then investigated whether the upregulation of miR-340 had an effect on the CDDP sensitivity of HepG2 cells. First, we observed the proliferation capacity of HepG2 treated in different ways. Figure 4A shows that compared with the Control and mimic-NC groups, the cell proliferation ability of the miR-340-5p mimic group was lower. It suggested that the overexpression of miR-340-5p significantly inhibited the activity of CDDP-R HepG2 cells. We then examined the apoptosis and cloning of the cells. Compared with the Control and mimic-NC groups, the apoptosis rate of the miR-340-5p mimic group increased and cloning was inhibited. Figure 4B and 4C more fully display that CDDP-R HepG2 was less active and more prone to apoptosis after transfection with miR-340-5p overexpression. In brief, a high expression of miR-340-5p could reverse the CDDP sensitivity of HepG2 cells.
Inhibition of CDK6 could reverse the sensitivity of HepG2 cells to CDDP
The above experimental results indicate that CDK6 is abnormal in CDDP-R HepG2 cells. We further investigate whether CDK6 silencing has an effect on the CDDP sensitivity of HepG2 cells. We transfected CDDP-R HepG2 cells with CDK6 silencing plasmid. CDK6 expression was substantially inhibited in the si-CDK6 group. The data in Figure 5A reflected that the transfection of si-CDK6 plasmid was successful. Next, we used CCK-8 to detect cell proliferation in Figure 5B. Compared with the si-NC group, proliferation of CDDP-R HepG2 cells in the si-CDK6 group decreased. The clone number of the si-CDK6 group decreased. The experiment of Figure 5C and 5D confirm that inhibition of CDK6 reduces the activity of CDDP-R HepG2 cells. Figure 5E is the result of the MiR-340-5p Inhibits CDK6 in HepG2 Cells 61 Fig. 2. Expression of miR-340-5p and CDK6 in HepG2 cells. A. CDDP-R HepG2 had low expression of miR-340-5p and high expression of CDK6 at the mRNA level. B. CDK6 was highly expressed in CDDP-R HepG2 at the protein level. * -compared with the CDDP-S group, p<0.05. Fig. 3 A-F. miR-340-5p targets CDK6 expression. A -The TargetScan website showed miR-340-5p had interaction with CDK6. B -The dual-luciferase reporter assay proved that miR-340-5p could target CDK6. Luciferase activity in 293A cells was significantly inhibited by the CDK8-WT + miR-340-5p mimic group. C -miR-340-5p was overexpressed in the miR-340-5p mimic group. D -The mRNA expression of CDK6, CyclinD1, and CyclinD2 in HepG2 was inhibited by miR-340-5p. E -The protein expression of CDK6, CyclinD1, and CyclinD2 was inhibited by miR-340-5p. F -The over-expressed miR-340-5p of HepG2 cells had a higher cell cycle staying in the G0/G1 phase, while the proportion of cells in the S phase was significantly reduced. * -compared with the Control group, p<0.05. # -compared with the mimic-NC group, p<0.05.
flow cytometry cycle test. Compared with si-NC, the ratio of G0/G1 cells in the si-CDK6 group increased, while the ratio of S-phase cells decreased. After inhibiting CDK6, the cells stayed in the G0/G1 phase. Finally, we conducted a TUNEL assay, and the data showed that compared with si-NC, the apoptosis of CDDP-R HepG2 cells in the si-CDK6 group was considerably increased. In short, when CDK6 was inhibited, it could affect the CDDP sensitivity of HepG2 cells by changing the cell cycle.
Discussion
Incidences of liver cancer are increasing year by year. Surgical resection supplemented with chemotherapy is the main treatment for early patients (CHENG et al. 2018). Cisplatin chemotherapy works by interfering with cell metabolism and DNA replication (LU et al. 2016;OH et al. 2014). Since the efficacy of chemotherapy is mainly affected by the natural or acquired drug resistance of tumor cells, the research on the mechanism of cisplatin resistance and MiR-340-5p Inhibits CDK6 in HepG2 Cells 63 Fig. 4 A-C. Overexpression of miR-340-5p reverses CDDP sensitivity in HepG2 cells. A -A CCK8 assay was used to detect cell proliferation. Cell activity was decreased by miR-340-5p interference. B -Annexin V-FITC and PI were used to evaluate the apoptosis rate. The apoptotic rate of CDDP-R HepG2 cells transfected with overexpressing miR-340-5p was increased. C -The Clonogenic assay was used to detect the number of cloned cells. The cloning ability of HepG2 cells was inhibited by miR-340-5p. * -compared with the Control group, p<0.05. # -compared with the mimic-NC group, p<0.05. Fig. 5 A-E. Silencing CDK6 could reverse the sensitivity of HepG2 cells to CDDP. A -CDK6 siRNA transfection efficiency. B -A CCK8 assay was used to detect cell proliferation. C -A Clonogenic assay was used to detect the number of cloned cells. HepG2 cell cloning ability was inhibited after CDK6 silencing. D -A TUNEL assay was used to detect the apoptosis rate (H100, Scale bar=100 ìm.). HepG2 cell apoptosis increased after CDK6 silencing. E -Cell cycle was detected by flow cytometry. The ratio of G0/G1 cells in the si-CDK6 group increased, while the ratio of S-phase cells decreased. * -compared with the si-NC group, p<0.05.
the strategy of reversing drug resistance have always been hotspots in the field of cancer research (TIAN et al. 2017). miRNAs could directly affect the target gene by recognizing and complementing the 3NUTR end of the target gene, down-regulating the expression of the target gene, thereby exerting its biological function (BA et al. 2019;ZHANG et al. 2019). This topic mainly explores the expression level of miR-340-5p and the CDDP-R HepG2 cells.
Recent studies have reported that CDK6, a key protein that regulates the cell cycle, could be specifically bound by SUMO1 in the SUMO family, thereby regulating the tumor cell cycle. CDK6 protein is degraded by ubiquitin, which significantly reduced the protein expression level, and then induced tumor cells from rapid proliferation cycle to slow proliferation cycle state (REN et al. 2019). In the bioinformatics prediction of miR-340-5p targeting CDK6 signal to reverse the molecular mechanism of cisplatin resistance in HepG2 cells, we found that miR-340-5p has species conservation. In the 5' UTR of miR-340-5p, 8 bases were completely complementary to the CDK6 mRNA3'UTR, and the free energy between the two was relatively low, so we speculated that CDK6 might be the target gene of miR-340-5p. In this study, by transiently translating si-CDK6 and miR-340-5p mimics, it was found that both CDK6 and miR-340-5p could affect the sensitivity of HepG2 cells to CDDP, and through computer biological information prediction, it was found that CDK6 may be the downstream target of miR-340-5p. In order to verify the results of bioinformatics detection, a luciferase report test found that the expression levels of miR-340-5p and CDK6 were synchronized, that is, the expression level of overexpressed miR-340-5p could inhibit the increase of CDK6 expression level. It further demonstrated that miR-340-5p could directly act on CDK6, that is, by directly targeting the 3'UTR region of CDK6, CDK6 could be inhibited, which results in the reduction of CDDP resistance and the increased chemotherapeutic sensitivity of HepG2 cells.
The cell cycle is divided into different time stages, G1, G0, S, and G2 and M. Cyclin D binds to cyclindependent kinases (CDKs) to the phosphorylate pRb protein, which plays an important role in regulating cell cycle progression . In this experiment, the concentration of CDDP-R HepG2-resistant miR-340-5p was changed by the transfection of the miR-340-5p mimic, and then fluorescent quantitative PCR and western blotting were used to detect the changes in the transcription and translation levels of the CDK6 gene and its downstream genes, CyclinD1 and CyclinD2. After the overexpression of miR-340-5p in CDDP-R HepG2 cells, the mRNA and protein expressions of CDK6 and its downstream genes, Cy-clinD1 and CyclinD2, were effectively inhibited. The above studies fully show that miR-340-5p has a certain targeting relationship with CDK6, and miR-340-5p could target the CDK6 signal transduction pathway to partially reverse the occurrence of cisplatin resistance in HepG2 cells. Since this research is still in the basic research stage, there are still a lot of problems waiting to be overcome before clinical application. Under the effect of CDDP, the apoptosis of hepatoma cells increased and proliferation ability decreased. In future work, we will clarify the expression characteristics and laws of CDK6 and CyclinD1 in HepG2, and explore the mechanism of CDK6 protein action and its role in the malignant transformation and radiotherapy resistance of liver cancer, so as to provide new methods and ideas for the future treatment of liver cancer.
In conclusion, miR-340-5p was significantly down-regulated in CDDP-R HepG2 cells. Overexpression of miR-340-5p leads to enhancing CDDP sensitivity. CDK6 is the direct target of miR-340-5p, and high expression of CDK6 improves CDDP sensitivity. Overall, our results indicated that miR-340-5p played a critical role in regulating CDDP chemosensitivity by targeting CDK6 in HepG2 cells. In this study, we discussed the expression pattern and biological effects of miR-340-5p in the CDDP chemosensitivity of HepG2, which may provide a new perspective for the treatment of liver cancer.
Conflict of Interest
The authors declare no conflict of interest. | v3-fos-license |
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} | pes2o/s2orc | Effect of Roasting and Kneading on Antioxidant Activity and Consumer Acceptance towards Asiatic Pennywort Tea
. Nowadays, population have more age and longer life than ancient people which the World Health Organization (WHO) reported that the proportion of the global population will become ‘Aging Society’ during healthier lifestyles, new advance technologies especially in medical manufacture. This study was aimed to study attitude, behavior and opinion of elderly people towards herbal tea products and to optimize preparation, procedure and process of Asiatic Pennywort tea. According of the study, there are 100% of Thai people ever consume herbs and herbal tea, they consume as beverage. Half of consumer surveys are no chronic health conditions. Safety of product is the most factors that effect to purchasing decision. The consumers are agree (73%) in nutrition value that presented on label as it’s clamed on the package. Result from this study shown that roasted and kneading process is highest mean score from consumer’s preference 7.07±1.51 (P<0.05) that this method is significantly different in total phenolic content and Ferric reducing antioxidant potential assay but not in DPPH radical scavenging which is highest mean of second significantly level.
Introduction
There are more than 7 billion living humans on earth and the rate of population in each country is increasing that is determined by births and deaths. Asia is the most populous region in the world or 60% of world population which China is the highest population in the world and follows with India. Rank of Thailand is 19 which have 68 million humans. United Nations Population Fund (UNFPA) reported that the world population has less amount of children population that means elderly population are increasing. The world society will change to Ageing Society. Thailand has chance to be Aging Society because the rate of elderly population is 5.7% in 1984 and grow up to 9.6% in 2003 which can expect that Thailand will has elderly population to 14.7% in 2019. Elderly definition is an old person who has age more than 60 years old. When entering old age, your senses (taste, smell, touch, vision, and hearing) become less acute and have some trouble in daily life. Herbs and spices have used as a tradition flavor enhancement for each characteristics and their medicinal properties. Herbs and spices are small plant that does not produce woody persistent tissues and has medicinal properties. Herbs and spices can use different parts of plant through processes such as roasting, smoked, fermented or fresh which consume herbs and spices in small amounts can help in bioactivities in human body which can provide health benefits for consumers [1][2][3][4]. Thai people have consumed herbal plant as medicines. Herbal plant can use in many ways. Tea is dried leaves of Camellia sinensis plant that usually consume by infuse in hot water. Camellia sinensis plant contains all kinds of great properties, including vitamins (E, C), caffeine and antioxidants. There are several types of tea that made from Camellia sinensis plant. In each types of tea made from different parts such as green tea, black tea, white tea and oolong etc. and different processes such as roasted, air dried and fermentation to create the specific flavors. Herbal tea will be separate from tea. Herbal tea can call "tisanes" (tea zahn) that is commonly used plants from herbs or spices but normal tea use Camellia sinensis plant. Herbal tea is the beverage that can get from using parts of herbs or spices then pass though air dried or fermentation process by pass through soaking, brewing or boiling process as normal tea. There are several herbs that are popular in Thailand and contain several benefits with human health. However, there are the most popular one is 'Asiatic Pennywort' or Centella asiatica or Gotu kola. Thailand has 4 herbs popular products which is Gotu kola (Asiatic Pennywort), Tumeric, Phlai (Zingiber cassumunar) and Black galingale (Kaempferia parviflora Wallich. ex Baker.) that is Thailand's Signature of herbal products. Asiatic Pennywort is important herbal medicinal plant used for many benefits [5]. Ancient people was use in treating all disease for long time such as wound healing and eczema. [6][7] Asiatic pennywort contains biological activities compounds for human health, during it contain antioxidant [8][9][10][11] that can help in anti-inflammatory [12] and memory enhancing property [10][11]. The main active constituents of Centella asiatica are triterpenes [13], polyphenols [14] and triterpenes [15] such as asiatic acid, asiaticoside, medecasic acid and madecassoside that resistant with antioxidation reaction [16]. Asiatic acid in the triterpenes component responsible for stimulating collagen synthesis of human fibroblasts [17]. Free radicals have been played an important role in ageing capable of damaging many cellular components [18]. Antioxidant will protect oxidative stress reaction. Reactive species (ROS) is low stable molecule, it need an electron to make itself more stable. So, reactive species will bring electron form other cells as a cycle that effect to mitochondria membrane and cell will damage. Protection against free radicals can be enhanced by intake of dietary antioxidants which may be major importance in disease prevention. Therefore, the aim of this project was to study the effect of roasting and kneading on antioxidant activity and consumer acceptance of Asiatic Pennywort tea.
Study attitude, behavior and opinion of elderly people towards tea products
There were 100 people participated in this survey. Questionnaire was developed as a tool to gather the information which it was divided into 6 parts which were Part 1: Consumer's behavior about herbs and tea, Part 2: Consumer's attitude and purchasing behavior, Part 3: The factors influence purchasing decision. this part was asked based on their important factors by rating level of importance which used 1 referred to not at all importance and five referred to extremely importance, Part 4: Food consumption behavior based on their knowledge which ask based on their thinking with ability and expectation from products, Part 5: General Information included questions regarding demographic and socioeconomic position of participants such as age, gender, education, house members, income, occupation and chronic health conditions.
Procedure
Asiatic Pennywort was purchased from local markets in Bangkok, Thailand. The Asiatic Pennywort was graded that the leave should not have yellow color, bite from insects and free from diseases and pesticides. Fresh Asiatic Pennywort was wash with tap water or rinse before use to clean any soil and dust. For roasted process, after cool down the leaves sample was roasted in the pan at 45 °C for 25 minutes. After that, the leaves were unfolded before put in hot air oven at 80 °C for 2 hours.
Sensory testing and sample preparation
There were 30 elderly people with age between 50 -75 years old participated in the test whose can be male or female. 2 grams of Asiatic pennywort dried leave was put in tea bag. Brew tea with 150 ml of 90 °C water. The herbal tea bag was soaked for 5 minutes. After that, the herbal tea bag was shaken up and down for 10 times and serve at 65°C -70°C.
Chemical analysis
The Asiatic pennywort tea was determined amount of antioxidant activities. The basic methods were used. The modified Folin-Ciocalteu method [19], DPPH radical scavenging [20] and Ferric reducing antioxidant potential assay [21] were used for evaluating antioxidant activity.
Antioxidant activity by determine total phenolic content
The tea sample The tea sample 20 µl of 10 mg/ml was mixed with 1.58 ml of distilled water and 100 µl Folin-Ciocalteu phenol reagent [22]. The samples were stood for 8 minutes 30 seconds at room temperature. Then, add 300 µl saturated sodium carbonate solution and incubated in dark place at room temperature for 30 minutes. After that, the test samples will be observed the optical density (OD) at 765 nm. The data was calculated as microgram garlic acid equivalent (µgGAE/ml). The experiment was done in triplicate [23].
Antioxidant activity by DPPH radical scavenging activity
DPPH radical scavenging activity assay [20]. This assay uses to show free radical scavenging activity between DPPH solvent (2, 2-diphenyl -1-picrylhydrazyl) and an antioxidant. Tea sample 2 µl was mixed with 2 ml of methanol. Then, DPPH was mixed with 100 ml of methanol. After that, volume of DPPH reagent was varied with the volume of tea sample. The varied sample was leaved in the dark place for 30 minutes. The test samples was measured the optical density (OD) at 517 nm. The result will be expressed as percentage reduction of DPPH. All measurements should analyze in triplication and three replications.
Where A 0 is the initial absorbance and A C is the value for add sample concentration. The tea sample was used for 2 µl mixed with FRAP reagent 1,000 µl, mix well. The mixture was kept in the dark place at room temperature for 30 minutes. After that, the mixture solution was measured the optical density (OD) at 593 nm. The value will be calculated as mmol Fe 2+ /mg of sample. All measurements should analyze in triplication and three replications. FRAP reagent is mixture between sodium acetate buffer pH 3.6, Iron chloride and 2,4,6 tripyridyl-s-triazine.
Statistical analysis
Data of survey information were processes using SAS version 9.4 (Copyright © [2017] SAS Institute Inc., Cary, NC, USA.), Randomized Complete Block Design (RCBD) was applied as experimental design. The data in this experiment was analyzed by using ANOVA (analysis of variance) and Fisher's LSD test(p<0.05) for multiple comparison. Sensory testing was used about liking score with 9 point hedonic scale.
Consumer behavior
They consumed Thai herbs (66%) and consumed as beverage (42%), as medicines (31%) and eat as food in meals (27%). The highest herbal tea that consumers had ever consumed is Chrysanthemum tea (16%), Ginger tea (14%) and Lemon glass tea (13%). They like to consume They tea from tea bag (34%) and consume only 1 time in a month (23%) which no specific the time to consume herbal tea (45%), in morning (26%) and afternoon (21%). Consumers purchase herbal tea at hypermarket the most (37%), and in each time will purchase more than 75 baht (41%) for herbal teas. The consumers agreed (73%) in nutrition value that presented on label as it's clamed on the package which they think that their body will absorb the nutrients from herbal teas only 50 percent (39%), 25 percent (33%) and 75 percent (24%). The reason of consumers consume herbal tea is contain medical benefit (28%), make them feel fresh (23%) and relaxing (18%). There are significant different between factors effect on purchasing decision (p<0.05). The most important factor is a safety; it means that they like to safe in consume the tea. Next factors are certification, nutrition, and taste. It means that consumer want to consume good products which should has approved from FDA or other warranties due to their age. The taste is also significantly important in the same level as medical properties. Taste relate to perception of consumer that should be good and more benefits together. Moreover, they are not concern in organic product or source, the reason might be the properties contain in herbs are mostly the same benefits. Package, Net weight and Brand of herbal tea products also not affect to purchasing decision. Paper tissue has used for package of tea both inside or outside for tea infusion. Plastic bottle has used as package of tea bottle that easy to consume than tea infusion or when you want to consume tea immediately. Volume or weight is not affected to their decision. Elderly people is not care much in the volume because they are more concern in taste than others factors. From survey found that they are consume only 1 time in a month. Brand and trend are lowest factors for elderly people; it means that famous products, social products or popular products are not effect to their purchasing decision. Nowadays, several was produced and lunched by small group of villagers or they can make it by themselves. Table 2: Liking score of Asiatic Pennywort tea from 30 consumers in 10 attributes.
Process
Overall liking * Color Greeny aroma * Roasted aroma Sweet Roasted also reduce greeny aroma that occur in the tea products [24][25].
Chemical analysis
Herbs are rich in phenolic compounds which are active compound and act as antioxidant properties. Asiatic Pennywort also has active compounds such as triterpenes [13] and polyphenols [14] which contains high total phenolic contents. Total phenolic content was determined with standard garlic acid in term of µg GAE/ mg dried weight. The highest antioxidant activity represented by amount phenolic compound that is 62.7 ± 32.8 µg GAE/mg dried weight which use Roasted & kneading process. This process is most significant different but not significant different from another 2 processes that is Not roasted & kneading and Not roasted & not kneading (p<0.05). Thermal process is a big influence in active compound which depend on their magnitude, duration and different heating methods. Thermal process lend to degradation of phenolic compounds. For example, roasting barley at 280 °C for 20 seconds can decrease 8% of phenol content [26]. For DPPH assay is significantly different in all processes that the highest mean is Not roasted & kneading process 0.3 ± 0.1 % DPPH radical scavenging (P < 0.05). Using convection oven drying with spearmint can decrease antioxidant activities ICCMP 2018 antioxidant activities approximately 60% which heating go to inactivate enzyme. [27]. Kneading was used to macerate the cells of leaf which can activate enzymes (mixing of chemical with enzyme) inside the leaf and can be easily being extracted during brewing. [28] For Ferric reducing antioxidant potential assay, there are not significantly different in all processes which the highest mean score is Not roasted & not kneading process 1.9 ± 0.2 mmol Fe 2+ /mg (P < 0.05).
Conclusion
The obtained result in this project was used to study habit, attitude and decision of elderly people. The process that will be selected to produce the Asiatic Pennywort tea depend on what is the most concern that if we concern at highest in both testing from sensory analysis from consumer and active compound, Roasted & kneading process will be selected to produce the Asiatic Pennywort tea. This process is get most significant different in mean scores for sensory, total phenolic content and ferric reducing antioxidant except DPPH radical scavenging has highest mean score from second significant. | v3-fos-license |
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} | pes2o/s2orc | A Novel, Stable, Helical Scaffold as an Alternative Binder — Construction of Phage Display Libraries
Figure 2. Electrophoretic analysis of virus domain purification. M — molecular weight marker, T — total bacterial lysate after sonication, S — soluble fraction of bacterial lysate, FT — flow through after binding to glutathione resin, E1-E5 — elution fractions after glutathione resin purification, U — uncleaved fusion of virus domain with GST, C — fraction after rTEV cleavage, GII — fraction after cleaved GST removal by glutathione resin, S1, S2, S3 — fractions after size exclusion purification (Superdex 75). Molecular weight of fusion proteins — around 33 kDa, GST — 27 kDa, virus domains — 6 kDa. The final SDS/PAGE analysis were perfomed on a tricine gel to enable visualization of small protein fragments.
InTRoducTIon
The most important proteins showing specific molecular recognition are antibodies.The ability to interact with almost any antigen makes them genuinely universal binders.Immunoglobulin-based molecules represent the fastest growing class of biopharmaceuticals used both in therapy and diagnostics.However, because of their numerous limitations such as the large size, presence of disulphide bonds and low conformational stability, alternative scaffolds are badly needed (Binz et al., 2005;Skerra, 2007).Several attempts have been made to introduce alternative binders, from small helical domains (Nord et al., 1997;Nygren & Uhlen, 1997;Nygren, 2008) and repetitive modules of Designed Ankyrin Repeat Proteins called DARPins (Binz et al., 2004;Tamaskovic et al., 2012) to the Ig-like β-structure scaffold of fibronectin type III domain (Koide et al., 1998;Koide et al., 2007).The main advantage of these novel binding proteins is their robust structure, high stability and efficient bacterial expression.They have also been proven to exhibit binding properties comparable to those of antibodies (Wikman et al., 2004;Lendel et al., 2006;Orlova et al., 2006;Zahnd et al., 2006;Zahnd et al., 2007;Friedman et al., 2008;Milovnik et al., 2009).
The specific binding molecules are selected from combinatorial libraries based on a technology that enables linking phenotype with genotype.The combinatorial library can be presented using either conventional phage display method (Smith, 1985;Smith & Scott, 1993) or in vitro based technologies like mRNA and ribosome display (Amstutz et al., 2001).Phage display has limitations in the library size (due to finite bacterial transformation efficiency) but, on the other hand, is much less demanding than the in vitro technologies.The main key to the success of phage selection is the size and quality of the library used for selection of specific binders (Ling, 2003).
Since the sequence diversity generated through randomization at several amino acid positions may lead to selection of variants with highly destabilized structure, it is accepted that scaffolds with favorable stability properties should be used as a template for phage display libraries (Nygren & Uhlen, 1997).
One of the first proposed alternatives to immunoglobulins was domain Z from staphylococcal protein A, called affibody.This 58-aminoacid three-helix bundle protein, despite a flat paratope, has been proven by phage display to bind several targets with high specificity and affinity (Nygren, 2008).These favorable binding properties of the affibody encouraged us to look for a structurally similar scaffold and apply it to construct phage libraries.
In this publication we investigated the potential of Measles virus phosphoprotein P sequence that folds into a three-helix bundle structure as a protein scaffold for phage display libraries.We tested the expression level, solubility and denaturation parameters of this protein.
We used this sequence to construct several phage libraries with different randomization types and translocation pathways.The libraries were fully characterized and initial selection was performed.
MATeRIAl And MeTHodS
Strains, vectors and enzymes.Escherichia coli strain DH10 was used for standard cloning, strain BL21(DE3) CodonPlus-RIL (Stratagene, USA) for expression of virus domains.Phage propagation was carried out in E. coli XL1-Blue (Stratagene).E. coli MC1061F' strain used for electroporation was obtained by standard mating protocol of E. coli MC1061 (Biorad) and XL1-Blue strains.Helper phage M13-VCS was obtained from Stratagene.
Restriction enzymes, ligase and polymerase were obtained from Fermentas, oligonucleotides from Metabion and GeneLink.The Gateway system (Invitrogen) was used for cloning of virus domains in vectors pDONR201 and pDEST15.
The Mumps virus domain DNA sequence was generated by PCR with primers MumA, MumB and MumC.Sequences encoding virus domains with flanking regions containing attb1 and attb2 sites together with an rTEV cleavage site were obtained by standard two-step PCR with primers shown in Table 1 (Supplementary Materials at www.actabp.pl).After purification (PCR purification kit, A&A Biotechnology), the PCR product was first cloned into the pDONR201 vector using the Gateway recombination system (Invitrogen).After restriction analysis the cloned genes were transferred into the expression vector pDEST15 by LR reaction (Invitrogen).The sequence of the coding region was checked by sequencing with the pGEX5 primer (Agowa, Germany).
Expression was done in E. coli BL21 (DE3) RIL strain in standard LB medium, till OD 600 =0.7 and then induced with 1 mM IPTG for 12 h at 30°C.
Bacterial pellet was resuspended in buffer A (50 mM Tris pH 8, 200 mM NaCl, 5 mM EDTA) sonicated (5×5 min with 5 min breaks) on ice, and centrifuged.Soluble fraction was applied to equilibrated Glutathione Superflow resin (Amersham Biosciences) for 3 h.Extensive washing was performed with buffer A and elution with 20 mM GSH buffer.Collected fractions were analyzed by SDS/PAGE.Fractions with the fusion protein were pooled and dialysed against buffer B (50 mM Tris pH 8, 150 mM NaCl, 2 mM DTT, 1 mM EDTA) and digested with rTEV protease.Digested protein was bound to glutathione resin (1 h 4°C), the unbound fraction was concentrated (Amicon) and applied to size exclusion chromatography (Amersham Biosciences, Superdex 75) to get rid of the rTEV protease and traces of GST protein.Fractions containing virus domains were pooled and stored at -80°C in buffer A. Purity of the protein was confirmed by SDS/PAGE (Laemmli, 1970) and mass spectra (Applied Biosystems AB 4800+ MALDI TOF/TOF).
Biophysical characterization.The folded state of the proteins was confirmed by CD spectra recorded in the wavelength range 200-270 nm in 10 mM Tris buffer pH 7 at 293 K on a Jasco J-715 spectropolarimeter.Thermal stability was measured by following changes in ellipticity at 222 nm, using 10-mm cuvette with a slit width set to 2 nm and a response time of 8 s.Transition was monitored at a constant rate of 1 deg/min.
Three different pH conditions were applied: 10 mM Tris pH 7.0, 20 mM sodium phosphate pH 5.6, and 20 mM glycine pH 2.9.Chemical denaturation of the protein at 15 μM concentration was performed by incubation in various concentrations of GdmCl in Tris buffer at 20°C for 12 h followed by CD signal measurement (222 nm) in a 10-mm cuvette.Data were analyzed using GraphPad Prism software assuming a two-state reversible equilibrium transition .
Construction of libraries.Primer sequences are shown in Table 2 (Supplementary Materials at www.actabp.pl)and the assembly scheme is presented in Fig. 1.For the NNC and HWC type of randomization two main primers (A and B for each library) were hybridized and then elongated with Klenow fragment exopolymerase (1 h, 37°C).The product was extracted from agarose gel (Gel-out, A&A Biotechnology) and used for two subsequent PCRs in order to obtain a full length randomized sequence with proper restriction sites.Ctype libraries, based on conventional pelB signal peptide, were cloned into pComb3H vector using NcoI and SpeI restriction sites.BamHI and EcoRI restriction sites were used for cloning of S-type libraries employing SRP translocation pathway.In order to obtain a highly diversified set of final sequences, gradient PCR was applied.Randomized DNA fragments for the C4 and S4 libraries were obtained through amplification of the long primer BIBCS4GL in a standard PCR with the Cfor and CRev, SFor and SRev primers, respectively.
Ten micrograms of final, cleaned-up product and 20 µg of vectors were digested at 37°C overnight.Ligation of purified digests was performed overnight at 17°C with T4 ligase.The mixture was subjected to phenol/ chloroform extraction and ethanol precipitation.DNA was dissolved in 100 µl of sterile water and used for electroporation in ten shots.
Electrocompetent cells were prepared according to a standard protocol (Wojcik et al., 2010).Transfromants were grown on ten big (25-cm diameter) plates with 2xTY medium supplemented with 1% glucose and antibiotic.Serial dilution in 2xTY medium was done to estimate the size of the library.Cells were scrapped from plates and libraries were kept at -80°C as a glycerol stock.The library was grown in 2×TY medium with an- tibiotic and infected with helper phage M13-VCS.The phage was purified from bacterial supernatant by precipitation with PEG according to the standard protocol (Russel M, 2004).To allow functional display the phage was further amplified in the XL1-Blue strain and purified as described above.The Mv3α-displaying phage particles at the concentration of 1×10 13 cfu/ml were stored at -80ºC, in PBS supplemented with 10% glycerol.
Characterization of libraries -size, quality, display.Thirty-six random clones from each library were isolated and sequenced (Agowa).PCR was also performed on random 20 clones from each library.To analyze the display of Mv3α phage particles were analyzed by Western blotting with use of anti-pIII antibody (MobiTec).10 10 phages were subjected to SDS/PAGE and then transferred to PVDF membrane by a standard semi-dry protocol.The membrane was blocked with 5% BSA in TBS overnight, then incubated with a 1:1000 dilution of a primary antibody, washed and incubated with secondary anti-mouse HRP-conjugated antibody (Jackson ImmunoResearch) at the 1:10 000 dilution.SuperWest signal (Pierce) was used for signal detection.For SRP libraries mouse anti-FlagM2 (Sigma) antibodies were used.
Test selections.Human S100A7 was expressed as a fusion with GST from the pDEST15 vector and purified as described for the virus domains.Purity of the antigen was confirmed by SDS/PAGE and mass spectra analysis.For solid surface selection the target protein was immobilized on immunotubes overnight at 4°C in 100 mM NaHCO 3 pH 9.6 at the concentration of 50 µg/ml.Tubes were blocked by 1-h incubation with 2% MPBS (PBS buffer containing 2% powder milk) at room temperature.For the first round of selection 10 13 phage particles were used, after 2 h of incubation (with rotation) unbound phage was removed by washing five times with PBST (PBS containing 0.1% Tween) followed by five washes with PBS.Bound phage was eluted with 1 ml of 100 mM triethylamine for 8 min and neutralized with 0.5 ml of 1 M Tris/HCl pH 8. Eluted phage particles were used for infection of exponentially growing E. coli XL1-Blue for 40 min at 37°C.Centrifuged bacteria were grown overnight on large 2TY agar plates with 33 µg/ml chloramphenicol and 1% glucose.Bacterial lawn was scrapped from the plates and used for inoculation to OD 600 =0.1 of 200 mL of 2TY broth containing chloramphenicol and 1% glucose.After reaching OD 600 =0.5, bacteria were infected with 10 10 helper phage at a final concentration of 10 10 , after centrifugation bacteria were resuspended in 2TY broth containing chloramphenicol, kanamycin and 0.1 mM IPTG and grown overnight at 30°C. 10 12 purified phage particles were used for subsequent rounds of selection followed by ten washing steps with PBST and PBS each.Five rounds of panning were conducted.
For selection in solution the antigen was biotinylated with EZ-link Sulfo-NHS-SS-biotin (Pierce) according to the manufacture's instructions.The level of biotinylation was estimated by mass spectrometry.All tubes used for selection were blocked with 0.1% gelatin in PBS.Before each round of panning negative preselection was performed -phage particles were incubated with preblocked streptavidin-coated magnetic beads Dynabeads (Invitrogen) for 1 h at RT with end to end rotation.For selection the phage was incubated with the biotinylated agent in 0.1% gelatin PBS, followed by 15-min incubation with Dynabeads to capture the phage-antigen complexes.After washing the Dynabeads with PBST and PBS the bound phage particles were eluted by incubation in 100 mM DTT in PBS for 15 min at RT.The amount of antigen used was decreased from round to round, while the washing conditions were progressively more stringent.Binding to the antigen was performed at RT or 4°C, depending on the round.Details of the selection conditions are given in Table 3.
Polyclonal phage ELISA.Polyclonal phage ELISA was performed for both solid phase and in-solution selection.A pool of 10 10 phage particles after each round of panning was incubated for 2 h at RT with the immobilized antigen (directly -for MaxiSorp selection or indirectly via streptavidin -for Dynabeads).As a control, phage was incubated in wells with buffer alone or with streptavidin.After washing with PBST and PBS, anti-M13 HRP-conjugated antibodies (Amersham Biosciences) were added for 1 h at RT. Another step of washing was performed and the amount of bound phage was detected with TMB solution (Sigma).The reaction with HRP was stopped with 1M H 3 PO 4 (Sigma) and determined colorometrically.
ReSulTS And dIScuSSIon choosing scaffold
We applied MSDfold software to identify protein domains with high structural similarity to the protein Z specific affibody for which the crystal structure is known (PDB code 1lp1/a).We identified three sequences of phosphoprotein P from Measles, Sendai and Mumps viruses that form 50-aminoacid helical domains with no sequence similarity to the affibody.The coding sequences of the virus domains were cloned into the pDEST15 vector enabling expression with a GST tag.The proteins were purified with affinity chromatography on glutathione resin, then the GST-tag was removed using rTEV protease and the final purification step was performed by size exclusion chromatography.The expression level for all three domains was high and the proteins were soluble (Fig. 2 -Supplementary Materials at www.actabp.pl).All three proteins were monomeric as judged from their size exclusion chromatography profiles (data not shown).The yield was about 15 to 20 mg of purified protein per 1 liter of culture.The purity assessed by SDS/PAGE and mass spectra analysis exceeded 97%.
Stability studies of the three domains were performed by thermal and chemical denaturation monitored by the CD signal at 222 nm.In agreement with recent results (Kingston et al., 2008) the Mumps virus domain had a poor stability profile and formed a molten globule in solution at pH 7.0 (data not shown), therefore it was excluded from further studies.The stability parameters of the Measles protein at pH 7.0 could not be assessed by CD spectra, as the thermal transition was not com- pleted up to 95°C and T den value could only be estimated to be above 80°C (Fig. 3B).At this pH the Sendai domain showed a much lower T den of 47.2°C.The [GdmCl] 1/2 values obtained from chemical denaturation at pH 7.0 were significantly lower for the Sendai than for the Measles domain: 2.2 M and 4.2 M, respectively.In agreement, the stability expressed as ΔG H2O den was much higher for the Measles domain than the Sendai one: 6.2 kcal/mol and 1.9 kcal/mol, respectively (Table 4).
To assess the T den of the Measles protein more exactly we analyzed temperature-induced transitions at a lower pH: 20 mM Gly pH 2.9 or 10 mM phosphate pH 5.6 (Fig. 3 and Table 4).While at pH 5.6 the T den of the Measles protein could still not be precisely determined, it was well over 40°C higher than that for the Sendai domain.At pH 2.9 the difference of T den between the two domains was 12°C.We also observed that the stabilty of the Measles virus domain in the harsh conditions of pH 2.9 could be easily improved by adding NaCl to 200 mM (Fig. 3 and Table 4).
Thus, the three domains studied, despite their structural and functional similarity, revealed completely different stability properties, from the molten globule of Mumps domain through the medium stable Sendai protein to the remarkably stable Measles domain.The latter domain with its superb stability properties was chosen as the template for library construction.
design of libraries
The main concern in designing libraries was to keep a balance between the theoretical and achievable library size to allow the majority of library sequences to undergo selection.The binding potential of an affibody is based on 13 amino acids located at two of the three helices that form a flat binding surface.Although different from the antibody CDRs (Complementarity Determining Region), the affibody binding surface buries a similarly large surface area of about 1650 Å 2 in the complex with a target macromolecule and forms eight or nine H bonds (Lendel et al., 2006).The key aspect in library construction is to efficiently design sequence diversity, meaning a rational introduction of randomized positions with respect to the structural nature of the scaffold and the library size limited by the bacterial transformation efficiency.Thus, our main goal in the library design was to limit their theoretical size in order to obtain representative libraries.
Since full randomization of 13 positions gives an unrealistic number of amino acid sequences (about 8.2×10 16 ) we decided to randomize only eight amino acid positions that appear to be crucial for the interaction with a target according to the available crystal structures of affibody complexes (Wahlberg et al., 2003;Lendel et al., 2006).The eight randomized positions include those that are in the binding hot spot and form hydrogen bonds with the target molecule.By applying the hard randomization scheme NNC to those eight amino acid positions we limited the theoretical library size to 2×10 9 , which can be achieved by bacterial transformation (Table 5).We also used two soft HWC and KMT schemes of randomization (for details of the randomization schemes, see Table 5).
The soft randomization KMT allowing only four amino acids: Tyr, Ala, Ser, Asp was proposed based on the generation of low-nanomolar antibody mimics obtained from four amino acid code libraries by the Sidhu's and Kossiakoff's groups (Fellouse et al., 2004;Sidhu & Kossiakoff, 2007).It was also shown that such minimal diversity allows selection of high-performance single-domain binders (Koide et al., 2007).Using this scheme we randomized 11 positions which led to the library size of 4 11 (=4×10 6 ) amino acid sequences.A slightly higher diversity level was obtained applying HWC randomization that allows a different set of amino acids (mainly hydrophobic ones) but still includes Tyr that is well known to be important in protein-protein interactions (Fellouse et al., 2006).We intentionally excluded from our soft randomization schemes proline, which is highly unfavorable for helices, to avoid destabilizing effects.
It is known that stable, rapidly folding proteins may fail to display efficiently on the phage surface due to their premature folding in the periplasm.To overcome this limitation the SRP phage display system was introduced for construction of phage libraries based on highly stable proteins (Steiner et al., 2006).This has been proven to improve the level of protein display by using a cotranslational (as opposed to the conventionally used post-translational) translocation pathway (Steiner et al., 2008).Therefore we constructed our libraries both in a conventional way (pelB leader) and in the SRP phage display system.Depending on the translocation type and randomization scheme we named the libraries C for conventional phage display and S for SRP; followed by the number of different amino acids introduced in the randomized positions (e.g.C4, S4, etc.) The final concept of the design of the libraries is presented in Fig. 4 and Table 5.
construction and characterization of libraries
The Mv3α libraries described here are based on two vectors, pComb3H (Barbas, 1995) and pDST23 (Steiner et al., 2008), depending on the translocation pathway -conventional and SRP, respectively.Both phagemids allow display of a protein fused to the pIII coat protein of M13 phage.To construct libraries, a total of 20 µg of randomized, restricted DNA fragment corresponding to each type of library was ligated into a double-digested phagemid.Desalted ligation mixture was electroporated into freshly prepared electrocompetent E. coli MC1061F' cells.The main parameters influencing the procedure efficiency concerned the amount of electroporated DNA and the quality of electrocompetent cells.The highly electrocompetent strain MC1061F' provided high electroporation yield.A detailed protocol of library construction is available in the Methods section.
Quality of the libraries was assessed by PCR, DNA sequencing, Western blots and test selections.
The most significant feature of a phage library is the number of individual colonies.For all of the constructed libraries their size was estimated by serial dilutions after electroporation (Table 6).Plasmid DNA from 36 random colonies was sequenced for each library.Different colonies were subjected to PCR screening to confirm the correct size of cloned insert (around 170 nt).All the libraries had an outstanding level (over 80%) of correct sequences (Table 6) and a well-balanced codon composition (Fig. 5), regardless of the method of sequence randomization.Usually a multi-step DNA synthesis (as for libraries C6, S6, C15 and S15) results in unfavorable library properties, such as a high content of incorrect sequences and uneven codon bias.Owing to the highquality of the DNA primers used for library construction and optimized PCR conditions, we achieved a satisfactory standard of the libraries at the DNA level.
Western blotting
Initially all the libraries were constructed in the phagemid vector pComb3H that uses the conventional, post-translational translocation route.However, our preliminary experiments found a very low presentation level of the helical scaffold and poor selections results.It has been reported (Steiner et al., 2006) that stable, fast-folding proteins do not display efficiently via the sec pathway on the phage particle due to their premature folding.Therefore, we decided to use the SRP phage display system which employs cotranslational translocation of the presented protein.To compare the two different translocation pathways we subjected purified phages to Western blot analysis using anti-pIII antibodies for all libraries and also anti-Flag M2 antibodies for the S-type libraries.
From the ratio of intensities of bands corresponding to pIII and pIII-Mv3α we estimated that the SRP system allowed around five-fold improvement of the display yield (Fig. 6).The high stability of Mv3α protein affected its presentation efficency, which can be observed on the blots.We assume that the cotranslational translocation system prevents premature folding of the stable threehelix bundle and thus affords its better display on the phage particle.
Test selections
To confirm the binding properties of Mv3α we performed test selections.We decided to use human S100A7 (psoriasin), a cytoplasmatic protein highly upregulated in many types of cancer (Emberley et al., 2004;Kesting et al., 2009;Morgan et al., 2011).As the SRP system gave a better display we decided to run the test selection with the S4 library, both on immunotubes and in solution with biotinylated targets.The biotinylation agent containing a disulfide bond allowed specific elution conditions -by reduction in 100 mM DTT.We applied mass spectra analysis to estimate the biotinylation level.For the panning procedure the target protein with one to three sites of biotinylation was apllied.The enrichment ratio was satisfactory only for the selections with Dynabeads.To assess the accuracy of the selection procedure we performed polyclonal phage ELISA using phage pools after each round of selection (Fig. 7).We estimated the amount of phage bound to the target using anti-M13 HRP-conjugated antibodies.The antigen was immobilized directly on Maxisorb wells for analysis of solid surface selection or via streptavidin in the case of Dynabead panning.The experiment confirmed that selection in solution was significantly more effective.To avoid the enrichment of non-specific binders we optimized the panning procedure by applying negative preselection on streptavidin beads and using gelatin as a blocking agent (Table 3).This strategy led to the most satisfactory selection results that are presented here (Fig. 7).
The above-mentioned properties of the Mv3α libraries show that all crucial requirements for the construction of a functional phage library have been met.We achieved Figure 6.Western blot analysis of Mv3α libraries Purified phage particles were separated by SDS/PAGE, blotted onto PVDF membrane and detected with antibodies specific for protein III.For S-type libraries also anti-Flag antibody was applied.Wild type pIII is a 56-kDa protein, whereas the pIII fragment expressed from phagemid vector is a molecule of around 25 kDa, therefore the Mv3α-pIII fusion is around 31 kDa.Phage pool after each round of panning was applied for the binding assay towards S100A7.Y axis shows OD 495 difference between probe and control well.For solid-phase selection well with buffer was used as negative control, for Dynabeads -streptavidin-coated surface.Stable, helical scaffold as an alternative binder an ultimate bacterial transformation efficiency (up to 10 10 ), which resulted in a favorable size of the obtained libraries.Moreover, the functional size of the libraries, which depends on the actual percentage of the correct sequences was unexpectedly high.The reported fraction of proper clones typically does not exceed 60%, which is sufficient to apply the phage library for selection procedure (Binz & Pluckthun, 2005).Our libraries were substantially better with over 80% of correct sequences.This limited the enrichment of defective molecules and increased the chances of isolation of high-affinity binders.The balanced codon bias of the randomized positions, as observed in the Mv3α libraries, also contributed to the positive outcome of the panning experiments.
The Mv3α libraries based on a vector containing a signal sequence for SRP translocation gave a better display level than the libraries constructed in the conventional phage system.So far, the SRP phage display has successfully been applied for construction of DARPin (Steiner et al., 2006) and fibronectin type III domain libraries (Koide et al., 2007;Wojcik et al., 2010).In this paper we showed that three-helix bundle domains are yet another group of proteins which can be efficiently presented on filamentous phage via the cotranslational route.
Our preliminary panning experiments showed that partial protein denaturation on the solid surface limits the enrichment of specific binders.Alternative use of a biotinylated target allowed us to perform selection in solution.The efficiency of biotinylation was strictly controlled: an excess of biotin on the protein surface can cause its partial unfolding or mask the potential binding sites.Using negative preselection on streptavidin-coated beads before each selection round we eliminated the straptavidin-specific clones.These precautions were necessary for proper enrichment, as confirmed by polyclonal phage ELISA.As for any phage library, a comprehensive analysis of panning conditions needs to be performed.Our preliminary results of the S100A7 selection of the S4 Mv3α library can be applied to other libraries and to different antigens.
In conclusion, we introduced a novel, stable, helical scaffold for construction of phage libraries.Different types of randomization and translocation systems were tested.We characterized six Mv3α libraries and tested the panning conditions.The results of this work demonstrate the feasibility of isolating specific Mv3α binders which, owing to their favourable properties, should become a useful affinity tool.
Figure 1 .
Figure 1.Assembly of randomized dnA fragments for construction of phage libraries Complementary sequences used for hybridization of oligonucleotides A and B are marked in red, restriction sites used for cloning in orange.After hybridization (based on custom oligonucleotides), three subsequent PCR reactions were performed to obtain final product, applied for restriction.
Figure 3 .
Figure 3. normalized denaturation curves of virus domains (A) Thermal denaturation of Sendai virus domain; (B) Thermal denturation of Measles virus domain; (C) chemical unfolding of both domains.For curve analysis GraphPad software was applied.Changes in ellipticity were monitored at 222 nm wavelength, the protein concentration in solution was 6 µM.For thermal denaturation experiments, different buffer conditions were applied -as indicated by colors on the graphs.Details of experiment are given in Methods section.
Figure 4 .
Figure 4.The spatial structure of Mv3α (1oksA) Eight positions chosen for NNC randomization are highlighted in red.The three green ones are additional sites for a limited diversification of a total of 11 positions.The structure was generated by the Chimera software.
Figure 5 .
Figure 5. codon bias of constructed libraries Frequency of occurrence of each amino acid in randomized positions is shown.Colors correspond to the character of amino acid: green -nonpolar, aliphatic; blue: polar, uncharged; purple: polar, charged; grey: aromatic side chain.The chart is based on the sequencing results of random clones from each library type.
Figure 7 .
Figure 7. Polyclonal phage elISAPhage pool after each round of panning was applied for the binding assay towards S100A7.Y axis shows OD 495 difference between probe and control well.For solid-phase selection well with buffer was used as negative control, for Dynabeads -streptavidin-coated surface.
Table 3 . In-solution panning conditions.
The concentration of target protein was decreased gradually to favor high affinity binders.Every other round the binding was performed overnight and the number of washes were increased after each round.
Table 4 . Thermodynamic parameters of Measles and Sendai domain unfolding
Both thermal and chemical denaturation parameters are presented.Details of experiments are described in the Methods section.
Table 5 . Types of randomization and theoretical size of designed libraries
The randomization scheme is described by the degenerated nucleotides, where each letter represents different set of nucleotides: N -A, C, G or T; K -G or T; M -A or C; H -A, C or T; W -A or T.
Table 6 . Analysis of constructed Mv3α libraries
Number of individual colonies was calculated by serial dilution after electroporation.The level of correct sequences was verified by sequencing 36 random clones from each library. | v3-fos-license |
2020-06-25T09:06:51.950Z | 2020-05-01T00:00:00.000 | 225889358 | {
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} | pes2o/s2orc | Investigating Synergistic Effects Of Surfactants And Nanoparticles On Emulsion Stability
A challenge of making oil production viable is emerging with time because most of the oil reserves have been exploited using primary and secondary recovery methods. Chemicals such as surfactants have been used to increase oil production through a method called chemical enhanced oil recovery. However, the application of this method is experiencing difficulties because of excessive use of surfactants that not only has negative effect on the economics of the project, but also poses severe environmental concerns. Another method that is being widely proposed is to apply emulsion flooding to enhance oil production. In order to maximize the impact of this method and as a result achieve higher oil recovery factor, it is vital to maintain the stability of the emulsion used. One of the claimed methods to improve the stability of emulsion is the application of synergistic effect between nanoparticles (NPs) and surfactants. This article aims on investigating the stability of emulsion using bottle test when applying the synergistic effect between NPs and surfactants with varying concentrations of the NPs, and surfactant charge. An anionic surfactant - sodium dodecyl sulphate (SDS) and a cationic surfactant - cetyltrimethylammonium bromide (CTAB) have been utilized in this study. Nano-silica was selected as the NPs used in this study. It was found that synergistic effect is more prominent between SDS and nano-silica with decrease in emulsion phase height percentage (measure of emulsion stability) going down to 22% compared with 43% for combination of CTAB and nano-silica, and 51% in case of using only surfactants. This has proved that the synergistic effect is beneficial in enhancing the emulsion stability, which can be implemented in the application of emulsion flooding where the stability of the emulsion is crucial.
Introduction
Natural methods are losing the ability to produce oil from a majority of the reservoirs that have reached maturity level. Hence, it becomes economically unviable to recover hydrocarbons by the primary drives. To maintain production at the targeted level, additional support is usually supplied such as injecting gas or water into the reservoir (namely gas/water flooding), which are known as the secondary recovery methods. Nevertheless, through a combined efforts of the primary and the secondary recovery techniques only 35 -50 % of oil in place can only be recovered [1].
After using primary and secondary recovery methods, the role of enhanced oil recovery (EOR) comes into action to recover the oil left behind. Rather than that only providing the external energy to push the oil, through applying EOR techniques, few of the physical and/or chemical properties of either/both the oil left behind or/and the displacing fluid might be altered in favor of increasing the production level. EOR methods are categorized according to the operating mechanism they are functioning to enhance the oil recovery. The three main types are thermal EOR, miscible EOR, and chemical EOR.
Chemical EOR is subcategorized based on the chemical substances involved in the modification of the equilibrium of the reservoir. This method involves the usage of surfactants, polymers, and other chemicals such as alkali. It can also be implemented through a suitable combination of those chemicals, as in surfactant-polymer, polymer-alkaline and alkaline-surfactant-polymer (ASP) flooding mixtures [2].
Emulsion consists of mutually two insoluble and immiscible fluids, like water and oil. It is a disperse colloidal system that is thermodynamically unstable. One of the two constituents is the continuous phase that surrounds the dispersed phase that is present in the form of finely dispersed spherical droplets. Emulsions in petroleum systems is generally comprised of oil and water as the interacting fluids [3][4][5][6][7][8][9].
Microemulsion possesses special characteristics that enables it to be considered as a promising technology to be utilized in significant application in areas like EOR [3,10,11]. Due to the ultra-low interfacial tension (IFT) values that could be achieved between the contacting oil and water microphases of emulsions, water-in-oil (W/O) emulsions stabilized with NPs or surfactants could be considered as an option to look after for applying EOR in harsh condition reservoirs [2,12,13].
Emulsion flooding could achieve the purpose of its utilization in enhancing the oil recovery through several mechanisms. First, it can alter the rock wettability towards wettable by oil by modifying the capillary forces. Second, it has the effect of partially blocking the larger pores in the rock by the dispersed emulsion phase. Third, it can assist in the modification of the relative permeability, which results in reduction of water mobility [11]. In order for the emulsion to maintain the effectiveness of its function and fulfill the target of its usage as an EOR flooding method, it is essential to maintain the stability of the emulsion for as long as possible; as it is noted that emulsions are thermodynamically unstable systems.
Emulsions could be formulated with the mixture of oil and another phase, which can be either water or any other immiscible aqueous solution with the supply of sufficient energy by mechanical shear. In general, the small tiny droplets of the dispersed phase, formed in the emulsification process like water in W/O emulsion, tend to coalesce after some time to self-resolve the emulsion [14]. In order to reduce the mechanical energy required for the dispersion process, which is quite high, emulsifiers are used to lower the interfacial free energy and interfacial tension (IFT) [3]. Moreover, emulsifiers function as stabilizers once the dispersed phase has been formed against coalescence [7]. They are aimed to accumulate at the interface of both phases and create a protective layer in the form of tough elastic film which remains unbroken with the collision of the droplets [6,8]. Emulsifiers can be surfactants, polymers, or even solid particles. In brief, in order for emulsion to be created, there is a need of oil, water, and emulsifier, and energy [9].
Surfactants stabilize emulsions by adsorbing at the interface and reducing the IFT, decreasing the size of the dispersed phase droplets, , increasing surface elasticity, and possibly increasing surface viscosity. These effects makes oil-water separation difficult and enhances their stability, which is favored in EOR application [3,6,15].
Emulsion system could be stabilized by adsorbing particles at the water-oil interface and are known as pickering emulsions [10,[16][17][18][19][20]. There are many particles that are used for stabilizing pickering emulsions such as clays, silica, etc. Droplets of the dispersed phase are hindered from coalescence through the steric hindrance via the adsorption of monolayer film of the solid at the interface [10,20]. The uniqueness of the form of emulsion resides in the fact that the adsorption energy is relatively irreversible and the particle to particle interactions are considered strong [19]. Harsh subsurface conditions requires excellent physical and chemical stability which are possessed by the NPs. They adsorb on the fluid/fluid interface, which leads to the formulation, rheological enhancement and improvement in their stability [13]. Low IFT is usually needed for the adsorption of NPs to the liquid/liquid interface. One way to achieve that is the addition of surfactants to lower the IFT and therefore attract particles to the interface. Thus, it is advised to exploit the synergy between NPs and surfactants to lower the IFT and increase the adsorption energy. As a matter of fact, the synergistic effect of adding surfactant to the nanoparticle dispersion is considered to be an effective way of spontaneously generating emulsions for the EOR application [21]. Hence, the stability of emulsion could be improved by applying this method.
There are many methods which are used to investigate the stability of the emulsions. Among them are the phase separation methods. These methods depend on measuring the amount of phases that have been separated from the emulsion and resolved; which can be either by natural separation induced by gravity segregation or by external forcing like centrifugation. Bottle test is an example that is implementing phase separation method to investigate emulsion stability [22]. This test is widely used in the industry to measure and monitor emulsion stability [23,24].
The amount of the dispersed phase separated from the emulsion is measured as a function of time. This can be used as a measure of emulsion stability. For example in the case of W/O emulsion, the amount of water separated is measured as a function of time. The higher the amount of dispersed phase separated from the emulsion is an indication of how loose the emulsion is (higher possibility to separate into oil and water), and smaller the "resolved" volume of dispersed phase (with respect to the original volume), the more stable the emulsion is. This is usually reported as Emulsion Separation Index (also referred to as Emulsion Stability Index and abbreviated as ESI) [22,25]. ESI is defined as follows: (1) In some case of stable emulsions, where the separation process takes a longer time, a single experiment could also be used to indicate the stability of the emulsion.
Experimental
This study focused on the utilization of anionic (SDS) and cationic (CTAB) surfactant. Nano silica was selected as the NPs to implement the synergy effect. A basic schematic diagram for emulsion preparation is shown in figure 1. Energy was supplied to the emulsion system via a high speed homogenizer (figure 2). Furthermore, since stability of the emulsion is an important aspect to be studied, the main theme of this study was to investigate the stability of the emulsion through phase separation method using bottle test.
After the emulsion preparation process ended and the foam settled down, bottle test was started. The emulsion samples were transferred into test graded bottles with capacity of 25 mL each (figure 3) and preserved stagnant throughout the allocated time for the investigation. The height of the emulsion and amount of the separated dispersed phase were recorded continuously for the first 150 minutes. Then the same recording procedure was followed for one month. The stability of the emulsion were reported as the emulsion phase height decrease percentage in comparison with the initial reading of the emulsion height. The emulsion samples were kept at room temperature and ambient pressure.
Results and Discussion
Light mineral oil and brine with salinity of 10,000 ppm were used to prepare the emulsion samples. Critical micelle concentration (CMC) analysis was conducted to decide the amount of surfactants needed to form emulsions. The amount of surfactant added into the emulsion samples were 1.0 g and 0.2342 g for the SDS and CTAB, respectively.
Different samples were prepared with varying NP concentrations (by weight) to perform stability analysis by bottle test. Based on previously conducted experiments on viscosity analysis by Abdul-Razzaq et al. (2019), an optimization set of NP weights was used for the bottle tests [26]. Based on the viscosity analysis, three ratios for each surfactant, which has resulted with peak and two tail viscosity values, were selected to conduct the stability bottle test through measuring emulsion phase height decrease percentage in comparison with the initial reading of the emulsion height. This can be inferred to the amount of water droplets being separated from the W/O emulsions as the height of the emulsion decreases with time. The higher the percentage, the higher the amount water separated and hence less stable the emulsion is. The weight ratios of nano-silica added to the SDS emulsion samples were 0.15, 0.5, and 2 respectively. In the case of using CTAB the weight ratios investigated were 0.5, 1.75, and 2.25 respectively. Figures 4 and 5 show the emulsion phase height decrease percentages with time in the case of using anionic surfactant (SDS) and negatively charged particles nano-silica. It is clearly shown that the amount of separated water (dispersed phase) from W/O emulsion increases with time. However, there are inherent differences in case of the samples with different weight ratios of nano-silica to SDS. However, it can be inferred that with the addition of nano-silica, the stability of the emulsion was enhanced. Figure 4 depicts the emulsion stability behaviour in case of SDS and nano-silica in the initial 150 minutes. It was observed that most of the water dispersed phase had resolved in this period for all NPs concentration used in comparison with figure 5, which is a continuation of figure 4 but for the time step of the subsequent 1 month. As described in figures 4 and 5, the sample without the addition of any nano-silica has experienced the maximum water separation with a decrease of emulsion phase height up to 51%. In comparison with addition of nano-silica of 0.5 times the SDS used by weight, it has resulted with a decrease in the emulsion height percentage up to 22% only. This can be inferred from the fact that SDS surfactant tends to lower the IFT between emulsion's constituents (water and oil) which aids the [20]. The adsorption of nano-silica on the dispersed phase droplets supports the droplet with additional steric hindrance which stops their coalescence in the incidents of collision [10,20].
The stability of emulsions prepared using cationic surfactant (CTAB) and negatively charged particles nano-silica is shown in figures 6 and 7. It is illustrated as a function of the emulsion phase height decrease percentage with time. It is clearly evident that the amount of separated water dispersed phase from W/O emulsion prepared with CTAB increased with time as well. However, the degree of the disassociation of oil and water differed with usage of different weight ratios of nano-silica to CTAB. In general, the stability of the emulsion was enhanced with the addition of nano-silica with a lower weight ratio of nano-silica to CTAB. Figure 6 illustrates the stability trend achieved with the utilization of CTAB and nano-silica in the first 150 minutes of the bottle test. In comparison with figure 7, which is a continuation of figure 6 but for a period of the following 1 month, most of the dispersed water phase got separated during the first 150 minutes ( figure 6) for all the NPs used ratios, similar to the case of SDS. Figure 6 and 7 demonstrate that the maximum dispersed water phase was separated from emulsion sample without nano-silica, which went up to 51%. The addition of nano-silica slightly improved the stability of the emulsion with the decrease in the emulsion height phase going down to 43% with weight ratio of nano-silica to CTAB being 1.75. This enhancement in the stability could be related to the fact that CTAB reduces the IFT between the water and the oil (emulsion's constituents), which helps the nano-silica to be adsorbed at the interface and hence form the emulsion's droplets [20]. As a result, steric hindrance was provided to the emulsion dispersed phase droplets that helps in reducing the coalescence process upon collision [10,20].
It is noticeable from figures 6 and 7 that when the weight ratio of negatively charged nano-silica to cationic surfactant CTAB was 2.25, the stability of the emulsion degraded to the same level as without addition of any NPs. The reason behind this is with excess amount of negatively charged nano-silica being presented to the system, there was a build-up in the negative charge. Together with the presence of the positive charged cationic surfactant (CTAB) in excess, the electrostatic attractive forces started to overrule and promote the coalescence of the water dispersed phase droplets. This finding was backed up with low value of viscosity achieved with this ratio which indicates that droplets started to coalesce and hence resulted with a less stable emulsion [26].
Our preliminary results also corroborate with the findings from the study conducted by Pei et al. (2015). They found that by adjusting the concentration of NPs, the synergy between NPs and surfactant could be proven and a desirable high viscosity can be achieved for emulsion samples [27]. In their study, they have used CTAB as the surfactant and silica NPs. This is directly related to the findings of this study that CTAB and nano-silica are improving the stability of the emulsion samples. However, with the application of higher concentration, the electrostatic attractive forces dominate the system and inhibit the stability.
Moreover, the viscosity of the emulsion plays a vital role in stabilization process [3,6,15,20,28]. When the viscosity of the sample is high, it provides resistance to diffusion, hence the possibilities of collisions decreases. This helps in declining the coalescence rate, reducing separation of the water dispersed phase in the W/O emulsion, and enhancing the stability of the emulsion. According to Abdul-Razzaq et al. to SDS was higher than other weight ratios, which indicates why this ratio presented a better stability behaviour in comparison with the other weight ratios of nano-silica to SDS [26]. In a similar case, the CTAB with a weight ratio of 1.75 exhibited higher viscosity than the other two weight ratios of nanosilica to CTAB under consideration. This explains the reason of getting better stability with 1.75weight ratio of nano-silica to CTAB [26].
It is observed that the rate of separation of the dispersed phase was decreasing with time. At the start, the separation rate was quite high (figures 4 and 6), which changed after 5 minutes from the commencement of the test to a lower rate. Then, the rate have slowed down again after 1 hour from initiating the test. This is inferring that the amount of the water dispersed phase in the emulsion was decreasing with time, which affected the tendency of the droplets to collide, coalesce, and separate out. After two days there was almost no water separating out from the emulsion and the height of the emulsion phase remained almost constant (figures 5 and 7). Figures 8 and 9 are showing snapshots of the bottle tests conducted for emulsion samples with the application nano-silica with a different weight ratios to SDS and CTAB respectively at the beginning of the bottle test as well as the above mentioned time steps.
In a comparison between figures (4 and 5) and figures (6 and 7), it is clear that in general the synergistic effect of nano-silica was more evident with anionic surfactant (SDS) rather than with the cationic surfactant (CTAB). The charge of the surfactant and electrostatic forces developed in the system plays pivotal role with the stability of the emulsions [21]. With the emulsion formulation with SDS and nano-silica, the prominent electrostatic force in the system is the repulsive forces. This repulsive forces prevents the water droplets of the dispersed phase to collide and coalesce due to the increase in the thickness of the diffusive layer in the electrical double layer (EDL) system developed around the droplets [29].
On the other hand when CTAB was used, the resultant electrostatic force in the system is the attractive forces between negatively charge nano-silica and CTAB. The contraction of the diffusive layer in the EDL system developed surrounding the water dispersed phase droplets is induced by the attractive forces prominent in the system. This reduction in the diffusive layer thickness give way to water droplet in W/O emulsion to collide, coalesce and separate out [29]. Therefore, the stability for this system is not as high as in compassion with system where the repulsive forces are the prominent such as with case of using anion surfactant (SDS) and negatively charged nano-silica. In a study conducted by Tikekar et al. (2013), they have discussed that the aggregation of emulsion droplets could be attributed to partial neutralization of electrostatic negative charge on silica particles [29]. This fact can be related to the current study where the CTAB plays the exact role on neutralizing the negative charge of the nano-silica. Hence, reducing the effectiveness of the nano-silica in stabilizing the emulsion droplets. In another study conducted by Qiu (2010), synergy was proven between NPs and surfactants in enhancing the emulsion's viscosity [30]. However, the mentioned study utilized different surfactant category. Nonionic Triton X-100 surfactant was used together with CAB-O-SIL TS 530 NPs. It was found that with increasing NPs concentration, the viscosity of the emulsion sample was increasing as well. This improvement in the viscosity with application of the NPs and surfactants, can be directly related to the enhancement of the emulsion stability which is investigated in the current study.
Conclusion
The synergy effect is clearly observed between surfactants and nanoparticles in enhancing the stability of emulsion samples. Anionic (SDS) and cationic (CTAB) surfactants were used to investigate the stability of the emulsion with the use of negatively charged nano-silica via bottle test. It has been observed that SDS performed better due to their electrostatic behaviour and interaction with nanosilica in forming emulsion dispersed phase droplets and providing the repulsive forces to prevent their coalescence. The emulsion samples' stability tested in this study have shown promising improvement with the usage of model light mineral oil whereby providing the basis for synergistic use of nano-silica and charged surfactants in enhancing the oil recovery through emulsion flooding. | v3-fos-license |
2018-04-03T05:01:32.960Z | 2015-08-27T00:00:00.000 | 41144234 | {
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} | pes2o/s2orc | Evaluating SAXS Results on Aqueous Solutions of Various Bacterial Levan utilizing the String-of-Beads Model
Polysaccharide levan is a homopolymer of fructose and is an important component of plants, yeast, fungi and some bacterial biofilms. In this paper we report on the structural properties of aqueous solutions of bacterial levan utilizing smallangle X-ray scattering and light microscopy. In addition to commercially available levan isolated from Zymomonas mobilis and Erwinia herbicola, we also studied levan isolated and purified from the biofilm of Bacillus subtilis. The smallangle X-ray scattering data were analyzed by the string-of-beads model that revealed qualitative differences in the structure of levan molecules. Levan can be represented as a semi-flexible chain that interacts intraand inter-molecularly and therefore forms various suprastructures on larger size scales. Increasing the concentration of levan makes the levan structure more compact, which was observed on the nano as well as on the micro scale. The structures with most homogeneously distributed polymer local density were found in B. subtilis levan solutions.
Introduction
Levan is a water soluble polysaccharide composed of fructofuranosyl rings, connected by β- (2,6) glycosidic linkages and occasional branching through β-(2,1) glycosidic linkages.2][3][4][5] Structure of levan is stable from pH 4 to pH 11, at concentrations of inorganic salts from 20 to 30% and temperatures up to 70 °C. 6Levan is widespread -it is found in plants, yeast, fungi and bacterial biofilms. 7Biofilms are multicellular microbial communities embedded in extracellular polymeric matrix (EPS-matrix) and levan can represent a major polymer component, as for example in B. subtilis subs.subtilis strain NCIB 3610 biofilms, grown in a sucrose rich liquid medium. 5,8Biofilms based on levan can play a key role in the development of plant diseases, 9 dental ca-ries and periodontal disease. 10,11Levan shows extremely low intrinsic viscosity and exhibits a rather atypical nongelling behavior in aqueous solutions in comparison to most other common polysaccharides. 3,12At high concentrations of up to 8 wt % levan can nevertheless support rather robust biofilms. 12Levan from Bacillus sp.behaves like a newtonian fluid up to 30 wt % solution and is soluble even up to 60 wt % solution. 3,13Modelling attempts to resolve the structural details of such polysaccharide systems based on small-angle X-ray scattering (SAXS) results are very scarce. 5,14n the present study we focus on the detailed interpretation of SAXS data obtained for 1, 2, 4, 6, and 8 wt % aqueous levan solutions of three bacteria: Bacillus subtilis (BS), Zymomonas mobilis (ZM) and Erwinia herbicola (EH).In our previous structural and rheological study we have shown that the mole fraction of branching Benigar et al.: Evaluating SAXS Results on Aqueous Solutions ... points was (10.5 ± 0.7) % in B. subtilis levan, (11.0 ± 0.7) % in Z. mobilis levan, and (10.2 ± 0.3) % in E. herbicola levan.The intrinsic viscosity value for B. subtilis levan was (0.35 ± 0.04) dL/g, for Z. mobilis levan (0.36 ± 0.01) dL/g, and for E. herbicola levan (0.45 ± 0.01) dL/g.The hydration number for all three samples was around 6. Levan samples also showed very similar viscosities at low concentrations (1 wt %), while viscosities varied significantly at higher concentrations.In concentration range from 1 to 8 wt % all three samples showed elastic character.Another difference between studied levan samples was in the weight average molecular weight M w .The estimate for M w of B. subtilis levan was (31 ± 2) MDa, of Z. mobilis levan (226 ± 4) MDa, and of E. herbicola levan (280 ± 40) MDa.These results were consistent with the ones obtained by light microscopy.The largest particles of (0.5 ± 0.3) μm were observed in E. herbicola levan solution, followed by Z. mobilis levan particles of (0.45 ± 0.10) μm and B. subtilis levan particles of (0.35 ± 0.10) μm in diameter in 1 % (w/v) aqueous solutions.The SAXS data of different levans were analyzed and interpreted by the classical approach utilizing the Ornstein-Zernike 15,16 and an additional Debye-Bueche [16][17][18] term. 5nfortunately the classical approach failed to provide the information on the static correlation length, i.e. we could not satisfactorily describe the SAXS intensities in the innermost regime of the scattering curves. 5Therefore, in this work, we applied a more complex string-of-beads model [20][21] to describe experimental SAXS curves, and propose a new levan supramolecular structure in the aqueous solution.
1. Materials
In this study three different samples of bacterial levan were used.Two of them were commercially available levans isolated from Z. mobilis (Sigma Aldrich) and E. herbicola (Sigma Aldrich).The third sample was obtained from biofilm of B. subtilis subs.subtilis strain NCIB 3610.Detailed isolation and purification procedures are described by Dogsa et al. (2013) 8 and Benigar et al. (2014). 5The purity of isolated B. subtilis levan (98.3%) was comparable to purity of commercial levans isolated from E. herbicola (98.5%) and Z. mobilis (98.8%). 5Levan samples for SAXS measurements were prepared as 1, 2, 4, 6, and 8 wt % aqueous solutions utilizing strong vortex mixing.Levans originating from bacteria B. subtilis, Z. mobilis and E. herbicola are hereinafter referred to as BS, ZM and EH levans, respectively.
Light Microscopy
The microscopy was performed with an Axio Observer Z1 inverted microscope (Zeiss, Gottingen, Germany) equipped with Zeiss Plan-Apochromat 100x/1.40Oil immersion objective and MRm AxioCam camera using the differential interference contrast technique (DIC).The magnification was further enhanced by using 2.5 fold internal lens (optovar).For slide preparation 5 μL of sample was spotted on a clean glass slide and covered with a cover glass with a thickness of #1.5.The prevention of water evaporation from the sample on the slide was accomplished by sealing the gap between the slide and the cover glass.For flat-field correction the out-of-focus images, where uneven light distribution can be observed, were taken for each sample.The images were processed by Imagej software (ver.1.48b, W. Rasband, NIH, USA).Firstly, by normalizing sample image on out-of-focus image the flat-field correction was achieved.Then the enhance contrast function of imageJ was applied.The particles were manually measured by drawing straight lines over them representing their diameter.In this way at least 50 levan particles were analysed.In order to exclude the biased selection of the levan particles the horizontal and vertical lines across the full-size of the processed image were drawn and only levan particles on the lines were considered.
3. Small-Angle X-Ray Scattering Measurements
Levan samples were measured at 25 °C with a Kratky compact camera (Anton Paar KG, Graz, Austria).The camera was attached to a conventional X-ray generator (Kristalloflex 760, Bruker SAXS GmbH, Karlsruhe, Germany) equipped with a sealed X-ray tube (Cu K α X-rays, λ = 0.154 nm) operating at 40 kV and 35 mA.Monochromatic primary beam was attenuated by a Ni foil of appropriate thickness due to the limitations of the detector.In addition a software monochromator was used to register only the scattered X-ray photons within a predefined range of energies.The samples were measured in a standard quartz capillary with an outer diameter of 1 mm and wall thickness of 10 μm.The scattered X-ray intensities were detected with a PSD-50M ASA position sensitive detector (M.Braun GmbH, Garching, Germany) in the small-angle regime of scattering vectors given by (0.07 < q < 7) nm -1 , where q = 4π/λ • sin (ϑ/2).Each individual sample was measured for 24 to 48 hours to yield reliable measurement statistics.Prior to further analysis the scattering data were corrected for the empty capillary and pure solvent scattering, and normalized to an absolute scale using water as a secondary standard.
4. Interpretation of SAXS Data -»String-of-Beads« Model
For the interpretation of SAXS curves the string-ofbeads model developed by Dogsa et al. [19][20][21] was applied.In this model the polymer or the polymer segment is si-mulated as a linear chain of sequential spherical beads approximating monomers.The validity of this approximation was verified in our previous paper, 21 where we calculated the theoretical scattering curves of sugar trimers at atomic level and compare them to the theoretical scattering curves of superimposed structures made of three beads.In the case of levan, which is uncharged β-fructan, a basic monomer unit is fructose.The position of the bead in the polymer molecule, relative to its predecessor, is described by Θ (bond) and Φ (torsion) angles.Each pair of angles is set according to the probability p* (Θ p and Φ p ) for the random variation of the angles Θ and Φ between adjacent beads.For example, if p* = 0 the polymer forms a rigid helical structure.If p* = 1 all monomers have random Θ and Φ values and this translates to a random conformation of the polymer molecule (chain).To further vary the stiffness of a polymer chain an additional parameter, Θ plim , is used.This parameter sets the upper limit of the bond angle Θ in the simulation.In addition, one can also vary the size of the polymer chain N, i.e. the number of monomers per chain.For this study the chains with N ranging from 25 to 1000 monomers were modeled.
The algorithm firstly calculates the representative form factor, P -( q) -, of the modeled chain with size ξ S (= 2R g ), shown in Figure 1, for each set of the four shape parameters. 20,21Each representative form factor is combined with the term representing the intermolecular interactions into a total scattering function I(q) according to the following expression: 20 (1) where Δρ 2 is the scattering length density difference between polymer and solvent, 〈δϕ 2 〉 is variance of the volume fraction of the polymer, 〈ϕ 〉 is the mean volume fraction of the polymer, ν 0 is the single polymer chain or polymer segment volume, K is the constant proportional to the strength of the repulsive interactions, ξ is the correlation length over which the repulsion occurs, q is scattering vector, Ξ is correlation length that describes the average size of the Debye inhomogeneities (Figure 1), 24 which cause fluctuations in the local volume fraction of the polymer.Model equation is then fitted to the experimental SAXS data and only solutions with sufficiently good fits are taken into further consideration. 20While fitting, only one type of interaction is assumed to be presented in the system, i.e. either 〈δϕ 2 〉 > 0 and K = 0 or 〈δϕ 2 〉 = 0 and K > 0. Based on solutions of interaction parameters one can than simulate the suprastructure made of individual polymer chains or polymer segments. 20The volume fraction of the polymer is assumed to be log-normally distributed through space. 20,25n the present study we apply this modelling approach on semi-concentrated aqueous solutions of various
1. Small-Angle X-ray Scattering and Modelling Results
When applying the SAXS technique one has to be aware that due to the limited range of the scattering vector available by this technique, structural details from approximately 1 nm up to a few tens of nm can be obtained.This means that in the studies of the polymer systems SAXS technique can successfully reveal the structural information on the molecular level, whereas for the information on the macroscopic structure usually other methods need to be applied.In combination with an appropriate molecular model one can usually obtain geometrical parameters of the macromolecules in solution.SAXS technique has previously been successfully applied in the structural studies of various polysaccharides, 5,[19][20][21][26][27][28] while SAXS studies on polysaccharide levan are scarce. 5,14 Ithe present study we apply the string-of-beads model according to eq. ( 1) to the experimental SAXS scattering data of 1, 2, 4, 6, and 8 wt % BS, ZM, and EH levan samples.Since levan is an uncharged polymer, the parameter K, describing the strength of repulsive interaction was set to 0. Experimental SAXS data of 1, 2, and 8 wt % BS, ZM, and EH levan samples are shown in a double logarithmic plot in Figure 2. The symbols represent the experimental data and the full lines correspond to the best fits obtained by the string-of-beads model.As can be seen from the scattering curves the BS levan most notably differs from the other two levan samples.Such a relationship was observed also in the case of macroscopic rheological, sound velocity and hydration results, which suggests that the molecular and macroscopic structures are in levan strongly related.
The result of string-of-beads model is a set of all solutions satisfying the fit quality criteria 20 each of which comprises the values of the interaction parameters and the corresponding set of four shape parameters: Θ p , Φ p , Θ plim , and p*.The ranges of resulting parameters for levan samples are given in Table 1a and b.In addition, the 3D representation of the polymer structure parameters obtained from the best fits of 2 wt % levan are depicted in Figure 3.The cube contains points corresponding to all good fit solutions.A representative molecular structure of the polymer segment is depicted below the cube.It can be observed that none of the structures represents a true random coil as p*-values are much smaller than 1.In general, levans adopt semi-flexible chain conformations irrespective of concentration with p* between 0.1-0.7.However, the p*-values of BS are higher than p*-values of ZM or EH, which means that the BS modeled chains in comparison to ZM or EH adopt conformations that are more similar to those characteristic for random coil.At the same time other shape parameters (Θ p , Φ p , Θ plim ) do not vary significantly.Therefore the chain segments of BS levan structure are more flexible than the segments of ZM or EH levan, which is in agreement with the previous observation that BS levan had the lowest viscosity. 5This is a characteristic of polymers with small persistence length, 29 typically occurring in random coils.At this point we have to comment on the values of the parameter N in Table 1, which represents the number of beads in the string.The value of N does not represent the whole levan molecule (individual polymer), but rather its shorter entangled segments (Figure 1).
It is important to point that not all of the values of parameter N provide good fits -if the value was too large or too low no satisfactory fitting solution was obtained.The lowest values of parameter N, as well as ξ S were obtained for BS levan.Because the electrostatic interaction is not present in uncharged levan (K = 0), the second term in eq. 1 assumes that modeled segments are randomly distributed in space.Therefore the size of the segment, ξ S (Figure 1) is also the distance above which the individual segments become uncorrelated in space.This means that BS structure is not only the most random at the level of the individual chain segment, but also at the level of segment arrangements.This is in the agreement with our pre-vious DLS (dynamic light scattering) results, where the relaxation times were the fastest in BS levan. 5Increasing the levan concentration, decreases the segment size N, as well as segment ξ S .This can be explained by the observation that in these levan samples critical overlap concentration is reached for concentrations above 1 wt %, 5 which forces the levan molecules to adopt more dense structure.Because segments build suprastructures, one expects that segments will impact the behavior on the larger size scale.The correlation length of Debye-Bueche heterogeneities Ξ is lowest in BS levan and decreases with levan concentration in all levans.Supramolecular structures of 2 wt % BS, ZM, and EH levan are shown in Figure 4 and were simulated based on the log-normal distributions of levan polymer density (i.e.volume fraction of the polymer), which was obtained from 〈δϕ 2 〉/〈ϕ 〉 2 and 〈ϕ 〉.For example, subspaces ϕ 5% indicate the characteristic subspace levan distribution, which is found in 5% of the levan sample volume.The local polymer density in the high density ex- As the molecular suprastructures build macrostructures the behavior of levan was also studied under the microscope.
2. Microscopy Results
To complement the interpretation of SAXS data light microscopy was used.As shown already in our previous study 5 1% (w/v) BS, ZM, and EH levans in aqueous solution form spherical particles.In the present study we used the differential interference contrast microscopy (DIC) to investigate an extended range of aqueous solutions of 1, 2, 4, and 6% (w/v) BS, ZM, and EH levan.We also prepared 8% (w/v) levan solutions, but ZM and EH levan samples were too dense to observe individual levan particles.The microscopy images of 1 and 6% (w/v) levan solutions are depicted in Figure 5.As can be observed the number of BS levan particles was much smaller than the number of ZM and EH levan particles.The levan particles became smaller and more homogeneously distributed with increasing concentration.The effect was most noticeable in EH levan solutions and the least in BS levan solutions.Average sizes of BS, ZM, and EH levan particles are presented in Table 2.The average size of 1% (w/v) ZM and EH levan particles was similar, while the average size of 1% (w/v) BS levan particles was significantly smaller.Similar trend can be also observed for 2% (w/v) levans.This is in accordance with the observations that levans of BS have the highest p*-values and lowest number of monomers per segment, N, as obtained from SAXS (Table 1).Such segments have small radius of gyration and are more flexible.Therefore one could expect that superposing these segments results in smaller structures, which was indeed observed.However, with increasing concentration the difference in size between the three levan particles decreased and finally the average size of 6% (w/v) BS, ZM, and EH levan particles were comparable (Table 2).This agrees with our observation that with increasing concentration levan molecules become more compact on nanoscale.
Conclusions
Structural properties of different bacterial levan aqueous solutions were studied with SAXS technique.The results of modelling SAXS curves indicate that with increasing concentration levan molecules in aqueous solution become more compact.This phenomenon propagates from nanoscale to microscale of levan.Heterogeneity of the levan suprastructure decreases with increasing levan concentration.The most random and homogeneously distributed structures can be found in BS levan aqueous solutions.
Figure 1 :
Figure 1: Scheme illustrating two correlation length parameters occurring in levan samples: Ξ describes the average size of the Debye inhomogeneities, while ξ S (= 2R g ) represents the size of the simulated polymer segment.Each segment is represented in different color.Ξ on the scheme corresponds to 300 Å.The contours on the left-hand side scheme indicate the regions with the same local polymer density (i.e.volume fraction of the polymer).White color represents high density, black low density.
Figure 4 .
Figure 4. Molecular distribution of 2 wt % levan in space, calculated from interaction parameters presented in Table1and the best-fit shape parameters from Figure3.On the right-hand side of the figure index of ϕ represents the probability to encounter a characteristic subspace with the local ϕ in a levan sample.Assumptions: ρ(H 2 O) = 9.47 × 10 -6 cm -2 , ρ(levan) = 1.31 × 10 -5 cm -2 , levan mass density = 1.45 g/cm3 .The size of the monomer bead (5.2 Å) is depicted in proportion to the side-length of the subspace box (300 Å).
Table 1 .
a) Interaction parameters of the fits to the experimental SAXS data, and b) shape parameters for BS, ZM, and EH levans.The ranges of parameter values of good fits are given: is much higher in ZM and EH levan than in BS levan.On the other hand, in the low density extreme the local polymer density of BS levan is higher compared to the other two levans.Furthermore, BS levan molecules were the most homogeneously distributed, as the variations among subspaces were the smallest.ZM and EH levans had virtually identical density distribution.This is in agreement with calculated relative fluctuation of volume fraction of the polymer 〈δϕ 2 〉/〈ϕ 〉 2 (Table1a), which measures the heterogeneity of the system on the size scale of Ξ.It is lowest in BS and decreases with concentration in all levans. | v3-fos-license |
2021-10-21T16:14:23.316Z | 2021-09-07T00:00:00.000 | 239652927 | {
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} | pes2o/s2orc | Can Salt-Tolerant Sludge Mitigate the Salt Inhibition to Acidogenic Fermentation of Food Waste? Insight Into Volatile Fatty Acid Production and Microbial Community
For treatment of saline wastewater, the feasible approach to mitigate the salt inhibition is using the acclimated salt-tolerant sludge. The aim of this work was to verify if the use of the acclimated sludge (AS) also could alleviate salinity stress on acidogenic fermentation of food waste (FW) under saline environment. The responses of volatile fatty acid (VFA) production and the microbial community to salt stress were investigated. Results showed that VFA production was reduced by high salinity (30 g/L and 70 g/L NaCl) compared with the control (0 g/L NaCl), especially for groups inoculated with the AS, whereas inoculating with the non-acclimated sludge (non-AS) caused less reduction. The impact of salinity was seen on VFA production with accumulation of more propionic acid and acetic acid along with traces of butyric acid. Significant shift on microbial community composition occurred upon biomass exposure to salt. The microbial communities of the non-AS and AS groups at the same NaCl concentrations converged over time. The non-AS groups contained a more proportion of the phyla Bacteroidetes, Atribacteria and Chloroflexi at high salt levels. These findings demonstrate that the non-AS was more conducive to VFA production due to the presence of higher proportions of hydrolytic and fermenting bacteria.
Introduction
Recovering energy and nutrients from food waste (FW) not only constitutes substantial economic opportunity but is also an essential requirement for the sustainable development of human society. Considering the negative environmental impacts of landfilling, incineration, or composting of FW, anaerobic digestion (AD) has been proposed as a relatively cost-effective technology for renewable energy production and waste treatment of this high-moisture and energy-rich material [1][2][3].
Salt (e.g., NaCl), used as a type of food flavoring, is accumulated in FW in large amounts when the food is processed. The general mass fraction of NaCl in FW in China ranges between 2 and 5%. Na + is an essential element for the cell synthesis, growth, and metabolism involved in anaerobic digestion system. However, high concentrations of salt can result in cell plasmolysis and cell death due to a dramatic 1 3 increase in osmotic pressure. As a result, the organic compounds in saline wastewaters often are poorly biodegraded and seriously affected the efficiency of utilization of this valuable resource [4,5]. Experimental results have shown that a low level of NaCl (5 g/L) improves hydrolysis and acidification, but inhibits methanogenesis, whereas a high level of NaCl (15 g/L) seriously inhibits acidification and methanogenesis processes [6]. Low concentrations of NaCl (5-9 g/L) were also found to increase the production of polyhydroxyalkanoate (PHA), while higher concentrations (13-20 g/L) inhibited cell viability and decreased PHA content [7]. Short-chain fatty acids, also known as volatile fatty acids (VFAs) are important intermediates in the anaerobic digestion and production of PHA. VFAs have attracted a great deal of interests due to their wide range of potential applications, including the removal of biological nitrogen removal, synthesis of bioplastics, and bioenergy production [8]. The presence of salinity also affects the production of VFA. Zhao et al. [9] showed that VFA production increased from 367.6 to 638.5 mg chemical oxygen demand (COD)/g volatile suspended solid (VSS) with increasing concentration of NaCl from 0 to 8 g/L. However, further increases in NaCl resulted in severe inhibition of VFA production. Our previous study investigating the using of FW to produce VFAs under different NaCl concentrations found that a maximum VFA production of 0.542 g/g dry weight of FW occurred under a NaCl concentration of 10 g/L NaCl, whereas that under an NaCl concentration of 70 g/L was about 23% lower [10]. In addition, the time required to reach maximum VFA production increased with increasing NaCl concentration.
An approach to overcome these challenges posed salinity is to use a salt-adapted microbial consortia in the microbial degradation process [11]. However, most related previous studies have focused on the treatment of saline wastewater. The various studies on the treatment of saline wastewater through activated sludge have reported different performances due to the differences in processes used and wastewater types [12], with some studies determining that treatment efficiency increased in saline wastewater [13]. Pierra et al. [14] used sediment with a salinity of 67.4 g/L as inoculum within the treatment of wastewater through activated sludge. Their study achieved the highest yield of hydrogen under the highest NaCl concentration of 75 g/L, suggesting a natural adaptation of the sediment inoculum to salt. In addition, many studies have reported on the ability of halophilic microorganisms to continue growth and metabolism under hypersaline conditions [15,16]. However, no study to date have focused on VFA production from biomass using acclimated anaerobic sludge. Therefore, there is a need for improved understanding of the response of the microbial community to the high salt stress. This understanding can help in the design of an operating strategy to alleviate the inhibition of waste treatment by salinity.
The sequencing batch reactor (SBR) process is often preferred over the continuous flow process (CFP) within waste treatment due to lower energy consumption and enhancement in the selective pressures for biological oxygen demand (BOD), nutrient removal, and control of filamentous bacteria [17]. Batch processes are extensively used to produce specialty chemicals, in biotechnology, and to produce pharmaceutical and agricultural products. Therefore, the present study aimed to evaluate the effect of inoculum on acidogenic fermentation operated in batch mode under highly saline conditions. Duplicate batch reactors were operated at two different high NaCl concentrations of 30 g/L and 70 g/L to observe the impacts of non-acclimated sludge (non-AS) and acclimated sludge (AS) as inoculum on the product spectrum, the type of acidogenic fermentation, and the respective microbial community.
Substrates and Inoculum
FW, containing rice, noodles, vegetables, meat, and tofu, was compounded based on the characteristics of similar FW previously collected from a canteen at Zhejiang Gongshang University (Hangzhou, China). The ratio, source and pretreatment of the substrate were consistent with previous study [10]. Two kinds of anaerobic granular sludge were used as inoculum. The non-acclimated anaerobic sludge (non-AS) was taken from an up-flow anaerobic sludge bed (UASB) reactor at the Snow Beer Brewery in Hangzhou, China. To promote an active bacterial population, the sludge was incubated at ambient temperature with a nutrient solution before inoculation. The acclimated anaerobic sludge (AS) was taken from a lab-scale anaerobic reactor which was used to treat saline wastewater with a NaCl concentration of 30 g/L running for 156 days. Table S1 shows the main characteristics of FW and the two kinds of anaerobic sludge used in this study.
Batch Fermentation Tests
Laboratory-scale batch tests were conducted in brown 1000-mL wide-mouthed bottles with a working volume of 500 mL, capped with a rubber stopper. The initial substrate/ inoculum ratio was 4:1, i.e., 28 g of FW and 7 g of anaerobic sludge (dry weight). The reactors were inoculated with the non-AS or the AS, dosed with different quantities of NaCl to obtain material NaCl concentrations of 30 or 70 g/L. A reactor with non-AS and with no additional NaCl for acidogenic fermentation was used as the control (non-AS_0). Table 1 shows more details on the experimental design. The experimental temperature was maintained at 30 ± 2 °C and pH was 1 3 maintained at 6.0 by the addition of 4.5 M H 2 SO 4 or NaOH during the experiment, based on our previous study [18]. Redox potential (ORP) ranged from − 100 to − 200 mV [19]. All fermentation tests were conducted in duplicate. Fermentation tests were carried out for 21 days.
Analytical Methods
Sample contents of sugar, lipids, soluble protein, total suspended solids (TSS), volatile suspended solids (VSS), total organic carbon (TOC), lactate, and VFAs (C2-C5) contents of the samples were determined using methods previously described [10]. Aliquots of the fermentation broth were removed from each reactor at specified times during the fermentation process. These samples were centrifuged at 10,000 rpm for 5 min. The supernatant was then passed through a filtration membrane with a pore size of 0.45 µm, following which the solubility indices (besides for TOC, lactate, and VFA) were measured. TOC, lactate, and VFA were measured after filtering the supernatant through a filtration membrane with a pore size of 0.22 µm.
Bacterial Community Analysis
Samples were collected from all reactors on day 0, day1, and every other day thereafter. The methods used to characterize the bacterial community was consistent with the previous study [10]. In brief, samples were collected from all reactors at specific times during the fermentation process. Genomic DNA was extracted using a DR4011 kit (Bioteke, Beijing, China) according to the instructions of the manufacturer. The methods used to determine the quality (A260/ A280) and quantity (A260) of the extracted genomic DNA, to amplify the extracted DNA, and to evaluate the bacterial community have been described in our previous study [10]. Samples were processed through MiSeq high-throughput sequencing (Illumina, San Diego, CA, USA), following with the obtained sequences were aligned and grouped into operational taxonomic units (OTUs) with 97% similarity. Sequences were then phylogenetically assigned to taxonomic classifications and allocated to phylum, class, and genus levels. Hierarchical cluster analysis was performed using R version 3.1.3 (www.r-proje ct. org).
Solubilization and Utilization of Substrates
The anaerobic digestion process typically consists of three steps: (1) hydrolysis; (2) acidogenesis; and (3) methanogenesis. The degradation of complex polymers in FW such as lignocellulosic materials, lipids, and proteins to smaller molecules requires the most time during the AD process [20]. Soluble chemical oxygen demand (SCOD) is an important intermediate in the metabolic pathway of AD due to its influence on the yield of VFAs through linking hydrolysis and acidogenesis. As shown in Fig. 1A, although there was a difference in SCOD on day 0 of fermentation, that of the non-AS groups significantly exceeded those of the AS groups by day 7 (p < 0.05). SCOD in the non-AS groups increased rapidly on day 1, almost reaching the maximum value. The maximum value of SCOD was obtained in the AS groups until day 9. These results indicated that non-AS groups produced more soluble organic matter compared to the AS groups at the hydrolysis stage. It was assumed that many hydrolyzing bacteria are not NaCl-tolerant and are eliminated during the acclimation process.
Soluble substrates such as sugars and proteins are intermediates within a dynamic process in which they are simultaneously and continuously dissolved from FW and consumed to produce other products such as VFAs. As shown in Fig. 1B, soluble sugars in the non-AS groups were significantly higher than those in the AS groups by a factor of 2.4-3.4 during the early stages of fermentation. Therefore, it can be speculated that the non-AS groups contained greater quantities of soluble organic matter compared to the AS groups and that organic matter dissolved more rapidly in the non-AS groups. As shown in Fig. 1E, the experimental data were consistent with the first-order kinetics equation for the reduction of soluble sugar during fermentation (R 2 > 0.88).
The concentration of soluble sugar at any fermentation time can be calculated by where C 0 is the initial concentration of soluble sugar, t is the fermentation time, C t is the soluble sugar concentration at t time, and k 1 is the reaction rate constant. As shown in Fig. 1E, the salt concentration had a considerable inverse relationship with the degradation rate of soluble sugar. The rank of the reactor treatments in terms of the rate of soluble sugar degradation was non-AS_0 > non-AS_30 > AS_30 > AS_70 > non-AS_70. At a lower salt concentration, the rates of soluble sugar degradation of the non-AS groups exceeded that of the AS groups. This result can likely be attributed to larger abundances of hydrolytic and fermenting bacteria in the non-AS groups ("Microbial Community of Inoculums" section). The use of the non-AS as an inoculum resulted in the production of greater quantities of soluble sugar. At a high NaCl concentration of 70 g/L, there was a slightly higher soluble sugar degradation rate in AS_70 compared to that in non-AS_70, which was likely due to the former being better adapted to a high salt environment. As is show in Fig. 1C, soluble proteins increased with increasing concentration of NaCl. Interestingly, the contents of soluble protein in the non-AS groups during early fermentation exceeded those in the AS groups. However, these differences reduced as the fermentation process progressed, with finally for NaCl concentration of 30 g/L the AS groups contained even more soluble protein compared to the non-AS groups. It is likely that NaCl resulted in high extracellular osmotic pressure, thereby triggering the rupturing of non-AS cells to release proteins and resulting in an increase in soluble protein during the early fermentation stage. Later, the non-AS groups gradually adapted to the high NaCl concentrations, resulting in an acceleration of protein degradation rate. At the same time, high NaCl concentrations inhibited the degradation of soluble protein, thereby resulting in high concentrations of soluble protein being maintained in the reactors.
Proteins in FW are first degraded to amino acids and then to ammonium, VFAs, and other products. The level of ammonia nitrogen is generally used to assess the degree of protein degradation. As shown in Fig. 1D, the changes in ammonia nitrogen concentration indicated that under the NaCl concentrations of 30 g/L and 70 g/L, the ammonia nitrogen concentrations in the AS groups significantly exceeded those in the non-AS groups by a factor of 1.6-1.8. This result could be attributed to two possible factors: (1) under higher NaCl concentrations (30 g/L and 70 g/L), the AS increased the degradation of proteins; (2) the metabolism of amino acids in the AS groups was dominated by deamination pathway, thereby inducing the release of ammonia nitrogen. Table S2 presents the durations of ammonia nitrogen production in different reactors. The ammonia nitrogen release rate was as follows: AS_30 > AS_70 > non-AS_30 > non-AS_70 > non-AS_0. As reported in our previous result, the release of ammonia nitrogen increased linearly with fermentation time [18], regardless of whether AS or non-AS was used in this study. It was also found that using the non-AS as inoculum, at NaCl concentrations higher than 10 g/L, the metabolism of amino acids shifts from mainly deamination to decarboxylation [10], which caused the less release of ammonia nitrogen. However, the rates of ammonia nitrogen release in the AS groups exceeded those in the non-AS groups in this study, indicating that salt-tolerant sludge could promote the release of ammonia nitrogen. Figure 2A shows that VFA accumulation in the control group (non-AS_0) increased rapidly from day 3 to day 12 and reached a maximum of 25.1 g/L on day 12. The time required to reach maximum VFA production in all groups was delayed under NaCl concentrations of 30 g/L and 70 g/L. The maximum VFA production for non-AS_30 and AS_30 groups occurred on day 15, whereas a shorter duration was required to reach the maximum VFA production in the AS_70 group compared to that in non-AS_70 group. VFA production generally decreased with increasing salt concentration, with the rank of the reactor treatments according to VFA production being non-AS_0 (25.1 g/L) > non-AS_30 (24.4 g/L) > non-AS_70 (22.6 g/L) > AS_30 (21.0 g/L) > AS_70 (20.5 g/L). Interestingly, the VFA produced by the non-AS groups exceeded that of the AS groups. This result could be attributed to the fact that although AS was better adapted to high NaCl concentrations, the abundance of acid-producing fermenters was not significantly increased (see "NMDS Analysis of Fermentation Process" section).
Product Spectrum
The products of fermentation were different under different NaCl concentrations (Fig. 2). The control group (non-AS 0) mainly produced acetic acid up to a maximum concentration of 14.9 g/L, equating to 70.1% of the total VFAs produced (Fig. 2B). The maximum acetic acid produced by the non-AS group of 13.4 g/L was higher than that of the AS group of 10.8 g/L under a NaCl concentration of 30 g/L. However, the differences in acetic acid production between the AS and non-AS groups decreased with increasing NaCl concentration up to 70 g/L, with an acetic acid concentration in both groups of 8.98 g/L. In comparison, higher quantities of propionic acid and lactic acid were produced at higher salt concentrations ( Fig. 2C and E), consistent with the results of our previous results [10]. Propionic acids produced in AS_30, non-AS_30, AS_70, and non-AS_70 groups accounted for 51.5%, 48.8%, 60.7%, and 62.2% of total VFA, respectively. Therefore, salt concentration had a greater impact on acidogenic fermentation compared with that of inoculum, regardless of whether the sludge was salttolerant or not. Figure 2D shows the change in butyric acid for all groups under different NaCl concentrations. The FW in the reactors under high NaCl concentrations showed low production of butyric acid compared with that of the control. However, Sarkar et al. [21] reported the different results and found VFA production with accumulation of more butyric acid (3.04 g/L) and acetic acid (1.17 g/L) along with traces of valeric acid at 40 g/L NaCl. As shown in Fig. 2E, lactic acid production increased with the increase of NaCl concentration. Also, as the NaCl concentration increased, the residence time of lactic acid in the reactors prolonged. During the following days, the concentration of lactate fell below 1 3 detection limits and propionic acid increased, indicating that propionic acid was produced by lactate fermentation. These results suggested that the type of acidogenic fermentation of FW changed to propionic acid production as the NaCl concentration increased.
Microbial Community of Inoculums
Then we analyzed the differences in microbial community structure between the AS and non-AS groups. Figure 3 shows the differences in bacterial composition resulting from the non-AS and the AS as an inoculum.
Among the 15 phyla, nine showed extremely significant differences (Fig. 3A). Proteobacteria (39.7%), Firmicutes (27.5%), Bacteroidetes (12.6%) and Chloroflexi (6.85%) dominated in the non-AS, whereas Proteobacteria (40.3%), Synergistetes (14.1%), Bacteroidetes (13.2%), Nitrospirae (12.5%) and Firmicutes (8.54%) dominated in the AS. Significant difference in the abundances of Firmicutes, Chloroflexi, Synergistetes, Nitrospirae and Bacteroidetes was observed between the non-AS and the AS. Proteobacteria, Firmicutes and Bacteroidetes are obligate or facultative bacteria characterized by high hydrolytic capacities during anaerobic digestion and an ability to produce VFAs from organic compounds [22]. Firmicutes was found at relatively high and low abundances in the non-AS and AS, respectively. This result indicated that high NaCl concentration strongly inhibited the Firmicutes phylum [23,24]. Most species in phylum Chloroflexi are filamentous bacteria capable of degrading macromolecular organics [25]. The relatively high abundances of Firmicutes and Chloroflexi in the non-AS groups during the early stage of fermentation might result in hydrolysis rates far exceeding those of the AS groups for fermentation experiments. In contrast, the AS had a relatively higher abundance of Synergistetes (14.1%) than that in the non-AS (3.90%), indicating that enriched Synergistetes played an important role in the anaerobic fermentation of FW under high salt stress, consistent with the report of Zhang et al. [24]. Interestingly, the relative abundance of Nitrospirae increased in the AS. Nitrospirae contain nitrifying taxa which oxidize nitrite to nitrate (nitrite-oxidizing bacteria, NOB). Nitrospirae are ubiquitously present in natural and engineered ecosystems, including oceans, freshwater habitats, soils, saline-alkaline lakes, hot springs, wastewater treatment plants, and aquaculture biofilters [26]. Wan et al. [27] similarly found that phylum Nitrospirae was dominant in the hydrogen reactors of thermophilic alkaline fermentation.
NMDS Analysis of Fermentation Process
The differences in microbial community composition were evaluated by comparing the AS with the non-AS groups using a nonmetric multidimensional scaling (NMDS) analysis based on unweighted Unifrac full tree similarity distance (Fig. 4). As shown in Fig. 4, samples from the reactors with the same inoculum generally clustered more closely and were separated from each other. This revealed a strong difference in microbial community compositions among different groups due to different inoculum and salts. The same inoculated sludge also showed a certain regularity with increasing of NaCl concentration.
Changes in Microbial Diversity
Alpha diversity (α-diversity) is defined as the mean diversity of species in different sites or habitats within a local scale. Table 2 summarizes the results of microbial alpha diversity analysis. Chao 1 and Ace indices represent microbial richness, whereas Shannon and Simpson indices represent microbial diversity. Compared with the inoculum, microbial diversity and richness decreased under high NaCl conditions. 1 3 Fig. 3 Analysis of the differences in the bacterial communities between inoculums of non-AS and AS at the A phylum level and B genus level (*p < 0.05, **p < 0.01, ***p < 0.001) Fig. 4 A nonmetric multidimensional scaling (NMDS) ordination based on microbial community composition Table 2 Qualified reads, OTU counts, and alpha diversity estimates of microbial populations a Non-AS and AS represents the seed sludges of non-acclimated anaerobic sludge and acclimated sludge, respectively. The number 6, 15 and 21 represent the sampling day. The number 0, 30 and 70 represent the NaCl concentrations of 0 g/L, 30 g/L and 70 g/L, respectively Microbial diversity and richness decreased first in the control, following which they increased up until the levels of the inoculum. This result demonstrated that microbes in the control were selected through acclimation to new conditions. In addition, the non-AS_30 and non-AS_70 groups showed decreased microbial richness. Although microbial diversity also decreased in both groups, there was an increasing trend in microbial diversity from day 6 to day 15. These results showed although high salinity decreased microbial richness and diversity, the microorganisms showed a capacity to adapt to the saline environment. Microbial diversity and richness increased with time in the AS_30 group, while increased microbial diversity and decreased microbial richness were observed in the AS_70 group. Under the same NaCl concentrations, compared with AS groups, non-AS groups had higher microbial richness but comparable microbial diversity, which is in alignment with the VFA production.
Microbial Composition and Difference Analysis
At the phylum level, Firmicutes, Proteobacteria, Bacteroidetes, Atribacteria, Synergistetes and Chloroflexi were the most abundant phyla in five groups of non-AS_0, non-AS_30, non-AS_70, AS_30 and AS_70, and together they make up more than 95% of the total, as shown in Fig. 5A. However, Firmicutes (43.4%) and Bacteroidetes (27.7%) dominated the control group (non-AS 0). Reactors containing 30 g/L NaCl showed relatively higher abundances of Firmicutes (50.8-68.8%) compared to the reactors containing 70 g/L NaCl. But greater abundances of Proteobacteria (36.7-60.7%) were observed in the reactors containing 70 g/L NaCl, regardless of the inoculum used. Similar microbial communities were observed in reactors under the same salt concentrations. The abundances of Firmicutes, Proteobacteria, Bacteroidetes, Atribacteria and Nitrospirae were significantly different in five groups (Fig. 5B). It was worth noting that the abundance of Nitrospirae was remarkably higher in the AS_70 group, compared with the other four groups (p < 0.002). The non-AS groups contained a larger proportion of the phyla Bacteroidetes, Atribacteria, and Chloroflexi, which are able to degrade macromolecular organics to VFAs, especially at high salt levels (Fig. S2). This result is consistent with the fact that a little higher VFA production was observed in the non-AS groups. At the genus level, the microbial compositions of the five groups of non-AS_0, non-AS_30, non-AS_70, AS_30 and AS_70 were significantly different. The Bacteroidetes (Bacteroidetes phylum), Veillonella (Firmicutes phylum), Streptococcus (Firmicutes phylum), Mangrovibacter (Proteobacteria phylum) and Candidatus_Caldatribacterium (Atribacter phylum) were the most abundant in five groups of non-AS_0, non-AS_30, non-AS_70, AS_30 and AS_70 (Fig. 6A).
Beneficial bacteria of Bacteroidetes and Veillonella, capable of producing VFAs, were remarkably increased in the non-AS_0 group, compared with other groups, as shown in Fig. 6B. The abundances of Streptococcus, Proteus (Proteobacteria phylum) and Lactococcus (Firmicutes phylum) observably increased both in groups of the non-AS_30 and AS_30, while the non-AS_30 group had higher abundance of Proteus than the AS_30 group (Fig. 6B). The abundance of Mangrovibacter in the AS_70 group was extremely significantly higher than that in other groups (p < 0.0005). In the non-AS_70 group, the abundances of Enterococcus (Firmicutes phylum), Clostridiisalibacter (Firmicutes phylum) and Weissella (Firmicutes phylum) obviously increased.
Effect of NaCl concentration on microbial composition difference
As shown in Fig. 6B, the beneficial bacteria of Bacteroidetes and Veillonella significantly increased in the control group. The main by-products of anaerobic respiration by Bacteroidetes include acetic acid, iso valeric acid, and succinic acid. Veillonella spp., which is well known for its ability to ferment lactate, mainly appeared from day 6 to day 15 during the fermentation (Fig.S1), consistent with the degradation of lactate in the control reactors.
Under NaCl concentration of 30 g/L, the phylum Firmicutes mainly contained the genus Streptococcus (Fig. 6B), which was observed in the non-AS_30 and AS_30 groups (Fig. S3), particularly from day 0 to day 9, following which their abundance clearly decreased with time (Fig.S1). The genus Streptococcus encompasses Gram-positive, catalasenegative, facultatively aerobic and homofermentative cocci which produce l(+)-lactic acid as major end product of glucose fermentation [36]. The genus Proteus appeared mainly from day 9 to day 21, particularly in the non-AS_30 group (Fig.S1). Proteus spp. decompose organic substances and oxidatively deaminate amino acids, hydrolyze urea and exhibit proteolytic activity [37]. Beneficial bacteria of Lactococcus, which are homofermentative and are used to produce l( +) lactic acid from glucose, had a higher relative abundance from day 6 to day 9 in both the non-AS_30 and AS_30 groups (Fig. S1). Correspondingly, abundant production of lactic acid was observed from day 3 to day 9 (Fig. 2). Further, the microbial composition difference between AS_30 and non-AS_30 groups was analyzed (Fig. S2). The relative abundances of Candidatus_Caldatribacterium and norank_f_Synergistaceae were significantly higher in the non-AS_30 group. Candidatus_Caldatribacterium played a vital role in the anaerobic fermentation of carbohydrates to VFAs [38,39]. norank_f_Synergistaceae can improve the hydrolysis acidification process and the acetotrophic pathway [33]. This result supported the fact that more VFAs were produced in the non-AS_30 group. Community abundance on phylum level. A Microbial community bar plot with the relative abundance higher than 5%, B Kruskal-Wallis H test bar plot. The asterisk represents significance (*p < 0.05, **p < 0.01, ***p < 0.001) 1 3 Fig. 6 Community abundance on genus level. A Microbial community bar plot with the relative abundance higher than 5%, B Kruskal-Wallis H test bar plot. The asterisk represents significance (*p < 0.05) Under NaCl concentration of 70 g/L, the genus Mangrovibacter belonging to the phylum Proteobacteria dominated (Fig. 6B), which was detected in both the non-AS_70 and AS_70 groups (Fig. S4). Moreover, the abundance of Mangrovibacter in the AS_70 group (45.7%) was much higher than that in non-AS_70 group (22.3%). Members of genus Mangrovibacter are facultatively anaerobic and nitrogen-fixing bacteria which are slightly halophilic. The optimal NaCl concentration and temperature for growth of Mangrovibacter were 1% and 30 °C, respectively [40]. Li et al. [41] found that Mangrovibacter was the dominant bacteria in halotolerant aerobic granular sludge for treating saline wastewater with a salinity of 3%. In addition, the abundances of Enterococcus, Clostridiisalibacter and Weissella were significantly higher in the groups with 70 g/L NaCl (Fig. 6B). Růžičková et al. [42] reported that Enterococcus is a large genus of lactic acid bacteria and can adapt up to 6.5% NaCl. Clostridiisalibacter is a Gram-positive moderately halophilic strictly anaerobic and motile bacterial genus with an optimum at 50 g/L NaCl [43]. Weissella are obligately heterofermentative bacteria that produce CO 2 from carbohydrate metabolism, with lactic acid and acetic acid being the other major end products of sugar metabolism [44]. Several genera also were identified accounting for most of the differences in microbial community between the non-AS_70 group and AS_70 group, including Mangrovibacter, norank_c_Bac-teroidetes_vadinHA17, Thioclava and Nitrospira (Fig. S4). norank_c_Bacteroidetes_vadinHA17 was more dominant in the non-AS_70 group. Greater abundances of halophilic bacteria were found in the AS_70 group, including the genera of Mangrovibacter, Thioclava and Nitrospira, and less hydrolytic/acidogenic bacteria could cause low VFA production. Therefore, salt inhibition seems not to be dependent on the inoculum. A selection of Streptococcus and Mangrovibacter as a result of gradual increase of NaCl from 30 to 70 g/L was observed in both the AS groups and non-AS groups. So NaCl concentration had a greater impact on the acidogenic fermentation process, and if grown under identical saline conditions sludges had similar microbial populations.
Conclusions
The non-AS was more conducive to the hydrolysis and acidogenesis process of FW compared to the AS. The degradation of organic matter was inhibited in all groups under high NaCl concentrations. Although the AS can shorten the time required to reach maximum VFA production, VFA production could not be increased. Microbial diversity and richness decreased under high NaCl conditions as compared with that in the inoculum. However, the microbial community also presented an ability to adapt to the saline environment. The microbial communities showed clear differences in NaCl concentrations. Proteobacteria, Firmicutes, Bacteroidetes and Chloroflexi dominated in the non-AS, whereas Proteobacteria, Synergistetes, Bacteroidetes, Nitrospirae and Firmicutes dominated in the AS. The non-AS groups contained a larger proportion of the phyla Bacteroidetes, Atribacteria, and Chloroflexi, which are able to degrade macromolecular organics to VFAs, especially at high salt levels. Therefore, more VFA produced in the non-AS groups, while more salttolerant bacteria were found in the AS groups. Nevertheless, the NaCl concentration had a greater impact on the process of acidogenic fermentation. | v3-fos-license |
2018-04-03T02:02:04.843Z | 2018-01-08T00:00:00.000 | 3577198 | {
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} | pes2o/s2orc | Probiotic Lactobacillus sp. inhibit growth, biofilm formation and gene expression of caries‐inducing Streptococcus mutans
Abstract Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real‐time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH‐neutralized, catalase‐treated or trypsin‐treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH‐dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide‐dependent antimicrobial activities. All biofilm‐forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.
Introduction
Dental caries is a common chronic oral disease that can affect the health of adults and children [1]. A number of studies demonstrated correlations between poor oral health and heart diseases [2,3]. Dental caries is an endogenous disease that results from homoeostatic imbalance between the host and microbiota [4]. The shift of non-pathogenic micro-organism from commensalism to parasitism resulted from changes in the oral environment due to poor hygiene, smoking, systemic diseases and decrease in saliva flow [1,5]. Streptococcus mutans has been identified as a main contributor to dental caries [6].
The oral cariogenic biofilm formation occurs through phases that start by early colonization of pellicle by non-mutans Streptococci.
This phase creates a favourable area for the growth of Streptococcus mutans and initial biofilm formation [7]. Streptococcus mutans possesses virulence factors that contribute to caries formation such as: (i) Production of acid that damages dental hard tissues [8]; (ii) An agmatine deiminase system and F-ATPase encoded by the aguBDAC operon [9] and atpD gene [10], which are major components in acid-adaptive response that contribute to the aciduric characteristics. (iii) The ability to synthesize exopolysaccharides (EPS) from sucrose by the action of multiple glucosyltransferases (Gtfs) encoded by the genes gtfb, gtfc, gtfd, in addition to fructosyltransferase encoded by sacB (ftf) gene. The glucosyltransferase and fructosyltransferase enzymes catalyse the synthesis of extracellular glucan and fructan polymers from sucrose, respectively [11]. The EPS formed is thought to play dual roles in promoting microbial adherence to surface in addition to protecting embedded bacteria [12]. These virulence factors work under the control of quorumsensing systems. The two-component signal transduction systems (TCSTS), including comCDE and vicRKX, are among the regulatory networks that regulate gene expression in response to stimuli from the surrounding environment and are thus essential for bacterial survival and virulence modulation [13,14].
Caries management strategies include the use of conventional physical removal of plaque and the reduction of bacterial population by chlorhexidine. Other interventions include maintaining the oral ecosystem by probiotics [15]. Probiotic bacteria are live micro-organisms that can confer health benefits to the host when administered in sufficient amounts [16]. Most probiotics are Gram-positive bacteria that belong to the genera Lactobacillus or Bifidobacterium [17].
Lactobacilli (LB) constitute part of the oral microbiota and can be linked to the oral health status of the individual [18]. They comprise about 1% of the cultivable oral microbiota. The most common LB strains isolated from oral microbiota include L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. fermentum, L. acidophilus and L. salivarius [19]. Isolated LB strains from subjects without dental caries have a significantly increased capacity to inhibit the growth of Streptococcus mutans compared with the strains isolated from subjects with active caries. Thus, probiotic LB does have a therapeutic anticaries potential [20][21][22]. Stamatova and Meurman [23].
There could be universal mechanisms by which probiotics impact oral pathogens. Generally, probiotics are believed to compete with pathogens for space and nutrients but have mostly unknown mechanisms of action. These may include impacts on the production of lactic acid, peroxide or bacteriocin in addition to possible immunomodulatory activities [24]. We hypothesized that Lactobacillus sp. inhibits the growth, biofilm formation and gene expression of Streptococcus mutans. We then studied the mechanisms by which the probiotic Lactobacillus sp. antagonizes Streptococcus mutans.
Preparation of spent culture supernatant (SCS)
The spent culture supernatant (SCS) for each Lactobacillus sp. strain was prepared according to Lin et al. [25], and then the supernatant was filtered using 0.45-lm filters (Millipore, Bedford, MA, USA). The supernatant was divided into four portions. One portion was left untreated, and the other three portions were treated to eliminate the effect of organic acids, hydrogen peroxide and bacteriocin. The effect of organic acid was neutralized by adjusting the pH of SCS to 6.5 with 1 N NaOH. The other two portions were treated with 1 mg/ml trypsin (Sigma-Aldrich, USA) and 0.5 mg/ml catalase (Sigma-Aldrich, C1345, USA) to eliminate the effect of bacteriocin and hydrogen peroxide, respectively [26]. Treated and untreated supernatants were stored at À20°C.
The agar diffusion method for antimicrobial screening of Lactobacillus sp
The antibacterial activity of Lactobacillus sp. on Streptococcus mutans was assessed using an agar diffusion method adapted from the one used by Cadirci and Citak [27]. Streptococcus mutans was incubated in Brain-Heart Infusion (BHI) at 37°C for 24 hrs. Melted BHI agar medium held at 45°C was inoculated with Streptococcus mutans at a concentration equivalent to McFarland 0.5 standard (1.5 9 10 8 CFU/ml). Wells of 7 mm diameter were filled by 100 ll of SCS. Inhibition zones were measured in millimetres after incubating the plates anaerobically at 37°C for 24 hrs. The same test was performed using Lactobacillus sp. whole bacterial culture (WBC) instead of SCS, with a turbidity equivalent to McFarland 0.5.
Antibacterial testing of treated and untreated SCS
To determine the antibacterial activity of the SCS, Streptococcus mutans was grown overnight at 37°C in BHI broth. The Streptococcus mutans culture was diluted with BHI broth medium to a turbidity equivalent to McFarland 0.5 (1.5 9 10 8 cells/ml). Then, 100 ll of the Streptococcus mutans suspension and 100 ll of untreated supernatants were added to the wells of 96-well microtitre plate in eight replicates for each Lactobacillus SCS (Greiner Bio-One, Kremsm € Unster, Austria). The plates were then incubated anaerobically at 37°C for 24 hrs. In control wells, the SCS was replaced by sterile MRS broth. The OD600 nm was recorded after incubation using microplate reader (Stat Fax â 2100) [28]. The same steps were repeated with treated supernatants to determine the change in antimicrobial activity after removing the effect of acidic pH, peroxides and bacteriocin.
The effect of Lactobacillus sp. SCS on Streptococcus mutans adherence in the 96-well microtitre plate by the volume of 100 ll and incubated at 37°C for 24 hrs. Culture supernatant was removed, and wells were washed with sterile saline. A volume of 100 ll of untreated supernatant was added in each well and incubated at 37°C for 24 hrs. Reduction in biofilm formation was determined as previously described [29].
Scanning electron microscopy (SEM) observation of dual-Streptococcus mutans-Lactobacillus sp. biofilm Streptococcus mutans and Lactobacillus sp. were cocultured overnight at 37°C in BHI and MRS broth respectively followed by dilution to a concentration equivalent to McFarland 0.5. A clean sterile cover slide was added to the wells of the six-well plate (Greiner Bio-One, Kremsm € Unster, Austria). In each well, 250 ll of the Streptococcus mutans suspension and 250 ll of one of the Lactobacillus sp. suspension were added to 1.5 ml of BHI broth (supplemented with 0.2% sucrose) and incubated anaerobically at 37°C for 24 hrs.
A monospecies culture of Streptococcus mutans biofilm was similarly prepared except that we replaced the Lactobacillus sp. culture with uncultured MRS medium. Cover slides were gently washed with phosphatebuffered saline (PBS) once, fixed and prepared for SEM observation (JSM-7600F, JEOL) according to a previously published protocol [30].
Extraction of total bacterial RNA
We studied the effect of Lactobacillus sp. filtered supernatant on Streptococcus mutans in the planktonic form and the biofilm form. Streptococcus mutans was grown overnight at 37°C in BHI broth and was diluted to McFarland 0.5. A volume of 250 ll Streptococcus mutans suspension and 250 ll of the SCS were added to 1.5 ml of BHI broth and were incubated anaerobically at 37°C for 24 hrs. In control wells, the Lactobacillus sp. supernatant was replaced by MRS broth [25]. After incubation, culture suspension was removed from wells for RNA extraction from planktonic bacteria. Cells adhering to the plate wells were washed twice by sterile saline and then dislodged and suspended in saline by scraping into a centrifuge tube. The total RNA was isolated from Streptococcus mutans planktonic and adherent cells using Direct-Zol RNA MiniPrep kit (Zymo Research, CA, USA) according to the manufacturer's instructions. The remaining DNA in RNA samples was treated by RNase-free DNase I (New England Biolab, MA, USA) to eliminate DNA contamination. Agarose gel electrophoresis of RNA samples verified its integrity. RNA concentration and purity were determined by the ND-1000 spectrophotometer (NanoDrop Technology; Wilmington, DE, USA). Finally, the SensiFast TM cDNA synthesis kit (Bioline, MA, USA) was used to reverse transcribe 1 lg of total RNA sample into cDNA.
Quantitative real-time polymerase chain reaction (qRT-PCR) and data analysis Using qPCR, we examined the effect of Lactobacillus sp. spent supernatant on the expression levels of ten target genes [gtfb, gtfc, gtfd, sacB, comC, comD, vick, vicR, aguD and atpD] involved in glucan production, fructan production, quorum sensing and acid tolerance in Streptococcus mutans. The primers used for amplification of comC, comD and sacB (ftf) genes were designed using the complete genome sequence of Streptococcus mutans ATCC 25175 obtained from the NCBI database (GenBank accession no. PRJNA179256) and used as the base for primer design. Primers for the qPCR used in the current study (Table 1) were synthesized by Invitrogen (Massachusetts, USA). Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed by Applied Biosystems StepOne TM Instrument using SensiFast TM SYBR Hi-Rox Master (Bioline, Massachusetts. USA). All reactions (20 ll) were performed using three technical replicates. Each reaction mixture contained 100 ng cDNA and 400 nM primers per reaction. The RT-PCR cycling conditions were as follows: one cycle with 95°C for 2 min.; then 40 cycles of denaturation at 95°C for 5 sec., annealing at 52-62°C (depending on primers used) for 10 sec., and extension and fluorescent data collection at 72°C for 20 sec. A dissociation curve was generated at the end of each reaction. In all qPCR runs, negative controls without template were run in parallel. The 16s rRNA gene (housekeeping gene) was selected as the internal control based on the results of BestKeeper â software tool [31]. The relative mRNA levels of genes of interest were determined and normalized to the expression of the housekeeping gene using the ΔΔCT value analysis [32]. The qPCR data were expressed as the fold change in expression levels of genes in Streptococcus mutans ATCC 25175 cells exposed to SCS of the four tested Lactobacillus sp. as compared to their levels in the untreated cells (calibrators). The changes in gene expression were tested in the Streptococcus mutans cells in the planktonic form and the biofilm-forming state.
Immunomodulatory effect of probiotic Lactobacillus sp
Human peripheral blood mononuclear cells (hPBMCs) from healthy volunteers were treated with SCS of Lactobacillus sp. as previously described by Wu et al. [30]. The concentrations of IFN-c and IL-10 were determined using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (CUSABIO, BIOTECH CO, USA). A written consent was obtained from each subject. The protocol was approved by the Ethics Committee of the Faculty of Pharmacy, October University for Modern Sciences and Arts.
Statistics
Experimental results were analysed for statistical significance using GraphPad Prism (GraphPad, San Diego, CA, USA). A one-way analysis of variance (ANOVA) was performed. Data comparisons were performed using either Dunnett's multiple comparison test or Tukey's multiple comparison test.
Agar diffusion assay
The zone of inhibition produced by whole bacterial culture (WBC) (concentration 1.5 9 10 8 cells/ml) was larger than that produced by spent culture supernatant (SCS) produced by equivalent concentration of cells. This indicates the higher antimicrobial effect of WBC as compared to the cell-free filtered supernatant. According to the zone of inhibition diameter, the highest antimicrobial activities of Lactobacillus sp. were observed with L. casei and L. reuteri, whereas the lowest antimicrobial activities were observed with L. plantarum and L. salivarius (Table 2).
Antimicrobial effect of treated and untreated Lactobacillus sp. supernatant against Streptococcus mutans
The untreated supernatants of the four Lactobacillus sp. showed strong significant inhibitory effect (Fig. 1) on the growth of Streptococcus mutans (P < 0.01). There was no significant difference in the potency of the inhibitory effect between the four samples (P > 0.05). After neutralizing the supernatant acidity, the antimicrobial effect was significantly reduced (P < 0.01) compared with untreated All results were significantly different from control (P < 0.01). supernatant, yet still showing significant reduction (P < 0.05) in Streptococcus mutans growth ( Fig. 2A, B, C and D). Lactobacillus salivarius was the only tested strain that showed significant reduction (P < 0.05) in its antimicrobial effect on Streptococcus mutans after addition of catalase (Fig. 2D) indicating that peroxides contribute in its antimicrobial effect against Streptococcus mutans.
Effect of Lactobacillus sp. filtered supernatants on Streptococcus mutans adherence and preformed biofilm Lactobacillus salivarius supernatant caused significant reduction (P < 0.01) in Streptococcus mutans adherence and preformed biofilm. Reduction percentages were 87% and 47%, respectively. The effect of L. casei supernatant was the least among tested supernatants on adherence as it showed no significant effect on the preformed biofilm. The L. plantarum and L. reuteri supernatant caused reduction in adherence with percentages of 81.7-80.5% and reduction in preformed biofilm with percentage of 26.5-24.7% (Fig. 3).
Scanning electron microscope
As shown in Figure 4, the Streptococcus mutans appeared to form a compact, island-like biofilm covered by large amounts of slime or network-like structures. Changes in exopolysaccharides (EPS) matrix structure and quantity were observed in biofilm formed by coculture of Streptococcus mutans and different Lactobacillus sp. strains. Moreover, we observed fewer bacteria and smaller microcolonies attached to the surface.
Analysis of qPCR results
We used qPCR to evaluate and compare the impact on Streptococcus mutans ATCC 25175 cells after exposure to four Lactobacillus sp. SCS (diluted 1:8 in BHI) overnight. The levels of expression of ten genes, that have been previously shown to be involved in virulence of the S. mutans in the planktonic and biofilm-forming cells, were compared to the control untreated cells prepared under the same conditions without tested SCS. The selected genes included four genes involved in the two-component signal transduction systems (TCSTS) [comC, comD, vicK, vicR], four genes involved in EPS formation [three of which are involved in glucan formation (gtfB, gtfC and gtfD), one gene is involved in fructan formation (sacB (ftf))], and two genes associated with stress survival (aguD, and atpD).
As revealed by the one-way ANOVA, there was an overall significant reduction (P < 0.01) in the expression of most of the tested genes among the different groups, in both planktonic forms and biofilm-forming cells. Dunnett's multiple comparison test was used to assess the significance of the difference between gene expression levels in target genes of exposed and control groups. As shown in Figure 5, few genes showed no significant difference (P > 0.01) in expression as compared to the control under certain conditions. These genes are comC and gtfD in planktonic cells exposed to L. plantarum SCS, gtfC gene in planktonic cells exposed to L. salivarius SCS, and comC gene in the biofilm-forming cells exposed to L. reuteri SCS.
The effect of SCS of different Lactobacillus sp. was variable on all tested TCSTS system genes. L. salivarius supernatant caused upregulation of the vicK gene by threefold to 21-fold, in the planktonic and adherent cell forms, respectively. In planktonic cells, the expression of the comC gene, coding for competence-stimulating peptide, was up-regulated in the presence of L. salivarius supernatant only. Up-regulation of the same gene was observed in biofilm-forming cells treated with L. casei, L. plantarum and L. salivarius. The comD gene, coding for cognate histidine kinase receptor, was up-regulated in the planktonic cells exposed to SCS of tested Lactobacillus sp. except for L. casei. On the other hand, it was down-regulated in biofilm-forming cells except those exposed to L. salivarius supernatant.
Significant reduction in gene expression of glucan (gtfB, gtfC, gtfD) and fructan (sacB) forming genes was observed in the adherent Streptococcus mutans cells in the presence of all tested SCS. The effects of the same supernatants were variable on the planktonic cells, as they showed significant up-regulation (P < 0.01) in gtfB and gtfC genes in the following cases: high up-regulation in gene expression levels in presence of the supernatants of L. casei (30-fold change in gtfB gene expression), and L. plantarum (20-fold and 17fold change in gtfB and gtfC gene expression, respectively); moderate up-regulation in gene expression of gtfB and gtfC genes in the presence of L. reuteri (2.5-fold) supernatant. Significant up-regulation (P < 0.01) of the gtfD gene was observed in the presence of Stress response genes (atpD and aguD) were down-regulated in biofilm-forming cells in the presence of all tested SCS. In planktonic forms, these two genes showed significant reduction (P < 0.01) in expression except in two cases: The first is the atpD gene in the presence of L. plantarum and L. salivarius supernatants, and second is the aguD gene in the presence of Lactobacillus casei.
Immunomodulatory activities of Lactobacillus sp
The SCS of Lactobacillus sp. was incubated with hPBMCs isolated from healthy volunteers for 48 hrs. The production levels of the immunostimulatory IFN-c and immunoregulatory IL-10 cytokines were measured by ELISA. All Lactobacillus sp. standard strains stimulated hPBMCs to produce IFN-c higher than untreated controls. In contrast, IL-10 concentrations were reduced after treating hPBMC with Lactobacillus sp. supernatants (Table 3).
Discussion
Dental caries is one of the most common diseases worldwide. The oral microbiota is composed of over 700 bacterial taxa [33]. Under certain conditions, bacteria like Streptococcus mutans can be pathogenic and cause dental caries. Streptococcus mutans is a major contributor to dental caries development due to its virulence factors including the ability to synthesize extracellular polysaccharide and the ability to produce acidic metabolites [8].
Lactobacillus sp. constitute a main constituent of the microbiota in our oral cavity [34]. Lactobacillus sp. probiotics have been proven to be efficient in treating certain gastrointestinal disorders [35]. Lactobacillus sp. probiotics could possibly control dental caries using similar mechanisms that can play against Streptococcus mutans invasion strategies [8]. This is because Lactobacillus sp. were shown to be able to produce organic acids, hydrogen peroxide, bacteriocins and adhesion inhibitors [36].
The Lactobacillus sp. used in this study were L. casei subspecies casei (ATCC 393), L. reuteri (ATCC 23272), L. plantarum subsp. Plantarum (ATCC 14917) and L. salivarius (ATCC 11741). These strains were chosen because they caused reduction in dental caries in previous studies including: Lactobacillus casei [37,38], Lactobacillus reuteri [39][40][41], Lactobacillus plantarum [42] and Lactobacillus salivarius [43,44]. The precise mechanisms by which this happens are still unclear. Thus, the aim of the study was to assess mechanisms by which Lactobacillus sp. can control dental caries. We tested the effect of these four Lactobacillus sp. on the growth, adherence, biofilm formation and gene expression of Streptococcus mutans (ATCC 25175), in addition to the immunomodulatory effect.
The antimicrobial screening of the four tested strains of Lactobacillus using the agar diffusion method revealed differences in antimicrobial activity between different strains as determined by the size of the zone of inhibition. The highest effect was detected by L. casei, and L. reuteri, followed by L. salivarius and L. plantarum. Lactobacillus WBC caused higher antimicrobial effect on Streptococcus mutans than their corresponding SCS of the same Lactobacillus species. The difference in zone of inhibition caused by WBC compared to SCS may suggest that the presence of living metabolically active Lactobacillus sp. cells in WBC could result in the production of active antimicrobial agents in response to stimuli [45]. The tested Lactobacillus sp. strains caused significant reduction in the microbial growth of Streptococcus mutans in BHI broth, as determined by the change in OD 600. At the same time, there was no significant difference between different Lactobacillus species regardless of their metabolic pattern. Strict homofermentative organisms such as Lactobacillus salivarius, facultative heterofermentative organisms such as Lactobactobacillus casei and Lactobacillus plantarum, and obligate heterofermentative organisms such as Lactobacillus reuteri showed similar antimicrobial effects.
To determine the effect of organic acids, hydrogen peroxide and bacteriocin produced by tested Lactobacillus sp., their effect was demolished by neutralization, catalase and trypsin addition, respectively. Neutralization of SCS to pH 6.5 significantly reduced the antimicrobial effect of the tested SCS. The low pH is an important factor for growth inhibition, and it is important for the production of bacteriocin [46]. Streptococcus mutans is an acidogenic bacteria, that is produce organic acid as end product for sugar fermentation, and it is an aciduric bacteria, that is can tolerate acid in the plaque environment, hence, it can survive under acidic conditions [47]. The acid tolerance genes, such as atpD and aguD genes, allow Streptococcus mutans to carry out metabolic processes at low-pH values [48]. The observed reduction in gene expression of atpD, aguD, in Streptococcus mutans, can decrease its acid tolerance, which can lead to bacteriostasis and eventual death [49]. Anticaries agents such as the natural compounds a-mangostin and catechin epigallocatechin gallate can down-regulate the atpD and aguD genes [10,50].
It was observed that neutralized SCS caused lower reduction in microbial growth than untreated SCS, but yet neutralized supernatant still showed significant reduction in Streptococcus mutans growth when compared to control. This suggests the influence of other antimicrobial agents such as hydrogen peroxide, bacteriocin, [51] and biosurfactant [52] that contribute with acid to growth inhibition.
Some Lactobacillus sp. have the ability to produce hydrogen peroxide, which can be toxic to organisms lacking hydrogen peroxidescavenging enzymes such as Streptococcus mutans [53]. Adding catalase to Lactobacillus sp. supernatant caused reduction in the antimicrobial effect against Streptococcus mutans, but the significant reduction was observed only with Lactobacillus salivarius (ATCC 11741). This indicates that hydrogen peroxide contribution in antimicrobial activity of the tested Lactobacillus sp. is low except for L. salivarius supernatant.
Streptococcus mutans has been shown to initiate a response to various adverse environmental stressors, including oxidative stress, and acidic pH, by actively producing competence-stimulating peptide (CSP) encoded by the comC gene [54]. In our study, the expression of the comC was up-regulated in biofilm-forming cells compared with the untreated control. This was in contrast to comD which was downregulated in biofilm-forming cells treated with L. casei, L. reuteri or L. plantarum. The antimicrobial testing of L. salivarius supernatant on Streptococcus mutans demonstrated the influence of supernatant pH and peroxide in the antimicrobial activity. Thus, the production of these stress factors by this strain might explain the significant up-regulation in comC and comD genes in both the planktonic and biofilm-forming Streptococcus mutans cells treated with this supernatant. Biofilm formed of Streptococcus mutans having single mutation in comC, comD and comE, or the triple mutation of comCDE showed different biofilm architecture in comparison with the wildtype strain [55]. This might explain the difference in biofilm formed by the coculture of Streptococcus mutans and Lactobacillus sp. as observed by SEM due to difference in comCDE expression.
Lactobacillus sp. can produce bacteriocin or bacteriocin-like polypeptides that have a small molecular weight of <10 kD. In our study, trypsin-treated supernatant showed no significant difference from the untreated SCS on Streptococcus mutans growth. This result indicates the low production of bacteriocin by Lactobacillus sp. This may not be the only explanation though because bacteriocin production by Lactobacillus sp. has been reported in several previous studies carried on L. casei [56], L. reuteri [57], L. plantarum [58] and L. salivarius [59].
Streptococcus mutans contributing to dental caries usually exists in the biofilm form inside the oral cavity. EPS of Streptococcus mutans contribute to dental caries by helping develop an oral biofilm in addition to forming a barrier against chemical agents. Therefore, therapeutic agents that target the biofilm can be the most suitable for dental caries prevention.
The SCS of the four tested Lactobacillus sp. caused reduction in Streptococcus mutans biofilm with variable degrees. The highest reduction observed was with the supernatants of L. salivarius (87% for Streptococcus mutans adherence, and 47% for Streptococcus mutans preformed biofilm). The vicR gene was down-regulated in planktonic forms and biofilm-forming Streptococcus mutans exposed to all tested SCS. On the other hand, the vicK gene was down-regulated upon the exposure of Streptococcus mutans to L. casei and L. reuteri supernatants. This down-regulation might explain the reduction in Streptococcus mutans adherence and preformed biofilm as demonstrated by the SEM results. The vicKRX Asterisks indicate statistically significant differences in the expression of each gene between treated samples and control, as analysed using the one-way ANOVA with Dunnett's post-testing for multiple testing (*P ≤ 0.01; ns, no significant difference). Error bars indicate standard deviation system has a significant influence on biofilm formation, and the null mutation in the vicK and vicR genes can cause aberrant biofilms which are easily removed [65]. The vicRKX system regulates the glucosyltransferase-encoding genes, and thus, mutation in this system can cause a significant decrease in gtfD gene expression, as well as increased expression of the gtfB gene [66]. Changes in the gtfB and gtfD gene expressions, due to a mutated vicRKX system, were observed in planktonic Streptococcus mutans in the presence of L. casei, L. reuteri and L. plantarum supernatants, which caused a reduction in both vicK and vicR genes. In biofilmforming cells, both gtfB and gtfD genes were down-regulated in the presence of all tested Lactobacillus sp. despite reduction in vicK and vicR genes. This could be attributed to the influence of factors other than vicKRX on the gtf genes such as: luxS (AI-2 autoinducer-coding synthesis), ropA (encoding for the trigger factor) and RegM (the catabolite-repression regulator in Streptococcus mutans) [67], in addition to biosurfactants produced by Lactobacillus sp. which could reduce gtfB, gtfC and gtfD expressions in Streptococcus mutans [68].
Gtf genes that code for glucosyltransferase enzyme are primary virulence factors for Streptococcus mutans and thus can be a selective drug target for prevention of cariogenic biofilms. The Lactobacillus sp. supernatant-induced altered gene expression indicates a promising anticaries effect. In vitro studies indicated that gtfB and gtfC were essential for the sucrose-dependent attachment of Streptococcus mutans cells to hard surfaces and for microcolonies formation, but gtfD was not essential [52]. In the biofilmforming bacteria, the expression of the three glucosyltransferase genes (gtfB, gtfC and gtfD) and the fructosyltransferase genes sacB (ftf) showed significant down-regulation as compared to the control group of untreated biofilm-forming cells. Glucosyltransferase S, encoded by gtfB, synthesizes insoluble glucan [67] and allows cell clustering [69]. Thus, mutant Streptococcus mutans strains, defective in gtfB, are less cariogenic than their parent strains [70]. The disruption of insoluble glucans synthesis can induce a reduction in biofilm formation, which can influence the pathogenesis [63]. Thus, the gtfB expression reduction could explain the highest antibiofilm effect produced by L. salivarius on Streptococcus mutans adherence (87% reduction) and on preformed biofilm (47% reduction). The promising results of L. salivarius supernatant on Streptococcus mutans biofilms may indicate its possible anticaries effect. Thus, restoring the oral microenvironment with L. salivarius might be effective in preventing the colonization of periodontopathic bacteria [30]. The difference in expression of gtfB and gtfC genes, in our study, indicates that there is no common promoter for them. Ullrich reported the potential presence of independent promoters for both genes [71]. Low-pH value increases the expression of the gtfBC gene but reduces sacB gene expression, which can lead to high-biomass biofilms [48]. The reduced Streptococcus mutans adherence and EPS formation in presence of SCS of L. casei, L. reuteri and L. plantarum despite the up-regulation in the expression of the gtfb genes could be attributed to reduction in enzymatic function rather than reduction in gene expression [63].
The effect of Lactobacillus sp. on the production of IL-10 and IFNc was studied. IL-10 is an immunosuppressive cytokine that is normally up-regulated in inflamed pulp by bacterial infection to prevent the spread of inflammation [72]. In our study, all tested Lactobacillus sp. supernatants inhibited IL-10 production. To the best of our knowledge, this is the first study to show reduced IL-10 production in response to Lactobacillus sp. This warrants further investigation. IFN-c is a pro-inflammatory cytokine that synergizes with TNFa in increasing the microbicidal capacity of macrophages [73]. The tested Lactobacillus sp. induced higher levels of IFN-c which can signify more robust innate and potentially adaptive immune responses at the site of infection.
The study showed that Lactobacillus sp. can inhibit tooth decay and control dental caries. This possible anticaries effect could be attributed to: (i) the inhibitory effect on Streptococcus mutans growth which were mainly due to organic acid generation and peroxide production; (ii) reduction in cell adherence and preformed biofilm; (iii) down-regulation in several Streptococcus mutans virulence genes including acid tolerance genes (atpD and aguD genes), EPS-producing genes (gtfBCD and sacB) and quorum-sensing genes (vicKR and comCD); (iv) immunomodulatory effect due to the induction of IFN-c production and inhibition of IL-10 production.
Author contributions
Dr. Reham Wasfi, Dr. Ola A. Abd El-Rahman, Dr. Mai M. Zafer and Dr. Hossam M. Ashour contributed to the design of the study, performance of experiments, analysis of the results and writing of the manuscript. | v3-fos-license |
2020-09-08T13:06:15.631Z | 2020-09-06T00:00:00.000 | 221524044 | {
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} | pes2o/s2orc | Development of a bioreactor system for pre‐endothelialized cardiac patch generation with enhanced viscoelastic properties by combined collagen I compression and stromal cell culture
Treatment of terminal heart failure still poses a significant clinical problem. Cardiac tissue engineering could offer autologous solutions for the replacement of nonfunctional myocardial tissue. So far, soft matrix construction and missing large‐scale prevascularization prevented the application of sizeable cardiac repair patches. We developed a novel bioreactor system for semi‐automatic compression of a collagen I hydrogel applying 16 times higher pressure than in previous studies. Resistance towards compression stress was investigated for multiple cardiac‐related cell types. For scaffold prevascuarization, a tubular cavity was imprinted during the compaction process. Primary cardiac‐derived endothelial cells (ECs) were isolated from human left atrial appendages (HLAAs) and characterized by fluorescence‐activated cell sorting (FACS) and immunocytology. EC were then seeded into the preformed channel with dermal fibroblasts as interstitial cell component of the fully cellularized patch. After 8 days of constant perfusion culture within the same bioreactor, scaffold dynamic modulus and cell viability were analyzed. Endothelial proliferation and vessel maturation were examined by immunohistochemistry and transmission electron microscopy. Our design allowed for scaffold production and dynamic culture in a one‐stop‐shop model. Enhanced compression and cell‐mediated matrix remodeling induced a significant increase in scaffold stiffness while ensuring excellent cell survival. For the first time, we could isolate HLAA‐derived EC with proliferative potential. ECs within the central channel proliferated during flow culture, continuously expressing endothelial markers (CD31) and displaying basal membrane synthesis (collagen IV, ultrastructural analysis). After 7 days of culture, a complete endothelial monolayer could be observed. Covering cells aligned themselves in flow direction and developed mature cell–cell contacts.
Abstract
Treatment of terminal heart failure still poses a significant clinical problem. Cardiac tissue engineering could offer autologous solutions for the replacement of nonfunctional myocardial tissue. So far, soft matrix construction and missing large-scale prevascularization prevented the application of sizeable cardiac repair patches. We developed a novel bioreactor system for semi-automatic compression of a collagen I hydrogel applying 16 times higher pressure than in previous studies. Resistance towards compression stress was investigated for multiple cardiac-related cell types.
For scaffold prevascuarization, a tubular cavity was imprinted during the compaction process. Primary cardiac-derived endothelial cells (ECs) were isolated from human left atrial appendages (HLAAs) and characterized by fluorescence-activated cell sorting (FACS) and immunocytology. EC were then seeded into the preformed channel with dermal fibroblasts as interstitial cell component of the fully cellularized patch. After 8 days of constant perfusion culture within the same bioreactor, scaffold dynamic modulus and cell viability were analyzed. Endothelial proliferation and vessel maturation were examined by immunohistochemistry and transmission electron microscopy.
Our design allowed for scaffold production and dynamic culture in a one-stop-shop model. Enhanced compression and cell-mediated matrix remodeling induced a significant increase in scaffold stiffness while ensuring excellent cell survival. For the first time, we could isolate HLAA-derived EC with proliferative potential. ECs within the central channel proliferated during flow culture, continuously expressing endothelial markers (CD31) and displaying basal membrane synthesis (collagen IV, ultrastructural ABBREVIATIONS: 3D, three dimensional; AA, atrial appendage; B(P)EL, bovine serum albumin (BSA) polyvinyl alchohol essential lipids; CD, cluster of differentiation; CM, cardiomyocyte; CPC, cardiomyocyte progenitor cell; CSC, cardiac stem cells; DAB, 3,3 0 -diaminobenzidine; DAPI, 4 0 ,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified eagle medium; EC, endothelial cell; ECM, extracellular matrix; EGM-2, endothelial growth medium 2; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; G 0 , storage modulus; G 00 , loss modulus; G*, complex shear modulus; Heart transplantation is still considered the gold standard treatment for patients suffering from terminal heart failure (Carrier & Perrault, 2014). For years, Eurotransplant has reported increasing numbers of patients on the waiting list facing a nearly constant pool of available donor hearts (Eurotransplant International Foundation: Annual Report, 2014). Cardiac tissue engineering could provide a solution to this problem of organ shortage. Different strategies have been pursued to create functional heart muscle tissue primarily implementing either cell-or scaffold-based approaches (Haraguchi, Shimizu, Yamato, & Okano, 2012).
When considering scaffold-based strategies, collagen type I has proven an ideal scaffold for cardiac tissue engineering on numerous accounts (Perea-Gil, Prat-Vidal, & Bayes-Genis, 2015). Moreover, mechanical dehydration of collagen hydrogel could create a stiffened network to match the alternating strains imposed on the intercellular matrix by cardiomyocyte (CM) contractility. Following a distinct physicochemical model described by Hadjipanayi et al. (2011), plastic compression (PC) of collagen gels not only greatly enhances mechanical properties like stiffness and tensile strength but also has been shown to positively influence cell migration (Serpooshan et al., 2013) and proliferation of epidermal, corneal, and urothelial cells in complex 3D models (Ajalloueian, Zeiai, Fossum, & Hilborn, 2014;Hu et al., 2010;Mi, Chen, Wright, & Connon, 2010). Most groups so far used fixed weight plates with a maximum force generation of approximately 1.4 kN/m 2 (Serpooshan et al., 2013) for compression. There is a proportional interrelation of collagen pore size and fibroblast-induced gel contraction (Serpooshan, Muja, Marelli, & Nazhat, 2011) favoring higher compression rates for stable shape retention. Furthermore, according to Ghezzi, Muja, Marelli, and Nazhat (2011) increasing collagen fiber density exerts a positive effect on fibroblast growth induction. Hence, an elaborate PC protocol with high compression rates is justified for ideal scaffold construction.
Another major issue to date in cardiac tissue replacement is the immediate and long-term sustenance of engineered tissue upon implantation. For example, Kawamura et al. (2012) reported limited cell survival in human-induced pluripotent stem cell (hiPSC)-derived CM sheets after transplantation in a pig model of chronic myocardial infarction. Therefore, instant nutrient and oxygen supply through an intrinsically preformed vascular network is mandatory for complex tissues exceeding 200 μm in thickness (Benavides et al., 2015). Two domains must be considered when dealing with in vitro prevascularization: (1) tube/network formation and (2) endothelial lining. Regarding (1), capillarization can be accomplished by selforganizing effects of admixed endothelial cells in coculture (Sekine et al., 2008;Shimizu, 2014) or micropatterning within suitable scaffolds (Choi et al., 2007;Zheng et al., 2012). However, direct surgical anastomosis with a human host vasculature requires the provision of a larger connecting vessel inside the transplant. Vollert et al. (2014) successfully generated endothelialized, perfusable channels of up to 500 μm in diameter within their engineered heart tissue using alginate spacer tubes. Other groups followed a top-down approach by reendothelializing decellularized porcine small bowel segments yielding full-size vascular networks which could be attached to host vessels via arterial and venous stubs (Andree et al., 2014;Mertsching et al., 2009;Schanz, Pusch, Hansmann, & Walles, 2010). In terms of (2), endothelial cell (EC) choice of origin is fundamental for graft-host and cell-cell communications. ECs are known to play a decisive role in allograft rejection, for example, via monocyte recruitment and T-cell stimulation (Al-Lamki, Bradley, & Pober, 2008), while at the same time exhibiting a certain tissue-specific heterogeneity concerning organ maintenance and cytokine responsiveness (Molema, 2010;Nolan et al., 2013). These data suggest an advantage using autologous, cardiac-derived ECs to optimize the cellular interplay for tissue engineered heart patch transplants.
We assumed that human left atrial appendages (HLAAs) could be harnessed for sufficient EC supply. These remnants of embryonic development can be accessed and excised easily during open or endoscopic heart surgery granting an additional benefit in reducing the incidence of postoperative cerebrovascular events, at least in patients with a low CHA2DS2-VASc score (Kato et al., 2015). In the past, mostly cardiomyocyte progenitor cells (CPCs) have been harvested from atrial appendages (AAs) fueling multiple animal and in-man studies. CPCs were tested for their potential of local cardiac repair after myocardial infarction in rats (Sakai et al., 1999), minipigs (Fanton et al., 2015), and humans (Chugh et al., 2012) yielding some promising results over the past two decades. In contrast, no efforts have been made so far to isolate stromal and EC populations of AAs, let alone introduce them to tissue engineering. Because coculturing of different cell types is known to exert positive effects on their self-organization potential (Czajka & Drake, 2015), it is conceivable that a proper mixture of endothelial and non-endothelial "support cells" could promote the growth and maturation of engineered cardiac tissues and a developing vascular network within.
In the present study, we aimed at building a scalable "cardiac- 2.2 | Cell isolation and differentiation 2.2.1 | Human dermal fibroblasts HDF were isolated as previously described by Moll et al. (2013). In brief, skin biopsies were washed with phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA), connective tissue and fat were removed, and tissue was cut in strips of even size. Tissue was digested in dispase (2 U/ml; Thermo Fisher Scientific, Langenselbold, Germany) for 16 to 18 h at 4 C. Epidermal and dermal layers were separated with tweezers; dermal layers were cut into smaller pieces and digested for 45 min at 37 C in collagenase (500 U/ml; Serva, Heidelberg, Germany). Pieces were plated in Dulbecco's modified eagle medium (DMEM; high glucose; Thermo Fisher Scientific) with 10% FCS (Bio&SELL GmbH, Feucht, Germany). Outgrowing cells were cultured and split with Trypsin (Thermo Fisher Scientific) at some 80% confluency.
| Primary cardiac cells
HLAAs were excised during routine cardiac surgery with patients' consent and processed within 8 h. Cells were isolated as previously described (Messina et al., 2004;Smith et al., 2007)
| hiPSC-derived CMs
hiPSC-derived CMs were kindly provided by the Edenhofer group (Kadari et al., 2015). Cells had been differentiated from AR1034ZIMA hiPSC clone 1 and cultured in RPMI1640 (Thermo Fisher Scientific) with 2% B-27 supplement (Thermo Fisher Scientific), β-mercapoethanol F I G U R E 1 Fabrication of collagenous scaffold. (a) Compression of collagen I by a linear motor within a custom-made bioreactor. (b) Schematic drawing of fabrication process. Dissolved collagen I with interstitial cells was cast into the bioreactor core module with topped cylinder and a central needle placeholder. After solidification, plastic compression was performed. The patch was then transferred to dynamic culture. A peristaltic pump was used to induce a constant medium flow of 1 ml/min. After 24 h of primary culture, the needle was removed, and endothelial cells were seeded. (f.c. 100 μM; Thermo Fisher Scientific), L-ascorbic acid (f.c. 0.5 mg/ml; Sigma-Aldrich), and 1% P/S.
| Fabrication of cell-seeded PC collagen scaffolds
Collagen I was isolated following a protocol from Dieterich et al. (2002) After 24 h, the medium reservoir was detached and stored at 37 C.
The lower compartment of the reactor was closed, the central placeholder removed, and noncytotoxic silicone tubing was attached. HAAEC suspension was injected in a "pump and suck" manner retaining some 400 μl of a 1 * 10 6 cells per milliliter suspension inside the channel. The tubing was closed and cells were allowed to settle for 1 h. The bioreactor was turned upside down for another hour to ensure ubiquitous EC attachment. The whole seeding procedure was performed twice to ensure adequate EC seeding density. After reconnection to the roller pump system patches were incubated under permanent perfusion conditions for a maximum of 7 days. Half of the reservoir medium was changed twice a week. At the end of the culture period, patches were gently removed from the bioreactor and further processed.
| Examination of PC effects on mechanical properties and cell survival
To comprehensively cover viscoelastic properties of our PC collagen scaffold, we chose to determine storage and loss moduli (G 0 and G 00 ) over Young's modulus. Rheological characterization was performed on freshly compressed patches (0D) and on patches cultured for 8 days (1 day of initial scaffold remodeling + 7 days of alleged channel endothelialization). Patches for both time points were manufactured with and without interstitial cells (HDF and control, respectively). For each setup (0D, 0DF, 8D, and 8DF), three patches were examined.
G* thereby closely resembles a Maxwell model of viscoelasticity with a serial setup of a pure viscous dampener and a pure elastic spring.
To determine the impact of compression on embedded cells, we digested freshly compressed patches (10 ml collagen, 5 ml GNL, and
| Qualitative MTT test of whole scaffolds
Viability testing of scaffold-incorporated cells was done via a qualitative MTT test. The cultured scaffold was taken out of the reactor and transferred into a 1 mg/ml MTT in VascuLife ® VEGF-Mv solution.
After 90 min of incubation at 37 C, the scaffold was washed thoroughly with PBS+ until no MTT residues were visible.
| Transmission electron microscopy
For transmission electron microscopy (TEM) samples were fixed as previously published by Helmprobst, Frank, and Stigloher (2015) for 1 h in 2.5% glutaraldehyde. Preparation of fixed patches followed the same protocol. Briefly, samples were washed, incubated with 2% OsO4, and contrasted with 0.5% uranyl acetate as described by Reynolds (1963).
| Human AAs are a reliable EC source for later autologous applications
To establish our prevascularized scaffold model, ECs were isolated from HLAAs. HLAA were excised during surgery, processed, and cells taken into culture (Figure 3a). Processed biopsies gave rise to WCM, a mixture of different cell types. We performed magnetic-activated cell sorting (MACS) for CD31 with WCM harvested from each specimen individually to obtain pure ECs. The resulting cell populations, CD31-negative cells and HAAEC as well as the unsorted WCM, were further investigated by FACS and immunofluorescence staining. Ki67 staining was applied to analyze the proliferative potential of all three cell populations from different donors (Table 2)
| DISCUSSION
In this study, we were able to devise a bioreactor system for combined PC of collagen I and subsequent 3D dynamic cell culture in a single integrated setup.
F I G U R E 2 Rheological examination of patches: (a) Vibratory rheological strain curves before and after 8-day incubation period, with and without fibroblasts. Two time points were examined: directly after compression (0D) and after 8 days of culture (8D). Shear modulus (G*; III) was calculated from elastic storage (G 0 ; I) and loss (G 00 ; II). (b) Bar chart of G 0 , G 00 , and G* at 0.2 % strain. 0D, 0DF, and 8D patches showed no significant difference regarding G 0 or G 00 . 8DF patches showed a significant increase in overall stiffness (*p %3C 0.05). (c) Collagen I has been broadly used for several tissue engineering applications. It is one of the most prominent proteins in various connective tissues throughout the human body and thus highly biocompatible with low immunogenicity. However, collagen I gels tend to be quite susceptible towards mechanical stress due to a high water content. Brown, Wiseman, Chuo, Cheema, and Nazhat (2005) (Brown et al., 2005;Cheema & Brown, 2013) or with a weighted plate (Drechsler et al., 2017;Hu et al., 2010;Witt et al., 2019). Our standardized, semi-automatic compression strategy yielded a considerably higher (about 16 times) compressive force than in previous reports by using a motor-driven approach instead of fixed weight plates (Serpooshan et al., 2013). While our method was able to limit further collagen shrink- (Ghezzi, Marelli, Muja, & Nazhat, 2012). Consequently, we also tested for surgical handling in a hands-on test of "cut and sew" by an experienced cardiac surgeon ( Figure S3).
As stated earlier, the complex interplay of all cardiac cell types is required to create a unique environment of closely related mechanic and paracrine networks. Thus, balanced coculture models are the premise for developing a fully grown myocardial patch. We postulated a cell-specific resistance towards mechanical stress during our PC cycle.
Results of cell survival analyses indicated two distinguishable groups:
While interstitial cells such as HDF, CD31-negative cells, and CM displayed high compression resistance, vascular-associated cells, that is, HAAEC and pericytes, were more sensitive. This matches general cell culture findings: EC often exhibit a more sensitive behavior regarding the processes of medium exchange, cell passaging, and freezing/thawing than more resilient cell types. High survival numbers of CM could be explained by their unique physiological properties.
Due to the demands of permanent contraction and relaxation CM exhibit a suitable cytoskeleton and membrane configuration to withstand substantial mechanical strains (Sequeira, Nijenkamp, Regan, & van der Velden, 2014). However, they are sensitive towards collagenase A treatment, becoming obvious after correction of survival rates for digestion effects.
Isolation of cardiac-derived cells, namely, cardiac stem cells (CSC),
has gathered more and more attention as first clinical trials of stem cell therapy for myocardial infarction have been registered (Gyongyosi, Haller, Blake, & Martin Rendon, 2018). In our work, we focused on HLAA-derived cells. HLAAs are a remnant of embryonic development attached to the left atrium (Al-Saady, Obel, & Camm, 1999;Regazzoli et al., 2015). So far, murine LAA were used for isolation of cardiac progenitor cells (CPC) (Leinonen et al., 2013). Approaches in human, for example, isolation of a new subtype of CSC, utilized right AAs (Koninckx et al., 2013). Preparation of HLAA gave rise to a cellular mix termed WCM which was divided in endothelial CD31-positive (Hahn & Schwartz, 2009). These findings suggest that proper medium flow and the resulting shear stress should positively influence growth and integrity of the endothelial layer. We chose a low flow velocity of 1 ml/min to avoid initial flush detachment of the ECs. This flow leads to a shear stress of some 0.77 dyne/cm 2 . Most methods published so far using microvascular networks with gravity-driven medium flow reported a shear stress range from 0.1 (Zheng et al., 2012) to 5 dyne/cm 2 (Chan et al., 2014;Chrobak et al., 2006). HAAEC in our model showed continuous proliferation over time and were metabolically active, suggesting appropriate dynamic culture conditions. Likewise, immunofluorescent 3D reconstruction of the channel highlighted adaptation of HAAEC to the applied perfusion conditions. Cells (ECM) as indicated by basal lamina residues. It is known that EC can express most components of the vascular basement membrane themselves, but the final assembly requires heterotypic cell-cell contacts (Davis & Senger, 2005).
| CONCLUSIONS AND OUTLOOK
We present a new method of manufacturing a large-scale collagenbased scaffold. Motor-driven PC of collagen I hydrogel yields a viscoelastic scaffold, more likely to resemble in vivo tissue stiffness. A traversing endothelialized channel and successful seeding of various cell types under dynamic culture conditions make this patch a versatile tool for complex tissue engineering efforts. Even though our choice of collagen work-up and cells aimed at constructing a cardiac repair patch, our principal strategy is not limited to this specific tissue type.
The method could be adapted for a different organoid framework design. Possible modifications include but are not limited to changes in compression cycle length and force development, cell composition, and cytokine application due to the modular architecture of our bioreactor system.
ACKNOWLEDGMENTS
This work has been funded by the Federal Ministry of Education and Research (funding numbers 01EO1004 and 01EO1504). We acknowledge support by the K-Centre VascAge (COMET program-Competence Centers for Excellent Technologies) funded by the Austrian Ministry for Transport, Innovation and Technology (FE).
Open access funding enabled and organized by Projekt DEAL.
CONFLICT OF INTEREST
None declared.
ETHICS STATEMENT
Direct humanization for autologous transplantation without necessity of animal-based transplants. All tissues explanted under ethical vote of the university clinic Wuerzburg number 182/10.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of this article. | v3-fos-license |
2017-10-17T02:15:21.986Z | 2016-11-02T00:00:00.000 | 7042665 | {
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} | pes2o/s2orc | Detecting structural variances of Co3O4 catalysts by controlling beam-induced sample alterations in the vacuum of a transmission electron microscope
This article summarizes core aspects of beam-sample interactions in research that aims at exploiting the ability to detect single atoms at atomic resolution by mid-voltage transmission electron microscopy. Investigating the atomic structure of catalytic Co3O4 nanocrystals underscores how indispensable it is to rigorously control electron dose rates and total doses to understand native material properties on this scale. We apply in-line holography with variable dose rates to achieve this goal. Genuine object structures can be maintained if dose rates below ~100 e/Å2s are used and the contrast required for detection of single atoms is generated by capturing large image series. Threshold doses for the detection of single atoms are estimated. An increase of electron dose rates and total doses to common values for high resolution imaging of solids stimulates object excitations that restructure surfaces, interfaces, and defects and cause grain reorientation or growth. We observe a variety of previously unknown atom configurations in surface proximity of the Co3O4 spinel structure. These are hidden behind broadened diffraction patterns in reciprocal space but become visible in real space by solving the phase problem. An exposure of the Co3O4 spinel structure to water vapor or other gases induces drastic structure alterations that can be captured in this manner.
Background
Since the advent of high voltage electron microscopy, electron beam-induced damage in the bulk of crystalline materials has been extensively studied (e.g., [1]). In the traditional picture, it is understood that the dominant interaction of the electron beam with the sample causes atom displacements by knock-on events. Remarkably, the rapidly improving performance of electron microscopes operating in the mid-voltage range, between 20 and 300 kV [2][3][4][5], has made it possible to obtain atomic resolution images with single atom sensitivity at voltages well below the abrupt threshold values for atom displacements from their bulk lattice sites. Therefore, one is tempted to speculate that atom displacements are not longer of concern. Indeed, recent experimental investigations show that bulk materials can stand long time periods of electron irradiation before any beam-induced sample alteration is recognized [6]. However, it would be inaccurate to conclude that electron beam-induced damage is now absent while observing the atomic structure of materials. Indeed, unobtrusive damage processes remain very active in the mid-voltage range. In particular, concerns arise in investigations that aim at detecting single atoms because the electron dose needed to create sufficient contrast is very large. Importantly, scientifically attractive investigations fall into this category. For example, atomic-resolution electron tomography [7][8][9][10], the imaging of single molecules [11], and the determination of structure-function relationships in catalytic processes [12,13] all demand resolution and sensitivity down to the level of single atoms. However, it will be pointed out in this paper that the detection of single atoms requires the Open Access *Correspondence: [email protected] 1 Molecular Foundry, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA Full list of author information is available at the end of the article application of electron doses as large as 500-10,000 e/Å 2 that are often delivered within 1 s during image acquisition while-at the same time-it becomes evident that electron dose rates above ~100 e/Å 2 s can already be too large if it is attempted to maintain structural integrity of small catalytic metal oxide electrocatalysts, for example [12]. This obvious gap between tolerable and needed electron dose (or rate) to detect single atoms is addressed in this contribution.
Advanced structural and chemical imaging with atomic resolution relies on the extraordinary performance of aberration-corrected electron microscopes that are equipped with high-brightness electron sources. These are required to provide the large beam currents (dose rates) [2,5,14] needed to generate interpretable contrast from electron scattering at single atoms. The total electron dose must be large because such scattering events contribute very little contrast to atomic resolution images from crystals, which are typically dominated by the much larger contrast of atom columns. However, the atomic structures of defects, interfaces, and surfaces are all preferentially affected by beam-induced object alterations because the binding energies of relevant atoms are significantly lowered compared to those of bulk lattice sites in crystalline solids. As a consequence, unintentional sample alterations are often obscured by a lack of sensitivity, the choice of forgiving detection modes, the absence of time resolution, and other factors [16], such that they are easily overlooked and may trigger misleading conclusions. Consequently, a variety of strategies are now deployed [3,5,15,17,18] that aim at overcoming the various aspects of electron beam-induced sample alterations at the single atom level in atomic resolution electron microscopy.
As a guideline, one must expect that relevant object distortions will occur during investigations of nanostructured soft and hard matter, where a rich variety of defects, interfaces, and surfaces allows forging new material properties that rapidly emerge worldwide. Furthermore, in environmental electron microscopy, liquids and gases are easily ionized along the path of the electron beam [19], which can lead to unexpected interactions of materials with their surroundings. This paper utilizes Co 3 O 4 catalysts as an example to summarize emerging capabilities that take advantage of controlling electron doses and rates to understand how crystal structures can be maintained or altered in a controlled fashion. Figure 1 illustrates how samples are altered by accelerated mid-voltage electrons in broad beam high resolution transmission electron microscopy (HRTEM) and in focused beam scanning transmission electron microscopy (STEM). A situation is depicted in which the illuminated sample area matches the imaged sample area. In HRTEM, this match is achieved by a Nelsonian illumination scheme [20] (Fig. 1a), and in STEM (Fig. 1b), it is naturally set by the scanning range. In this case, both detection modes can be compared. It is well established that electrons from the mid-voltage range preferentially remove atoms in surface proximity [21][22][23]. However, unlike the abrupt onset of knock-on damage in bulk materials, this onset is continuous and occurs at much lower energies because of the distribution of reduced binding energies of atoms at surfaces, interfaces, or defects [24,25]. In fact, surface effects entirely dominate if the sample dimensions shrink to the range of singledigit nanoparticles, where entire particles can become unstable in the electron beam [20,26].
A summary of beam-induced sample alterations
In Fig. 1, altered sample surfaces are symbolized by irregular white bands. An image of a thick sample acquired in broad beam mode (Fig. 1a) typically captures the entire illuminated sample volume, including bulk and surface contributions. Current densities can be chosen as small as 1 atto Ampere per square Ångstrom (aA/Å 2) , which delivers only six electrons to each square Ångstrom of the sample in a second (e/Å 2 s). Such minimal current densities are typically utilized to capture images in cryomicroscopy because they prevent radiation-sensitive organic tissue or molecules from degrading during their observation [27,28]. In contrast, a cone shaped STEM beam produces images of thickness slices from the sample. Conditions can be chosen to capture the interior of samples, thereby showing a pattern that is only marginally distorted by blurred, unfocused contrast contributions from the sample surfaces [14], as depicted in Fig. 1b. As a result of the cone shaped illumination, the current density at the sample surface can be smaller compared to its value in the focused beam spot and surface damage can be reduced. For example, a 50 pA probe, with a beam semi-convergence angle of 25 mrad that is focused 100 Å deep into the sample to form a spot of 1Å diameter, would spread over an area of ~20 Å 2 at the crystal surface. Thus, the current density at the sample surface is reduced to ~2.5 pA/Å 2 , which is equivalent to 1.5 10 7 e/Å 2 s. Buban et al. [3] matched the total electron dose of STEM experiments to the needs of cryo-microscopy by choosing a ~1 pA beam current, in combination with a short dwell time of ~1 μs. Both the blurred contrast contributions from surfaces-if present-and the selective amplification of contrast from heavy atoms can make high angle annular dark field (HAADF) STEM images appear more stable than their TEM counterparts, but at the cost of a reduced information content concerning sample thickness and strain contrasts (e.g., dislocations). Upon prolonged beam exposure, however, the unavoidable sputtering of surface atoms reduces the sample thickness such that both techniques simultaneously capture bulk and surface contributions in single images (Fig. 1c, d). In any case, electron microscopy certainly allows for a damage-free acquisition of images even from organic matter if the total electron dose is kept below ~20 e/Å 2 [1,3,4] and contrast remains sufficiently high, which is easily achievable in the low magnification range.
Concerns regarding beam-induced sample alterations arise if it is attempted to understand structure and chemical composition of materials at atomic resolution with single atom sensitivity. For example, Fig. 2a shows atomic resolution HRTEM images of a ~5 nm sized Co 3 O 4 catalyst imaged along its [114] zone axis orientation with total electron doses between ~10 and ~100,000 e/Å 2 . Bright spots in the images mark the location of atom columns that are detectable in all images but signal-to-noise (S/N) ratios naturally increase with the total electron dose. Certainly, it is not obvious from the images how many electrons are needed to reliably identify scattering contributions from single atoms. S/N ratios for the detection of single atoms can be estimated from the literature and their magnitude is indicated in Fig. 2b). For the aberration-corrected TEAM0.5 microscope, it was reported that a single gold atom with its atomic number Z of 79 (Z 79 ) can be detected in HRTEM images with a S/N ratio of 2 [14], if a total electron dose of ~2300 e/Å 2 is applied at 300 kV. A voltage reduction to 80 kV improves on contrast by a factor of ~2 [29], which allows shot noise (N/√N) to be decreased by a factor of 4 to maintain the same S/N ratio. Therefore, the total electron dose for the detection of a single gold atom at 80 kV decreases to ~575 e/Å 2 . Single carbon atoms in graphene are typically observed with a S/N ratio of 2 at 80 kV using a total dose of ~20,000 e/Å 2 [30]. Ignoring contributions from inelastic scattering, this dose difference for the detection of single carbon and gold atoms with S/N ratios around 2 can quantitatively be expected because electron scattering depends on the atomic number (Z); it obeys a Z 0.66 rule in the bright field (BF) HRTEM mode [31] and a Z 1.7 rule in the STEM high angle annular dark field (HAADF) mode [32]. In HAADF Fig. 1 Schematics of electron beam-induced object alterations in the mid-voltage range. Damaged areas are dominantly generated in surface proximity and symbolized by wide diffuse lines. HRTEM (a, c) and STEM (b, d) detection modes are considered. a, b Initial situation for thick samples. c, d Structure after a prolonged exposure to the electron beam (e-beam) or thin sample. The ongoing sputtering process removes atoms (black dots) from the top and bottom surfaces at different rates. In HRTEM mode, the beam is static, the semi-convergence angle is ~0.1 mrad and the dose rate can be varied between 1 atto A/Å 2 and 1 pico A/Å 2 . In STEM mode, the beam is scanned, the semi-convergence varies between 5 and 25 mrad and the dose rate typically changes between 1 and 100 pico A/Å 2 . The illuminated area is matched to the imaged area (camera). For more details see text STEM at 300 kV, the contrast from single, heavy atoms can be reliably identified from its contribution to the contrast of atom columns [8]. We measured S/N ratios between 2 and 4 at 300 kV for the detection of a single gold atom at a resolution between 0.5 and 1 Å for total electron doses between 35,000 and 70,000 e/Å 2 in single HAADF images. Similar doses are applied to single images to produce tomograms of tungsten atoms in three dimensions. 1 Certainly, these estimates cannot be more accurate than a factor of 2 or 3 but they provide very useful guidance for choosing total doses required for different experiments, as shown in this paper.
Thus, the current technology allows detection of single heavy atoms with comparable S/N ratios at 300 kV if 1 From Ref. [9], we estimate a total dose per image of ~30,000 e/Å 2 from the given acquisition parameters of two sub-images that were recorded with a probe size of ∼50 pm, a beam current of 70 pA, a pixel size of 0.405 Å and a dwell time of 6 μs. For an image series of 62 images the total dose adds up to 1,900,000 e/Å 2 . A S/N ratio of 2 for the detection of a single W atom can be estimated by extracting line profiles from the supplementary Figure 2, the pyramidal shape of the needle, which makes it roughly as wide as it is deep, and assuming that noise is dominated by the dark current of the detector since the electron counts are high. the total electron dose in HAADF STEM images exceeds the BF HRTEM dose by a factor of roughly 20. This difference originates from the less effective detection of only those electrons that are scattered to large angles in reciprocal space, where all information is lost about the real space localization of the scattering events. S/N ratios diverge even further if light atoms are considered because of the distinctly different Z-dependences of electron scattering in the HAADF and BF imaging modes. Single atoms can also be detected at atomic resolution by collecting secondary electrons (SE) [33]. However, in this case, the S/N ratio for the detection of a single uranium atom is even poorer than the contrast of a single uranium atom recorded in an annular dark field STEM mode.
Given the considerations discussed above, it is not surprising that it has become common to improve on the STEM detection efficiency by reducing the acceleration voltage and the inner angle of the dark field detector to capture more electrons [2] or by directly using BF detectors [34]. Nevertheless, the real space HRTEM mode remains not only the most effective detection scheme but also the fastest way to capture images of scattered [4] can be used to record images with the low dose rates that are used in biological sciences and the method allows to build up the needed total dose for the detection of single atoms by the acquisition of large image series electrons because of the parallel electron detection. Consequently, it is used in biological [28] and environmental sciences [35], where obtaining the largest contrast with the fewest number of primary electrons possible matters greatly.
Time constants differ greatly in both detection modes, since aberration-corrected HAADF STEM and BF HRTEM imaging modes build on distinctly different technologies. Typical parameter ranges are given in (Fig. 2c, d), respectively. In STEM, the relevant parameters are the local beam current and the dwell time, which set the total electron dose for probes that, can be focused to the diameter of a single atom column. With current equipment, beam currents of (6-600) × 10 6 e/Å 2 s are used and total doses are set by dwell times ranging from microseconds to milliseconds. Low dose images of radiation-sensitive matter can be obtained by combining a beam current around 1 pA with a dwell time of 1 μs to produce images of crystalline solids that are largely noise dominated [3]. In the other extreme, typical dwell times of 15-50 μs are used in combination with beam currents of approximately 50 pA to enable electron tomography with single, heavy atom sensitivity and atomic resolution. In the HRTEM mode (Fig. 2d), typical image exposure times of 0.1-1 s and dose rates between 1 and 50,000 e/ Å 2 s determine the total dose. Thus, time constants and dose rates differ roughly by up to six orders of magnitude if STEM and HRTEM modes are compared.
Further differences between the detection modes are caused by the convergence of the electron beam to form a local probe, which forces a serial image acquisition of thickness slices with an increased spread of the electron beam in z-direction because of electron channeling in zone axis orientations of the samples. Further, the detection of electrons in real space is most effective. For convenience, the Table 1 summarizes relevant differences between broad beam and focused probe detection modes.
It was recently demonstrated that electron beaminduced sample alterations can be effectively retarded by recording images with low dose rates and accumulating the needed total electron dose by acquiring image series that contain hundreds of frames [26]. Our implementation consists of retrieving exit wave functions form focus series of images to obtain in-line holograms that always capture the whole elastic scattering information unlike single images. In doing so, the contrast transfer function (CTF) starts oscillating with increasing defocus. Typically, we balance the third-order spherical aberration C 3 with the fifth-order spherical aberration C 5 to maximize contrast transfer close to 1 [36] and capture all elastically scattered electrons. With increasing defocus, CTF oscillations occur and half of all electrons contribute to the contrast formation since the average of a squared sinusoidal oscillation equals 1/2. In addition, the range of defocus values can be suitably adjusted to extend the low frequency limit to 4-5 nm if needed.
In this manner, best practices known from biological sciences are adopted for the acquisition of atomic resolution images [27]. Finally, the low-dose rate approach also allows for self-consistent electron tomography from single projections, which preserves pristine atomic structures most effectively [10].
Initially, electron beam-induced object alterations only cause small contrast changes in images since they relate to the loss of single atoms from the much stronger contrast of atom columns. Thus, they are hardly recognizable by visual inspection. Only the accumulation of damage can be readily observed. Therefore, a quantitative contrast analysis is not optional but mandatory, if genuine structure of surfaces, defects, or other radiation-sensitive matter, including catalysts is to be interpreted. As a minimum requirement, it should become good practice to list electron doses and rates with every published image. This paper presents detailed examples that highlight the pressing need to address beam-sample interactions in investigations of catalytic nanocrystals using the BF HRTEM mode, where primary beam currents and total doses can be tuned very effectively. The derived conclusions, however, are equally valid for broad beam and focused probe imaging modes and can be converted using the guidelines of Fig. 2.
Experimental details
The investigated Co 3 O 4 samples (space group 227, volume = 525 Å 3 , 56 atoms in the unit cell) are prepared by three different methods: a. A high throughput inkjet printing process of the (Ni-Fe-Co-Ce)O x composition space for active OER electrocatalysts [12], which includes a successive calcination step at 350-400 °C. b. A surfactant-assisted solvothermal method, as previously reported by Agiral et al. [37]. c. A plasma-assisted atomic layer deposition (ALD) method in which a thin polycrystalline film of CoO x catalyst is deposited at a substrate temperature of 100 °C [38].
In all cases, the detected electron nano-diffraction patterns of the as-prepared samples can be described by the spinel phase of Co 3 O 4 [39]. Different crystal structures were observed in surface proximity of the nanocrystals and after exposure of the material to gaseous environments at elevated temperature.
Low dose rate in-line electron holography is performed with the TEAM0.5 microscope, which can be operated between 20 and 300 kV [4,15]. Focus series containing up to 100 images are recorded with variable dose rates and reconstructed with the McTempas software [41] to produce electron exit wave functions, which are in-line holograms. In this process, the phase problem is solved using a Gerchberg-Saxton algorithm [40]. The Fourier transforms of the complex electron exit wave functions provide all depicted nano-diffraction patterns. Unlike a Fourier transform of real images, they do not exhibit Friedel symmetry and allow measuring sample tilt. The software is also used for image analysis and multislice calculations [41]. Doses and rates are calculated from counts on the CCD camera, which are calibrated by a known number of electrons that are emitted from the filament for operating voltages of 80 and 300 kV.
The electron transparent samples are produced by crushing the fabricated powders and dispersing flakes of the Co 3 O 4 catalysts onto grids for electron microscopy made from stainless steel. The ALD of the Co 3 O 4 is performed directly onto an electron transparent SiN x membrane [38].
An exposure of the samples to water vapor at a temperature of 400 °C is accomplished in an environmental electron microscope that is described in detail elsewhere [42].
Pristine and altered Co 3 O 4 surfaces
First, it is shown in Fig. 3 in which circumstances surface alterations occur. For this purpose, we consider phase images of the electron exit wave function reconstructed from focus series that include 50-100 image frames. Unlike single images, these reconstructed series reveal the crystal structure even if the structural information is overwhelmed by noise in the single low dose rate images that are shown in Fig. 2. Total and accumulated electron doses are chosen such that the reconstructed phase images of the focus series can be compared with usual acquisition parameters for HRTEM images. Relevant phase images are shown in Fig. 3a-c, which were recorded with 14, 65 and 460 e/Å 2 s, respectively. Figure 3d, f reproduce the experiment with dose rates similar to those of Fig. 3a, after applying a higher dose exposure, thereby allowing for a direct comparison of the irradiated material with the initial object structure. From subtle differences in Fig. 3a and d, it can be recognized that initially attached surface layers are removed from the catalyst and that its crystalline surfaces are restructured to a depth of one or two crystalline monolayers, as highlighted for a specific location by arrows. Thus, surface features can be lost without being noticed in single images, even if dose rates and total doses are moderate. Detecting the presence and genuine structure of such surface features is highly relevant for understanding the native catalytic surface activity. The inserted nano-diffraction patterns show minor changes of the crystal orientation that can become significant if the utilized dose rates are high enough to locally heat the samples [43].
A total electron dose of 950 e/Å 2 , such as used in Fig. 3a, would suffice to observe single heavy atoms, such as gold, with an S/N ratio around 2. A dose of 31,300 e/ Å 2 , as used in Fig. 3c, would allow detection of single carbon atoms. Similarly, high total doses are used in emerging approaches to electron tomography: an estimated electron dose of 90,000 e/Å 2 was used at 300 kV to record a single HRTEM image to reconstruct a tomogram of MgO with single light atom sensitivity. 2 In another study, a dose of ~30,000 e/Å 2 was used to capture individual HAADF images within a 62 frame image series to produce a tomographic reconstruction of a tungsten needle tip (Footnote 1). In this case, the accumulated electron dose approaches 2,000,000 e/Å 2 see (Footnote 1). Figure 3d, f show that one must expect that unprotected surfaces are significantly compromised by such imaging conditions. Consequently, a tomographic reconstruction from image series acquired with high dose rates will include uncontrolled time averages of a dynamic situation that can be confused with the genuine structure of nanocrystals. Tomographic reconstructions from single projections, in combination with acquisition of low dose rate images, can address this issue [10].
Since our in-line holography approach retards beaminduced structure alterations, the question arises: to what measurable extent can structural integrity be maintained? Reconstructed phase images from two successively recorded low dose-rate image series are shown in Fig. 4a, b. Careful image inspection certainly suggests that the method faithfully reproduces the structure of the catalyst and its surfaces in great detail, and reveals that its initial surfaces are rough at the atomic scale if recorded in low dose-rate conditions. Faint contrast differences are present in the outermost layer of the catalyst at the Co 3 O 4 /vacuum interface and we quantify their magnitude by extracting line profiles from an identical area that is marked by arrows in both images. Line profiles are compared in Fig. 4c. It is seen that intensity maxima occur in identical locations but with variable intensity and on a fluctuating baseline. Comparing these fluctuations with the literature data for phase changes is depicted in the inset of (a). The arrows and circles point to electron beam-induced surface alterations that occurred as a result of exposing the sample to an increasing number of electrons as listed. The sample was prepared by the high throughput printing process. b-f Are successive phase images of reconstructed wave functions that were recorded with the listed dose rates, total dose of the image series and accumulated electron dose caused by electron scattering at single atoms [44] (inset in Fig. 4c), it is seen that the fluctuations are very small: they are comparable with the signal from a single oxygen atom, while the larger contrast of the heavier Co atoms can already be recognized with a S/N ratio of 1-2. Such faint contrasts reveal that the Co 3 O 4 /vacuum interface is composed of a layer that holds incomplete unit cells that contributes to surface roughness. Since the unit cell of Co 3 O 4 is 8 Å large and contains 56 atoms, we probe for single atoms within a unit cell of Co 3 O 4 and can infer if single cobalt atoms are kept in place or not to better understand the chemical composition of surfaces. A possible loss of single oxygen atoms, however, would hardly be recognized because a total dose of 5100 e/Å 2 does not suffice to generate enough signals above noise to detect. In the TEAM0.5 microscope, a total dose of ~20,000 e/Å 2 is needed for the detection of light atoms, which was not targeted in this experiment. Surely, such capabilities can Fig. 4 Sequential low dose-rate phase images a, b of a spinel Co 3 O 4 particle. The mid-voltage range electron beam acceleration is 80 kV. A total of 160 images were recorded to produce the result. The sample was prepared by the high throughput printing process and exposed to technical air (N 2 , O 2 ) at 400 °C for 2 h. The inserted nano-diffraction patterns show crystal tilt away from the [211] zone axis. Dose rate, total dose and accumulated dose are indicated and arrows mark the location of line traces that yield the contrast (phase) profiles of c. For comparison, expected phase changes from single O and Co atoms are indicated in c, too be tuned to provide unique insight into atom dynamics and can be brought to good use in diverse applications.
Structural Co 3 O 4 alterations by electron beam-stimulated atom diffusion
Since the frequency of atom displacements at surfaces increases with the beam current and peaks at medium acceleration voltages, beam-induced object alterations become much more visible if atoms are rapidly displaced from their lattice sites using high dose-rate conditions. Commonly, individual atoms are not instantly lost after a displacement event, but remain in surface proximity and contribute to object alterations by diffusion, as already reported for another material system [44,45]. Before investigating single grains of the Co 3 O 4 catalysts, it is instructive to consider a dense monolayer of ~4 nm large grains that form a continuous layer on a SiN membrane [38]. Figure 5 shows two reconstructed phase images from an identical sample area that is exposed to a large total electron dose of 150,000 and 469,000 e/Å 2 , successively. Our image reconstruction makes use of 50 individual frames from each series; it took 2.8 min of recording time to capture all images. A visual inspection of the images in Fig. 5 directly reveal that significant electron beam-induced grain growth and grain reorientation is stimulated, as specified in the figure caption. Such sintering processes of nanoparticles were studied by in situ plan-view transmission electron microscopy as early as 1998 [46] at a lower magnification by raising temperature to stimulate the observed transformations. There, it was pointed out that grain reorientations occur by grain boundary migration, grain rotation, and surface diffusion, which are exactly the processes that we can now observe at atomic resolution using the electron beam as a stimulus. It is remarkable that each image by itself gives the impression of being static and radiation resistant in spite of the fact that a strongly dynamic situation is created by the large beam currents. Certainly, time averaged structures are displayed in Fig. 5, but they hardly show any image blur, even at the chosen resolution of one Ångstrom. The illusion of a static situation is fostered because atom diffusion is fast compared to the image recording time and the evolution of grain growth is not arbitrary, but is linked to specific lattice sites.
With these experimental results in mind, we now analyze electron beam-induced object alterations in the single Co 3 O 4 particle shown in Fig. 6. Its genuine crystal structure is shown in Fig. 6a, which is recorded with a low dose rate of 90 e/Å 2 s and a total electron dose of 4600 e/Å 2 that allows for the detection of single Co atoms but requires at least 2 O atoms to generate a suitable signal above noise. A coexistence of two image patterns A, B is pointed out in Fig. 6a. The image pattern B can be approximated by simulating a spinel structure of the [211] oriented Co 3 O 4 particle. However, structure A can only be partly be explained in this manner for the following reason: upon prolonged exposure of the crystal to a more intense electron beam, both patterns A, B disappear and are replaced by the pattern C (Fig. 6b, c), which can be approximated by simulating a [321] grain orientation shown in Fig. 6c. In fact, all reflections of the nano-diffraction patterns in Fig. 6 can be reproduced by simulating the kinematic electron diffraction patterns of the spinel structure in [211], [531], and [321] zone axis orientations. This match of the simulations with the experiment is shown by insets in Fig. 6 and suggests a beam-induced grain rotation by 10.9 degrees that occurs as a result of an increased beam current. The geometry of the rotation is shown in the stereographic projection of Fig. 6. However, the different pole axes orientations do not strictly appear sequentially as one would expect if a grain rotates from a [211] to a [321] orientation. Instead, characteristic reflections coexist over the entire rotation range, such as the three dominant reflections from the [211] zone axis orientation that are pointed out by arrows. They should be absent from the [321] diffraction pattern, even if the excitation errors are large because the particles are small. Thus, the question arises if the observed crystal rotation is purely geometrical or if atom diffusion contributes, too, because it is strongly stimulated in high dose-rate conditions, as shown above.
We address beam-induced atom diffusion in Fig. 7 by probing for rotation-induced intensity changes between the (0-22) and (0-44) reflections and their centro-symmetrically equivalent (02-2) and (04-4) reflections. This measurement is only meaningful because we consider the Fourier Transforms of a complex wave function, which is not forced to be centro-symmetric by mathematics. Reflection intensities are extracted along the dotted line in Fig. 7a. A visually equal intensity of the centrosymmetric diffraction pairs in this image characterize an exact [211] zone axis orientation, while the different intensities of the (−444) and (4-4-4) reflections capture a rotation of ~1° around the perpendicular crystal axis. In Fig. 7b, we compare quantitatively these line profiles from all three Fourier Transforms of Fig. 6a-c. A geometric crystal rotation by 10.9° must change the intensity ratio of centro-symmetric reflection spots drastically, while a constant reduction factor would point towards a changing volume fraction of a particular structural phase. Unexpectedly, both situations are observed simultaneously in Fig. 7b but concerning different sets of reflections: the relative intensities of the {220} and {440} reflections change their magnitude collectively to maintain their relative intensity closely, which excludes a large crystal rotation. On the other hand, different reflections emerge in Fig. 7b, such as the (1-57) reflection of the [321] crystal orientation because of the changing diffraction patterns that are shown in Fig. 6. This suggests the presence of a crystal rotation. Consequently, the measurement of Fig. 7b directly conflicts with a model of a beam-induced geometrical sample tilt, only.
Moreover, the measured crystal structure labeled A in Fig. 6a is incompatible with the simulated image of the Co 3 O 4 spinel phase in [211] orientation that is present in a different area of the sample. Thus, the nano-diffraction pattern of Fig. 6a contains all reflections that are allowed to appear in the existing crystal symmetry of the spinel structure but the occupation of lattice sites with atoms must differ locally. This is a manifestation of the phase problem that we solve by reconstructing the electron exit wave function [41]. In Fig. 6c, the contribution of a [211] pattern to a [321] orientated crystal remains visible in reciprocal space but is reduced beyond recognition in real space, which cooperates with the partial loss of intensity upon an increase of the dose rate in Fig. 7b. In addition, the {220} and {440} reflections are significantly broadened if compared to other reflections, as pointed out in Fig. 7a.
This coexistence of different diffraction patterns over a substantial range of the stereographic projection, the simultaneous existence of different crystal structures in real space images with similar diffraction patterns, and the unusual broadening of specific reflections strongly suggest that the initial grain consists of a spinel structure at its core but may be covered by an external region of surface reconstructions. Electron beam-induced atom diffusion starts altering the crystal structure and orientation if the total electron dose significantly exceeds ~5000 e/Å 2 s and the electrons are delivered at a rate larger than 100 e/Å 2 s. Such a model can accommodate all experimental results including a grain rotation that is partly driven by atom diffusion and partly by a pure geometrical rotation.
Our measurements point out comprehensively that pristine Co 3 O 4 structures, including their surfaces, can be reproducibly captured and analyzed in atomic resolution images with single atom sensitivity by keeping Fig. 5 Electron beam-induced grain reorientation and growth in a polycrystalline Co 3 O 4 film of ~4 nm thickness that was deposited by ALD on an electron transparent SiN membrane. The electron beam acceleration voltage is 300 kV. Dose rates and total doses are indicated. Reconstructed phase images are shown. A box marks a specific grain that helps relating both images to each other. Frames a and b list dose rates and the total electron dose for the series. Comparing frame a and b grains labeled A ([110 zone axis orientation) and C ([100] zone axis orientation) exhibit dominant grain growth because their zone axis pattern is almost maintained. Grains labeled B and D exhibit rotations, their respective zone axis pattern change the dose rates low and using the smallest amount of electrons that are needed to detect single atoms. Large doses and rates will produce images of a seemingly static structure that, however, is a time average of beam-stimulated structure alterations where atomic resolution is maintained. Other detailed considerations are possible and exciting but lie beyond the scope of this paper.
Conclusion and outlook
This contribution addresses electron beam-induced sample alterations in atomic resolution electron microscopy using acceleration voltages between 80 and 300 kV and beam currents that range from atto-Amperes/Å 2 to pico-Amperes/Å 2 . The significance of their control is highlighted by investigating Co 3 O 4 catalysts to show that acquisition of images from pristine catalytic surfaces can be accomplished at atomic resolution with single atom sensitivity. The contrast from single atoms is optimized by operating the TEAM0.5 microscope at suitably low voltage, by delivering electrons with rates below 100 e/ Å 2 s, and by capturing large image series that provide the needed number of scattering events to create sufficient contrast from single atoms without causing damage.
Exploiting the current technology and electrons accelerated by 80 kV, we estimate a S/N ratio of ~2 for the detection of one gold atom or one carbon atom if ~600 or 20,000 e/Å 2 are delivered, respectively, in the bright field phase contrast imaging mode. Beam-induced object alterations primarily affect atom sites possessing lowered binding energies, which are typically present at surfaces, interfaces, defects, or in radiation-sensitive amorphous materials and soft matter where they consistently cause undesirable contrast variations [15,47].
Electron beam-sample interactions cannot be described by strictly isolated knock-on events, where atoms are abruptly removed from the object without further impact. Instead, an increase of doses and rates stimulates physically meaningful processes such as atom diffusion, surface reconstructions, and sintering. Furthermore, they may trigger distinct relaxation pathways that otherwise can only be captured if the temperature is raised or additional charge is provided. In this manner, we find that Co 3 O 4 polycrystalline films exhibit a strong tendency to sinter and that single grains can assume previously unknown reconstructions that extend from the crystal surfaces into the sub-surface region of the catalysts on a nanometer scale. Beam-induced crystal reorientations are found to be affected by diffusion-stimulated atom rearrangements within the unit cell of the material.
Finally, it is pointed out that all our experiments are executed in the vacuum of the electron microscope, which is a high vacuum environment that, however, does not reflect the environment of functional catalysts, particularly when considering the impacts of temperature and pressure on structure. Figure 8 highlights the relevance of this aspect by comparing the structure of a spinel Co 3 O 4 catalyst that was exposed to dry air with one that was exposed to water vapor. It is seen that an exposure to water vapor leads to a substantial disintegration of the spinel structure into a variety of locally different structures that all exhibit a diffuse [211] diffraction pattern, which is indistinguishable from the one shown in Fig. 7a. This dramatic structure alteration can now be shown to be genuine and it complements similar findings from the past [48]. Better understanding of such interactions, which are of considerable relevance to the real world, will require elevating environmental electron microscopy to an adequate level of resolution and sensitivity under atmospheric pressure and humidity. Finally, we stress the importance of not only recording diffraction patterns, but to also solving the phase problem with with spinel structure fabricated by the high throughput printing process after an exposition to technical air at atmospheric pressure at 400 °C for 2 h. b Sample as in a but exposed in situ to 1 mbar of H 2 O vapor at 400 °C. c The related nano-diffraction patterns showing substantial peak broadening after the exposure to H 2 O vapor | v3-fos-license |
2016-05-12T22:15:10.714Z | 2016-03-09T00:00:00.000 | 695465 | {
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} | pes2o/s2orc | Influenza Vaccine Manufacturing: Effect of Inactivation, Splitting and Site of Manufacturing. Comparison of Influenza Vaccine Production Processes
The aim of this study was to evaluate the impact of different inactivation and splitting procedures on influenza vaccine product composition, stability and recovery to support transfer of process technology. Four split and two whole inactivated virus (WIV) influenza vaccine bulks were produced and compared with respect to release criteria, stability of the bulk and haemagglutinin recovery. One clarified harvest of influenza H3N2 A/Uruguay virus prepared on 25.000 fertilized eggs was divided equally over six downstream processes. The main unit operation for purification was sucrose gradient zonal ultracentrifugation. The inactivation of the virus was performed with either formaldehyde in phosphate buffer or with beta-propiolactone in citrate buffer. For splitting of the viral products in presence of Tween®, either Triton™ X-100 or di-ethyl-ether was used. Removal of ether was established by centrifugation and evaporation, whereas removal of Triton-X100 was performed by hydrophobic interaction chromatography. All products were sterile filtered and subjected to a 5 months real time stability study. In all processes, major product losses were measured after sterile filtration; with larger losses for split virus than for WIV. The beta-propiolactone inactivation on average resulted in higher recoveries compared to processes using formaldehyde inactivation. Especially ether split formaldehyde product showed low recovery and least stability over a period of five months.
[1] J. M. Wood, G. C. Schild, R. W. Newman, and Valerie Seagroatt, An improved singleradialimmunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines, Journal of Biological Standardization 1977 5, 237-247
Total protein
Total protein quantity was determined by using the Peterson colorimetric method [2]. (Membrane bound) proteins were first dissolved with deoxycholate, then the proteins were precipitated using trichloric acid and subsequently Lowry assay was performed. Materials Folin-Ciocaleu's Phenol Reagent 2 N, Sigma F-9252, Spectrophotometer [2] Peterson G.L., A simplification of the protein assay of Lowry et al, which is more generally applicable. Anal. Biochem. 1977, 83, p.346-356.
Ovalbumin
Ovalbumin concentration was measured using a direct sandwich ELISA (Enzyme Linked Immuno Sorbent Assay). The assay was performed according to the instructions of the supplier of the ELISA kit. Independent duplicates of two different dilutions were uses as samples. Samples containing the antigen were pipetted in ELISA plate wells coated with polyclonal anti-ovalbumin antibodies. Anti-ovalbuminelinked to Horse Radish Peroxidase was added, followed by washing away unbound substances. The addition of a substrate initiated the development of a blue color. This process was stopped by adding sulphoric acid; the color changed from blue to yellow. The absorption at 450nm was a measure for the quantity. A 630nm filter was used as reference. Materials Serazym Ovalbumin ELISA: Cat.No. E041C, Seramun Diagnostica GmbH Wolzig. ELISA-plate reader with filter 450nm and reference filter 630nm
Endotoxin
Endotoxin content was measured using the de Limulus Amoebocyte Lysate (LAL) test. Small amounts of endotoxine cause clotting of a of amoebocyte lysate from horseshoe crab. The test is semi quantitative and described in the European Pharmacopoeia [3]. The lysate sensitivity (λ) mentioned in the analysis certificate was confirmed with standard solutions. Equal amounts of lysate were added to multiple dilutions of the sample. To verify that the sample matrix is of no influence, samples were also spiked with the standard and tested. The verification samples should clot. After incubation at 37°C bromothymol blue was added to facilitate reading: if no clotting the stain was distributed equally and in case of a gel clot the stain solution flows to one side of the well.
Neuraminidase inhibition assay
The purpose of this test is to determine the presence and identity of neuraminidase in master seed virus (MSV), working seed virus (WSV) and monovalent bulk vaccine (MBV) using the neuraminidase activity and inhibition test. The test is based on the following principle: neuraminidase can release sialic acid from the substrate fetuïn. This sialic acid can react to build a colored complex, which is measured spectrophotometrically. The concentration of the antigen which gives an Optical Density (OD) of approximately 0.8 is used for the NI-test. In this test antisera, specific to A-(N 1 and N 2 ) and B-(NB) strains, are used to give an interaction with the present antigen, thereby inhibiting the development of a colored complex. Present antigens are bound to specific antibodies. Remaining unbound neuraminidase results, after releasing sialic acid, in a colored complex. The NI titer is defined as the serum dilution whereby 50% of neuraminidase activity is inhibited. Materials Fetuin, Neuraminidase Antiserum N 1 , Neuraminidase Antiserum N 2 , Neuraminidase Antiserum B, Control Sheep Antiserum (CS).
Residual Infectious Viruses
The purpose of this test is to confirm the absence of any residual infectious influenza viruses in the monovalent bulk vaccine (MBV) after treatment with beta-propiolactone (BPL). For each MBV, 10 fertilized eggs will be inoculated with 0.2 ml MBV into the allantoic cavity. After 3 days incubation, 0.5 ml allantoic fluid is harvested from the eggs and pooled. Again 10 fertilized eggs will be inoculated with 0.2 ml allantoic fluid from the 1 st pooled harvest. After 3 days incubation, the allantoic fluids are harvested and incubated with a fixed amount of turkey erythrocytes. If there are viruses present in the allantoic fluids, haemagglutination with the erythrocytes will take place. If haemagglutination is found, further passage in eggs will be carried out. Materials 11 days old fertilized chicken eggs, Turkey blood, Bovine Serum Albumin
Sterility
The purpose of this test is to determine the sterility of master seed lot (MSL), working seed lot (WSL) and monovalent bulk vaccine (MBV). The sample is filtrated through a membrane, flushed and placed in media. After incubation the media is examined for turbidity, which indicates the presence of micro-organisms. The advantage of filtration of the sample is based on removing factors that could inhibit the growth of micro-organisms. Furthermore, a bigger volume of sample can be tested for the presence of micro-organisms which increases the sensitivity of the test. Materials Pseudomonas aeruginosa Baccilus subtilis | v3-fos-license |
2020-05-07T09:17:13.818Z | 2020-01-01T00:00:00.000 | 218942257 | {
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} | pes2o/s2orc | CHARACTERISTICS OF Ca2+, Mg2+-DEPENDENT ATP HYDROLYSIS IN SPERM CELLS OF INFERTILE MEN
© 2020 O.I. Meskalo et al.; Published by the Ivan Franko National University of Lviv on behalf of Біологічні Cтудії / Studia Biologica. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://www.budapestopenaccessinitiative.org and Creative Commons Attribution 4.0 License), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. UDC: 615.25:612.616.2
Disturbances of fertilizing potential of spermatozoa are closely associated with dysfunction of ion-transporting ATPases, in particular Ca 2+ , Mg 2+ -АТРase. Reduced activity of tapsigargin-resistant and tapsigargin-sensitive Ca 2+ , Mg 2+ -АТРase leads to disruption of Ca 2+ -homeostasis and is characteristic for abnormal spermatozoa (pathoospermia). In order to study the peculiarities of action of Ca 2+ , Mg 2+ -АТРase, we determined the initial reaction rate, the maximum (plateau) amount of the reaction product and the characteristic reaction time. To determine these kinetic parameters of Ca 2+ , Mg 2+ -dependent hydrolysis of ATP catalyzed by Ca 2+ , Mg 2+ -ATPase, the dynamics of product accumulation of the ATP-hydrolases reaction was studied. The obtained curves were linearized in the coordinates {P/t; P}. Analyzing the changes in the activity of Ca 2+ , Mg 2+ -ATPase, the kinetics of primary-active transport of calcium ions through the plasma membrane and membranes of intracellular Ca 2+ -stores in saponin-permeabilized spermatozoa of infertile men was studied. It was shown that in normozoospermic samples, the transport of Ca 2+ ions through the plasma membrane is characterized by a higher capacity than through the membranes of intracellular Ca 2+ -stores, but it occurs with practically the same initial velocity and characteristic reaction time. It was found that in pathospermic samples, transport of Ca 2+ ions with the participation of both components of Ca 2+ , Mg 2+ -ATPase occurs less intensively and is characterized by a lower capacity compared to spermatozoa of men with preserved fertility. Specific changes in the kinetic parameters of Ca 2+ , Mg 2+ -dependent hydrolysis of ATP lead to inhibition of tapsigargin-resistant and tapsigargin-sensitive Ca 2+ , Mg 2+ -ATPase activity and cause a decrease in fertilizing potential of spermatozoa.
INTRODUCTION
Infertility is one of the most important medical and biological problem. According to statistics 15 %, of married couples face this problem during the reproductive period. Nowadays, large-scale studies are being conducted all over the world to investigate the causes of impaired reproductive function and to develop methods that restore fertility [1]. Approximately 50 % of the infertility cases are related to male factor which has a tendency to increase. Defective functions of spermatozoa is the main cause of male infertility [11,13]. The most common abnormal parameters are low motility (asthenozoospermia), low sperm count (oligozoospermia), or a combination of these abnormalities (oligoasthenozoospermia) [9,14].
Сalcium ions play a pivotal role in sperm physiology, specifically in sperm hyperactivation, chemotaxis and motility which depend on intracellular free calcium concentration [7]. For the normal functioning of sperm cells, it is necessary to change rapidly the intracellular concentration of calcium ions in response to certain stimuli. Ca 2+ ,Mg 2+ -AT-Pase (ЕС 3.6.1.38) plays a pivotal role in Ca 2+ extrusion from cytoplasm maintaining its concentration in low nanomolar range (10-100 nM). Total activity of Ca 2+ , Mg 2+ -ATPase consists of thapsigargin-resistant plasmatic membrane and thapsigargin-sensitive ATPase of internal Ca 2+ -stores. Our previous results showed that asthenozoo-, oligoasthenozoo-and leucocytospermic patients have significantly impaired thapsigarginresistant and thapsigargin-sensitive Ca 2+ , Mg 2+ -ATPase activity compared to healthy men [12]. Lowered activity of Ca 2+ , Mg 2+ -ATPase activity is likely to contribute to the disruption of Ca 2+ homeostasis, a hallmark of abnormal sperm cells [4].
The aim of present study was to study the properties of membrane-bound Ca 2+transport systems in spermatozoa of fertile (normozoospermia) and infertility men (oligozoospermia and asthenozoospermia).
MATERIALS AND METHODS
Reagents. The following reagents were used in the present study: ATP, ouabain, thapsigargin, EGTA (Sigma, USA), saponin (from Quillaja Saponaria Molina pract.; Acros organics, Belgium). Other reagents of the domestic production were of reagent grade or laboratory grade.
Donors and semen sample preparation. Human semen was obtained from 7 healthy volunteers and 12 infertile men with asthenozoospermia (AS) and oligoasthenozoospermia (OLAS) undergoing routine semen analysis for couple infertility at Lviv Regional Clinical Hospital (Ukraine). Control group consisted of healthy men with somatic fertility, normozoospermia (N) and confirmed parenthood (married for 3-10 years and having 1-3 healthy children).
Approval for study was taken from the Ethics Committe of Danylo Halytsky Lviv National Medical University (Ethical Committee Approval, protocol No 6 from March 29, 2017). Terms of sample selection meet the requirements of the principles of Convention of Europe Council on human rights, Helsinki Declaration on protection of human rights and biomedicine and the laws of Ukraine. All patients and healthy donors were matched by age and gave written informed consent to participate in research. Exclusion criteria: subjects who are currently on any medication or antioxidant supplementation were not included. In addition, subjects with infertility over 10 years, azoospermia, testicular varicocele, genital infection, chronic illness and serious systemic diseases, smokers and alcoholic men were excluded from the study because of their well-known high seminal reactive oxygen species levels and decreased antioxidant activity which may affect calcium level. Samples were obtained by the masturbation after 3-4 days of sexual abstinence and processed immediately upon liquefaction. The classical semen parameters of spermatozoa concentration, motility, and morphology were examined according to World Health Organization criteria (2010) [19].
Cell preparation. Biochemical studies were carried out in the Department of Medical Biology of Danylo Halytsky Lviv National Medical University. Sperm cells were washed from semen plasma by 3 times centrifugation at 3,000 ×g for 10 min in media which contained (mM): 120 NaCl, 30 KCl, 30 Hepes (pH 7.4). Protein concentration in the samples was determined by Lowry method using a kit to determine its concentration ("Simko Ltd"). Determination of ATPases activities was carried out in the permeabilized spermatozoa. The detergent saponin in a final concentration of 0.5% was added to sperm suspension for permeabilization of sperm membranes. Saponin interacts with membrane cholesterol, selectively removing it and leaving holes in the membrane [6].
Kinetic calculations. Studies of Ca 2+ , Mg 2+ -ATP-dependent hydrolysis of sperm ATP were performed in a standard incubation medium, that was modified by the time of incubation. The apparent kinetic parameters of Ca 2+ , Mg 2+ -dependent ATP hydrolysis are the initial reaction rate V 0 , the maximum (plate) amount of the reaction product P max and the characteristic reaction time was determined by linearization at {P/t; P}, where P is the amount of reaction product (P i ) and t is the incubation time [8].
Statistics analysis. Data are expressed as means ± standard error of the numbers of determinations. Differences between paired sets of data were analysed using paired Student's t-tests in Microsoft Excel. Differences were considered significant at p < 0.05 as the minimum significance level.
RESULTS AND DISCUSSION
For determination of the kinetic parameters of Ca 2+ , Mg 2+ -dependent ATP hydrolysis in saponin-permeabilized spermatozoa, the dynamics of inorganic phosphate (Р і ) accumulation in ATP-hydrolysis reaction wad studies. To do that sperm cells of fertile and infertile men were incubated in incubation medium during different time intervals (1-10 min). The experimental data showed that the curves of Ca 2+ , Mg 2+ -dependent hydrolysis of ATP in sperm cells tend to saturate (Fig. 1). The analysis of the obtained results shows that accumulation of Р і in reaction catalyzed by thapsigargin-resistant ATPase in the normozoospermic samples in time interval 0-2 min correspond to zero order reactions. In this time interval, the graph of dependence of the amount of Р і on incubation time is almost linear. Similar dependence is observed for thapsigargin-sensitive ATPase.
By linearization of the obtained data in the coordinates {P/t; P} (Fig. 2), the apparent kinetic parameters of the reaction of Ca 2+ ,Mg 2+ -dependent hydrolysis of ATP were calculated (see Table). As can be seen from the Table, the values of the apparent kinetic parameters of ATP hydrolysis catalyzed by thapsigargin-resistant and thapsigargin-sensitive ATPase Based on these results, we assume that in sperm cells of fertile men, Ca 2+ -transport by thapsigargin-resistant ATPase is characterized by a higher capacity compared to Ca 2+ -transport by thapsigargin-sensitive ATPase. These results are in disagreement with data reported in lymphocytes and cells of the submandibular salivary gland [3,17]. It was found that transport of Сa 2+ ions by Ca 2+ , Mg 2+ -ATPase of plasmatic membrane in healthy donors is slower and less intensive, but it is characterized by higher capacity in comparison with Ca 2+ , Mg 2+ -ATPase of endoplasmic reticulum. Our results might be explained by structural organization of spermatozoa. Since cytoplasm of spermatozoa has a restricted volume and sperm cells have highly polarized morphology, they pose a unique challenge to maintain Ca 2+ -homeostasis [15]. Standard components of Ca 2+signaling for somatic cell are present in spermatozoa in a slightly modified form [16]. The primary candidate for the intracellular Ca 2+ -store in spermatoza is the acrosome, since human sperm do not contain the endoplasmic reticulum [5]. The presence of thapsigargin-sensitive Ca 2+ -ATPase SERCA 2 mainly localized to the acrosome and mid-piece was demonstrated in mammalian spermatozoa [2,10].
The curves of Ca 2+ , Mg 2+ -dependent hydrolysis of ATP of sperm cells of the infertile men with asthenozoospermia and oligoastenozoospermia have a similar appearance, but differ from the values for the normozoospermic samples (Fig. 3). As follows from the data in the table, the values of apparent kinetic parameters of Ca 2+ ,Mg 2+ -dependent hydrolysis of ATP by thapsigargin-resistant ATPase in sperm cells from the infertile men differ significantly from these values in the normozoospermic samples. Specifically, the value of V 0 of ATP hydrolysis in astheno-and oligoasthenozoospermic samples is reduced 4 times compared to normozoospermic samples. The value of P max in pathospermic samples is twice lower in comparison with the normozoospermic samples. The τ values of reaction catalyzed by thapsigargin-resistant ATPase in spermatozoa from men with astheno-and oligoasthenozoospermia are 1.8-2.0 times greater than values in normozoospermic samples.
In the absence of a significant difference in the value of τ reaction catalyzed by thapsigargin-sensitive ATPase in normozoo-and pathospermic samples, the value of V 0 in astheno-and oligoasthenozoospermic samples is reduced 2.0 times compared to normozoospermic samples. The value of P max in pathospermic samples is 2.5 times higher than normozoospermic samples. Based on these results, we assume that in sperm cells of infertile men, the transport of Ca 2+ ions is less active and characterized by a lower capacity than in fertile men.
CONCLUSION
The analysis of the alterations in hydrolase activity of thapsigargin-resistant and thapsigargin-sensitive Ca 2+ ,Mg 2+ -ATPase showed that primary active transport of Ca 2+ ions is less intensive and characterized by lower transport capacity in sperm cells of the infertile men in comparison with fertile men.
COMPLIANCE WITH ETHICAL STANDARDS
Human Rights: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. | v3-fos-license |
2021-08-02T00:06:41.951Z | 2021-04-29T00:00:00.000 | 236576769 | {
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} | pes2o/s2orc | Metallo-stannosilicates as inorganic supports to immobilization of lipase from Thermomyces lanuginosus for biodiesel production
This study reports the application of metallo-stannosilicates as potential inorganic solid matrixes for enzymes immobilization and their use as a heterogenous catalysts in enzymatic transesterification reactions for the conversion of triacylglycerides into fatty acid ethyl esters (FAEEs). Several stannosilicates were synthesized and physicochemical characterized by X-ray powder diffraction (XRD), scanning electron microscopy with energy dispersive X-ray spectrometry (SEMEDS), Brunauer–Emmett–Teller (BET)-N2 surface area analysis and solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR Si and Sn nuclei) techniques. The experimental results for enzymes immobilization were promising, especially for a nickel ion-exchanged metallostannosilicate, which were able to immobilize 82 ± 6% of Thermomyces lanuginosus lipase and also kept a high enzymatic activity (42 ± 3 U mg). Systematic catalytic reactions for conversion of refined palm oil (Elaeis guineensis) using some of these stannosilicates enzymes complexes yielded 63.3 ± 0.7% of FAEEs. It is worth noticing that, when the transesterification reaction was performed with (a) the as-made stannosilicate without enzymes and (b) the equivalent amount of immobilized Thermomyces lanuginosus lipase in its free form, the FAEEs yield sharply decreased to < 5.0% and 6.3 ± 0.3%, respectively. This result is a clear evidence of a synergistic effect among the metallo-stannosilicates and the immobilized enzymes.
Introduction
The development of alternative biofuels as viable ways to replace or reduce the use of fossil fuels has stimulated the efforts of the scientific community. The energy demand arises from numerous factors, including environmental concerns and the depletion of fossil fuel resources that have stimulated the development of new alternative sources of more sustainable fuels. Among the various options, biodiesel is attractive because it is a sustainable and renewable form of energy 1 . Chemically, biodiesel consists of a mixture of fatty acid alkyl esters (FAAEs) and is predominantly produced, in industrial scale, by means of transesterification reactions using refined vegetable oils as triacylglycerides sources, together with short chain alcohols (methanol or ethanol) and homogeneous catalysts. The appropriate choice of triacylglycerides feedstocks and catalysts are the main challenges and have precluded a faster development of this industry.
Concerning the available triacylglycerides sources, the use of edible oils has raised ethical and economic questions, one of the reasons to search alternative lipid feedstocks that do not compete with food production 2,3 . There are several lipids feedstocks for biodiesel production, such as: palm, castor, soybean, cotton, peanuts, jatropha, sunflower (vegetable oils), besides the utilization of animal fats, nonedible and waste oils. Considering oils from vegetable origin, the palm tree (Elaeis guineensis) is an excellent option due of its low price, relatively high oil content (palm fruit contains approximately 40% of oil) and high productivity (2500-4000 kg hectare -1 year -1 ). Furthermore, palm oil is more saturated and has greater oxidation stability when compared to other vegetable oils, in addition to being an important alternative for the sustainable development of some Brazilian regions, mainly in the Amazon 4,5 . Another important component in the biodiesel production process is the short chain alcohol source. In Brazil, the use of ethanol is a viable option, since the country is the world's second largest producer of ethanol. However, most industrial biodiesel plants do not have the technology to synthesize biodiesel by applying ethanol as a solvent, since that the production of biodiesel via the ethylic route presents some obstacles, such as high alcohol consumption and difficulty in separating the final products (glycerol and biodiesel), which generates greater energy costs for this process.
In terms of catalyst technology, sodium hydroxide (NaOH), potassium hydroxide (KOH) and sodium methoxide (NaOCH3) are currently the main homogeneous catalysts employed in the biodiesel industry. Their disadvantages are associated with the impossibility of use low-quality feedstocks, with high contents of water and free fatty acids (FFAs) 6 . Although not currently applied in industrial scale for biodiesel production, heterogeneous acid catalysts could be an interesting alternative to solve this problem due the fact of being less sensitive to FFAs content and perform simultaneously the esterification and transesterification reactions. Moreover, the heterogeneous catalyst can be recycled (reused), there is none or very little amount of wastewater produced during the catalytic process and the separation of the catalysts from biodiesel and glycerol is relatively easy. Nevertheless, its application in the biodiesel industry is preclude due the necessity of employing longer reaction times, higher reaction temperatures and large alcohol to oil molar ratios [7][8][9][10] . The combination of all these factors has a negative impact in the final price of the biodiesel, therefore the search for new heterogeneous catalysts able to overcome these drawbacks is a technical and scientific challenge.
Zeolites and Mixed Octahedral-Pentahedral-Tetrahedral Silicates (OPT) materials are inorganic microporous framework oxides and can be an interesting option to overcome these challenges. Mixed framework OPT materials containing coordinated tetrahedra or octahedra, namely stannosilicates, is one of these materials subject of research. Stannosilicates and tin-zeotypes materials have been reported as ion exchangers, sorbents and catalysts for several types of reaction, as purification of natural gas with sulfur compounds, like hydrogen sulfides or oxysulfides 11 , and catalytic reactions for decomposition of propan-2ol and oxidation of cyclohexene 12 , Baeyer-Villiger oxidation reactions 13 , monosaccharide isomerization reactions 14,15 and application as heterogeneous catalysts for biofuels production 16 . Tin-based homogeneous and heterogeneous catalysts have been used in transesterification reactions, mainly using refined edible oils as feedstocks and methanol, typically providing high yields of fatty acid methyl esters (FAMEs) [17][18][19][20][21][22][23][24] .
The use of lipases (triacylglycerol acylhydrolases, E.C.3.1.1.3) as catalysts in the biodiesel production is also an attractive option due to their high specificity for the transesterification of triacylglycerides to FAMEs or FAEEs in comparison with the other conventional chemical catalysts employed in industrial biodiesel production. In comparison to the basic homogeneous catalysts, enzymes exhibit high selectivity and catalytic activity under mild operative conditions. In addition, lipases can also catalyze the transesterification of waste feedstocks that contain a high content of free fatty acids (FFAs) and water, therefore decreasing the probability of forming soap and emulsion 25 . Some examples of biodiesel production by enzymatic catalysis are the transesterification of soybean oil (Glycine max) by applying different lipases and experimental parameters (quantity of biocatalyst, reaction time, amount of water added and turnover of lipases) [26][27][28] , esterification reactions of oleic acid 29 and FAEEs production by enzymatic transesterification of triolein 30 . Besides all the advantages of the enzymatic process in comparison to the other ones, there are important drawbacks that preclude their large use in industrial scale process for biodiesel production. Between them are the enzymes expensive costs, the difficulty of its separation (recover of the enzymes and reuse in its free form), besides the possible deactivation of enzymes active sites by glycerol, a subproduct of triacylglycerides transesterification. In this context, the enzymes immobilization is a viable alternative for overcoming these obstacles [31][32][33][34][35][36] .
The use of supports for enzymatic immobilization is an effective way to combine the advantages of both heterogeneous and enzymatic catalysts. Enzymatic immobilization consists of locating or confining an enzyme on a solid support or matrix. The methods are basically classified as chemical or physical processes, and the choice of the appropriate support depends of physical-chemical characteristics, such as mechanical, chemical and microbial resistance, thermal stability, high loading capacity, adequate pore diameter and hydrophilic/hydrophobic behavior that allows the immobilization of the enzymes 37 . Several solid materials, such as ceramics, kaolinites, silica, cellulose, polymers and zeolites, have been used as supports for enzymes immobilization. Specifically, in the case of zeolites and mixed framework oxides, the aim of the immobilization is to create a zeolite-enzyme complex that can be applied as biocatalysts. It is clear from these studies 38-40 that both the zeolites and the enzymes can themselves alone catalyze the transesterification reaction: each one of them have their own particularity that determines the rate and yield of the final product.
This study is a sequence of previous work reported by the authors research group 16 concerning the application of a metallo-stannosilicate for biodiesel production using edible, nonedible and waste oils as feedstocks. In this paper, several stannosilicates were synthesized using different sol-gel chemical compositions.
The stannosilicates were physicochemical characterized and, afterwards, used as heterogeneous catalysts in the transesterification of refined palm oil (Elaeis guineensis) by ethanolysis reactions. Furthermore, these materials were also studied as potential inorganic solid matrixes for immobilization of Thermomyces lanuginosus lipase and tested in enzymatic transesterification reactions with the following aims: are metallo-stannosilicates feasible for use as inorganic supports for enzymes immobilization? What can be learned from catalytic results about the appropriated application of different catalysts in specific reactions for biodiesel production?
To the best our knowledge, metallo-stannosilicates were not previously explored as inorganic supports for enzymes immobilization and as heterogeneous catalysts in enzymatic transesterification reactions for biodiesel production by applying the feedstocks (refined palm oil and ethanol) used in this work.
Hydrothermal syntheses of stannosilicates
The stannosilicates were synthesized according to experimental procedures adapted from the literature 45,46 by hydrothermal crystallization in the Na2O-SnO2-SiO2 oxides system. Detailed experimental conditions of all the stannosilicates synthesized are summarized in Tab. 1. A typical synthesis for the material named stannosilicate I, using Sn 4+ (SnCl4•5H2O as the tin source) and gel composition of 1Na2O:SnO2:4SiO2:80H2O was performed at the following manner: 14.9 g of sodium hydroxide (NaOH) was dissolved in 20 g of water (solution A). A second solution (solution B) was prepared by dissolving 20 g of SnCl4•5H2O in 30 g of distilled water. This solution B was added to the solution A and homogenized under 500 rpm of stirring during 60 min at 25 °C of temperature (solution C). Finally, solution C was added to 27.4 g of Ludox HS-40 colloidal silica (40 wt.% SiO2 in water) and this final gel was also kept stirring for 30 min at 500 rpm. The sol-gel solution was transferred to a 125 mL teflon-lined stainless-steel autoclave (Parr Instruments Co., Illinois, USA) and kept at 200 °C for 7 days. Afterwards, the reactor was cooled down, the product collected by filtration, washed with distilled water and dried at 100 °C for 12 h. Table 1. Summary of the experimental procedures for the metallo-stannosilicates syntheses.
Gel composition Nomenclature
Step
Nickel ion-exchange experiments
Prior to the enzyme immobilization, all stannosilicates synthesized were subjected to nickel ion-exchange experiments. The experiments were made as follows: 30 mL of 0.5 mol L -1 NiSO4 solution (3.94 g of NiSO4•6H2O was dissolved in 30 mL of distilled water) was added into a teflon bottle. Afterwards, 1 g of the stannosilicates was added to this solution and kept stirring at 500 rpm for 60 min and 25 °C of temperature. Afterwards, the solution was heated at 80 °C for 24 h. After this period, the nickelstannosilicates were cooled down, collected by filtration, washed with distilled water and dried at 100 °C for 12 h.
X-ray diffraction, BET-N2 surface area and SEM-EDS
All the stannosilicates synthesized were characterized by XRD using a Rigaku Miniflex (Rigaku, Tokyo, Japan) operated at 40 kV, 15 mA and using a Ni-filtered Cu-Kα radiation (λ = 1.5418 Å) in the range of 2θ from 3 to 80° with goniometer rate of 2° (2θ) min -1 . Surface area measurements at 77 K were performed on a Micromeritics ASAP 2020 (Micrometrics Instrument Corporation, Norcross, USA) using the facilities of the BAM Federal Institute for Materials Research and Testing (Division 1.3. Structural Analysis, Berlin, Germany). Prior to the measurement, all samples were degassed at temperature of 300 °C and pressure of 5 × 10 -5 mbar for 3 h, and isotherms were processed by the BET (Brunauer-Emmett-Teller) method 47 . Scanning electron microscopy (SEM) and energy-dispersive Xray spectrometry (EDS) results were recorded on a FEI Inspect F50 (FEI Instruments, Oregon, USA) using the facilities of the Brazilian National Laboratory of Nanotechnology (LNNano, Electron Microscopy Laboratory, Campinas, Brazil). The electronic microscope is equipped with a Schottky field emission source, probe current at 200 nA and an electron beam with accelerating voltages between 0.2 and 30 kV.
Solid-state MAS NMR
Solid-state magic angle spinning NMR (MAS NMR) experiments were performed using the facilities of the BAM Federal Institute for Materials Research and Testing (Division 1.3. Structural Analysis, Berlin, Germany). The fully hydrated metallo-stannosilicates samples were characterized by 29 Si and 119 Sn Single-Pulse MAS NMR using a Bruker Avance 400 spectrometer (9.4 T). The data were analyzed and processed in the software TopSpin 3.6.2 version. All the experiments were carried out at room temperature and the samples were filled into zirconia rotors equipped with Kel-F caps (Bruker, Wissembourg, France) of 7 and 4 mm for 29 Si and 119 Sn nuclei, respectively. The 29 Si single-pulse MAS NMR spectra were obtained using radiofrequency pulses at the Larmor frequency of 79.5 MHz, MAS frequency of 6.5 kHz, 90° pulse length of 6.0 μs and recycle delay of 300 s. 1 H{ 29 Si} cross-polarization (CPMAS) experiments were also made by applying 90° pulse of 3.0 µs, cross-polarization time of 6.0 ms and repetition time of 3.0 s were used in order to maximize 29 Si signal intensities. A 50% amplitude CP ramp and TPPM15 (decoupling power level is the same as for the 90° 1 H pulse of 3.0 μs) was also applied. The 29 Si chemical shifts were reported relative to kaolinite as secondary reference (δ = -91.5 ppm). The 119 Sn single-pulse MAS NMR spectra were obtained using radiofrequency pulses at the Larmor frequency of 149.1 MHz, MAS frequency of 12.5 kHz, 90° pulse length of 2.5 μs and recycle delay of 600 s. The 119 Sn chemical shifts were reported relative to SnO2 (δ = -604 ppm) 48 .
Zeta potential of the stannosilicates and stannosilicates-lipases complexes
The zeta potential of the stannosilicates and stannosilicates-enzymes complexes were measured in a Nano Zetasizer ZS90 equipment (Malvern Instruments, Worcester Shire, UK). Prior to the measurements, 1 mg of the materials and 1 mL of deionized water were placed in Eppendorf tubes and stabilized during 30 min at 25 °C, and then transferred to a DTS1060 cell, equipped with golden electrodes. A wide angle (90°) laser Doppler velocimetry was used to measure the electrophoretic mobility and the ζ potential (zeta potential), expressed in mV, was calculated by Smoluchowski equation 49 .
Enzymes immobilization on the stannosilicates
The enzymes immobilization experiments were made in triplicate, according to adapted procedures from the literature 35,50 . The initial concentration of 2 mg mL -1 of the commercial lipases (enzyme Lipolase 100 L, from Thermomyces lanuginosus lipase) was added in a phosphate buffer solution, 20 mmol L -1 and pH 7, according to a proportion of 1:40 (25 µL of commercial lipase:1 mL phosphate buffer:50 mg of stannosilicates) and stirred at 300 rpm for 16 h at room temperature of 25 °C, followed by the separation of stannosilicates-lipases complexes via centrifugation at 12,000 rpm for 1 min. The products were washed twice with deionized water, dried at 25 °C overnight and stored at 4 °C. The amount of the enzymes immobilized was determined according to proposed by Bradford 51 and based on procedures adapted from the literature 52 .
Determination of percentual of enzymes immobilized on the stannosilicates
The percentual of Thermomyces lanuginosus lipase immobilized on the stannosilicates supports were determined by the method described Bradford 46 , which uses bovine serum albumin as standard and its absorption in the ultraviolet wavelength (λ = 595 nm). For these measurements, three different solutions were prepared, previously the measurements: (a) supernatant solution that remain after the enzymes immobilization reactions, (b) a solution with the same quantity of enzymes prepared for the immobilization reaction, diluted in the phosphate buffer solution (20 mM and pH 7) and (c) only the same amount of the phosphate buffer solution, which has its absorbance subtracted of the other solutions. Afterwards, a mixture of Bradford reagent (1-1,400 μg mL -1 protein, Sigma Aldrich, Germany) and the samples described above were mixed in the proportion of 1:20 (50 µL of solution:950 µL of Bradford reagent) and kept out of the luminosity for 20 min. The percentage of enzymes immobilized was calculated from the relationship 52 shown in Eq. 1:
Enzymatic hydrolytic activity of stannosilicates-enzymes complexes
The enzymatic hydrolytic activity of stannosilicateenzymes complexes was determined by titrimetric method 35,53 and following ACS specifications in the sigma standard enzymatic assays for lipases (triacylglycerol acylhydrolases, E.C.3. 1.1.3). Since that, the enzymatic activity is obtained by a hydrolysis reaction, one unit of lipase activity was defined as the amount stannosilicate-enzymes complexes releasing one mole of FFAs from a triacylglyceride source in 1 h of reaction at 40 ± 2 °C, expressed by the unity of U mg -1 (unit of lipase activity/mg of stannosilicateenzymes complexes). In a typical measurement, 10 mL of a mixture containing 20 mmol L -1 at pH 7 of phosphate buffer and a triacylglyceride substrate (refined soybean oil, Glycine max) was prepared in the volume proportion of 1:1.
This solution was thoroughly mixed and equilibrated at 37 °C. Afterwards, 30 mg of the stannosilicates-enzymes complexes was added and the reaction maintained at 37 °C for 30 min. Finally, the reaction was quenched by adding 3 mL of ethanol (95 %, Sigma Aldrich, Steinheim, Germany) and then cooled to the temperature of -4 °C by using a mixture of ice and ethanol for 10 min. The solution was centrifuged at 12,000 rpm for 1 min and the FFAs obtained as a result of the enzymatic hydrolysis reaction were neutralized by a titration with a solution of 50 mmol L -1 NaOH in the presence of thymolphthalein as indicator. One unit of stannosilicate-enzymes activity was expressed as micro equivalents of FFAs released from the triacylglyceride substrate in 1 h at 37 °C, calculated according to Eq. 2: NaOH = volume (mL) 1000 = conversion factor from 10 -3 equivalents to 10 -6 equivalents 2 = time conversion factor from 30 min to 1 h (unit definition) Mass of the solid = mass (mg) of stannosilicate-lipase complexes used in the hydrolysis reaction
Enzymatic transesterification of refined palm oil (Elaeis guineensis) for FAEEs production
Enzymatic transesterification of refined palm oil (Elaeis guineensis) by ethanolysis reaction catalyzed by free enzymes and stannosilicate-enzymes complexes were performed in 10 mL flasks, with an oil:ethanol ratio of 1:4, 3% of stannosilicates-enzymes complexes referred to the oil mass and temperature of 40 ± 2 °C for 48 h. In order to avoid the inactivation of the enzymes, the total amount of ethanol was equally divided and added stepwise over four different time intervals (0, 3, 6 and 12 h after the start of the reaction). Afterwards, FAEEs and glycerol were separated by centrifugation at 12,000 rpm for 1 min.
Heterogeneous transesterification of refined palm oil (Elaeis guineensis) for FAEEs production
Syntheses of FAEEs through the heterogeneous transesterification reactions were performed in triplicate (as performed for the enzymatic transesterification reactions) using refined palm oil (Elaeis guineensis) as triacylglycerides feedstock. In a typical transesterification reaction, 20.0 g of refined palm oil (FFAs content = 0.3 wt.%, acid value of 0.6 mg KOH g -1 ) was added in a 100 mL open glass reactor (catalytic reaction made at atmosphere pressure) with a reflux condenser, in a thermostatic bath equipped with a magnetic stirrer. Anhydrous ethyl alcohol was added according to alcohol:oil molar ratio of 1:30. The appropriate amount of catalyst (3% of heterogeneous catalyst referred to oil mass) was added and the mixture heated at 100 °C for 12 h, magnetic stirrer was set at 900 rpm and the reflux temperature adjusted of 10 ± 2 °C. After the reaction time, the mixture was filtered, the excess of alcohol was removed using a rotary evaporator (Büchi Rotavapor R-210, Flawil, Switzerland) and glycerol were separated by centrifugation at 12,000 rpm for 1 min.
Quantification of FAEEs yields by gas chromatography (GC-FID)
The measurements of FAEEs yields were made under the following experimental conditions: the measurements were made in a gas chromatograph (PerkinElmer, Massachusetts, USA) using a sample injection volume of 1 mL, helium as gas carrier at a flow rate of 2 mL min -1 and pressure of 83 kPa, injector and detector temperatures of 250 °C. The oven temperature started at 50 °C for 1 min, increased up to 250 °C at a rate of 5 °C min -1 . In a typical measurement, solutions of 20 mg mL -1 of the ethyl myristate (C14:0) and ethyl nervonate (C24:1) standards were prepared. Additionally, an ethylic nonadecanoate standard solution was prepared in a concentration of 10 mg mL -1 . The range of peaks integration were identified using the ethyl ester standards C14:0, C24:1 and an ethylic ester (C19:0) as the internal standard (IS). The FAEEs contents were obtained by integrating the peak areas from C14:0 to C24:1 and subtracting the nonadecanoate area, according to Eq. 3:
Physicochemical characterization of the stannosilicates
The XRD patterns of the stannosilicates synthesized according to sol-gel chemical compositions detailed in Tab. 1 are shown in Fig. 1a. The stannosilicate I presented a XRD pattern characteristic of a crystalline material and has structural similarity with the stannosilicate AV-10 54 . The stannosilicate AV-10 has an orthorhombic unit cell (a = 7.945 Å, b = 10.344 Å and c = 11.625 Å) and its gel composition (8.5Na2O:5.4SiO2:1.0SnO2:115H2O) contained lowest SiO2:Na2O ratio (≈ 0.6) in comparison with stannosilicate I (≈ 4.0). X-ray diffraction patterns of stannosilicate II (gel composition of 2Na2O:Sn2O:4SiO2:80H2O) indicate the formation of a new microporous material (XRD pattern not reported previously), while the stannosilicate V (gel composition of 5Na2O:Sn2O:10SiO2:80H2O) is similar to phase L 46 . The effects of the sol-gel composition were observed in the crystallization of these materials. The experimental evidences indicated that SiO2:Na2O ratio in the range of 5-10, as the ones used for the syntheses of stannosilicates III (2Na2O:Sn2O:10SiO2:80H2O) and stannosilicates IV (1Na2O:Sn2O:10SiO2:80H2O), induce the formation of tin oxide, clearly noticed in the XRD pattern of the stannosilicate III (Fig. 1a), as most of diffraction peaks were assigned to the SnO2 phase (JCPDS 41-1445). The 29 Si Single-Pulse MAS NMR results are shown in Fig. 2. The result for stannosilicate I (spectrum not shown) is similar to reported for stannosilicate AV-10 54 . The spectrum presents two resonances lines with chemical shift positions at δ1 = -87.0 ppm and δ2 = -88.7 ppm with relative intensities of 1:2 and are associated with the Si(2Si, 2Sn) environment. This similarity between both materials was proved recently by our research group 55 in a solid-state NMR study, where the stannosilicate I was used as a model compound for developing a solid-state NMR strategy to verify the connectivity of SiO4 and SnO6 polyhedral, despite the very low natural abundances of 4.68% for 29 Si and 8.59% for 29 Si and 119 Sn nuclei, respectively. The 29 Si{ 119 Sn} and 119 Sn{ 29 Si} REDOR (rotationalecho double-resonance) NMR, 29 Si{ 119 Sn} and 119 Sn{ 29 Si} REPT-HMQC (recoupled polarization transfer-heteronuclear multiple-quantum correlation) NMR and 2D 29 Si INADEQUATE NMR experiments using the crystallographic data of the sodium stannosilicate AV-10 (chemical composition Na2SnSi3O9•2H2O) proved that both stannosilicates consist of the same materials. The 119 Sn single-pulse MAS NMR spectra are shown in Fig. 3 therefore confirming the formation of a pure SnO2 phase 14 . The stannosilicates IV presented weak 119 Sn MAS NMR signals (spectra not shown). The 119 Sn single-pulse MAS NMR spectra of the stannosilicates I, II and V have shown chemical shifts in the range from δ = -660 ppm to δ = -720 ppm, which are assigned to the tin species in the Sn 4+ oxidation state 58,59 . The stannosilicates II and V ( Fig. 4b and 4c, respectively) are built up by micrometric plates, while an aggregate of spherical particles with particles size smaller than 100 nm were observed for stannosilicates III and IV ( Fig. 5a and 5b, respectively).
Enzymes immobilization and enzymatic activity of the stannosilicate-enzymes complexes
Prior to the enzyme immobilization, the stannosilicates were submitted to nickel ion-exchange reactions. The important role played by nickel in enzymes immobilization and enzymatic activity has been reported previously 34,35 and was also observed here for the stannosilicates supports nickel-exchanged. The amount of Ni 2+ incorporated on the stannosilicates were determined by EDS analyses and the results are shown in Tab. 2. The nickel ion-exchange reactions have not caused significative changes in the stannosilicates structures (Fig. 1b). The zeta potential measurements of the as-made stannosilicates and the nickel-stannosilicates are also presented in Tab. 2 and the results showed an increase in the surface charge after the nickel-exchange reactions (except for the stannosilicate III, which presented the lowest amount of nickel), as well as the percentual of immobilized enzymes (Tab. 3). The enzymatic activity of stannosilicates-enzymes complexes is also presented in Tab. 3: the as-made stannosilicate III enzymes and stannosilicate IV-Ni 2+ -enzymes presented the highest enzymatic activities of 52.3 ± 3.8 and 58.0 ± 3.2 U mg -1 , respectively. Table 2. Zeta potential (before and after nickel ion-exchange experiments) for the metallo-stannosilicates synthesized and nickel composition determined by energy dispersive X-ray spectroscopy (EDS).
Fatty acid ethyl esters production by transesterification of refined palm oil (Elaeis guineensis) using stannosilicates and stannosilicates-enzymes complexes as heterogeneous catalysts
Fatty acid ethyl esters yields obtained in the heterogeneous transesterification of refined palm oil (Elaeis guineensis) are shown in the Tab. 4. The asmade stannosilicates I, III and IV have shown no catalytic activity (FAEEs yields below 5%), while the catalytic conversion of triacylglycerides using the stannosilicates II (Na:Sn = 2.0 ± 0.2) and V (Na:Sn = 4.1 ± 0.7) have yielded 28.3 ± 0.2% and 60.8 ± 0.5%, respectively. The FAEEs yields using the stannosilicates-Ni 2+ -enzymes complexes as enzymatic catalysts are also presented in Tab. 4. The triacylglycerides conversion into FAEEs using the stannosilicates-Ni 2+ -enzymes complexes I, III and IV have yielded the following amounts of FAEEs: stannosilicate I Ni 2+ enzyme 63.3 ± 0.7%, stannosilicate III Ni 2+ enzyme 39.9 ± 0.3 % and stannosilicate IV-Ni 2+ -enzyme 38.5 ± 0.7 %. An example of gas chromatogram (GC-FID) for the highest FAEEs conversion (stannosilicate I Ni 2+ enzyme as biocatalyst) is illustrated in the supplementary material. It should be noticed that these metallo-stannosilicates in their as-made forms have shown no catalytic activity, nevertheless these positive catalytic results indicate their possible application as solid matrixes for enzymes immobilization.
The results indicated that the highest yield of FAEEs was obtained for the stannosilicate I Ni 2+ enzyme complex, which has 29 ± 9% of enzymes immobilized. In order to verify the possible occurrence of a synergistic effect as reported by other authors 34,35 , the equivalent amount of enzyme immobilized on this catalyst was used in its free form in the transesterification of refined palm oil (Elaeis guineensis) under the same experimental condition. The FAEEs yield of 6.3 ± 0.3% obtained in this reaction is a clear evidence of a synergistic effect among the nickel-stannosilicates used as inorganic supports and the Thermomyces lanuginosus lipase applied in transesterification reactions for biodiesel production. Table 4. Results for the FAEEs yields by the transesterification of refined palm oil (Elaeis guineensis) using the asmade stannosilicates and stannosilicate-Ni 2+ -enzymes complexes as heterogeneous catalysts.
Conclusions
Several metallo-stannosilicates were synthesized and characterized by XRD, SEM, BET-N2 surface area and solid-state MAS NMR ( 29 Si and 119 Sn nuclei) techniques. These materials were studied as potential inorganic solid matrixes for immobilization of Thermomyces lanuginosus lipase and also as heterogeneous catalysts in the transesterification of refined palm oil (Elaeis guineensis) by ethanolysis reactions. The results were promising, since that asmade and nickel-exchanged stannosilicates were able to immobilize from 5 ± 2% to 82 ± 6% of enzymes and presented enzymatic activity varying from 1.0 ± 0.4 U mg -1 to 58 ± 3 U mg -1 . Some of these as-made materials, when applied as heterogeneous catalysts, presented no catalytic activity. However, when applied as biocatalysts in enzymatic transesterification reactions, stannosilicate-Ni +2enzymes produced FAEEs yields varying from 38.5 ± 0.7% to 63.3 ± 0.7%. These results are an evidence of a synergistic effect among the stannosilicates and enzymes, contributing to the advancement of research in the field of biodiesel production since these results were not reported previously for this class of mixed framework oxides. | v3-fos-license |
2018-04-03T00:35:10.801Z | 1982-02-01T00:00:00.000 | 13057860 | {
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} | pes2o/s2orc | Environmental enrichment and neurotransmitter receptors
The extent of high affinity, specific binding of several pharmacological agents to brain membrane fractions derived from rats reared in an environmentally enriched or impoverished environment has been assayed. The binding of all labeled agents studied was not significantly altered in cerebellar, subcortical, or cortical membranes by the rearing condition. In the case of cortical membranes, binding of 3 H-benzodiazepine, 3 H-dihydroalprenolol, 'H-dihydroergocryptine, 'H spiroperidol, 'H-quinuclidinyl benzilate (QNB), and 'H-muscimol was not de tectably altered by exposure to these two housing conditions. These data suggest that the observed small but statistically significant increase in density of cortical synapses in rats kept in the "enriched" condition may not necessarily reflect a great increase in a specific class of synapse.
The extent of high affinity, specific binding of several pharmacological agents to brain membrane fractions derived from rats reared in an environmentally enriched or impoverished environment has been assayed.The binding of all labeled agents studied was not significantly altered in cerebellar, subcortical, or cortical membranes by the rearing condition.In the case of cortical membranes, binding of 3 H-benzodiazepine, 3 H-dihydroalprenolol, 'H-dihydroergocryptine, 'Hspiroperidol, 'H-quinuclidinyl benzilate (QNB), and 'H-muscimol was not detectably altered by exposure to these two housing conditions.These data suggest that the observed small but statistically significant increase in density of cortical synapses in rats kept in the "enriched" condition may not necessarily reflect a great increase in a specific class of synapse.
The effect of experience on the physical and chemical characteristics of the brain has been the subject of many studies, and several reports have suggested that the environment may modulate cerebral anatomy and biochemistry (Rosenzweig & Bennett, 1976;Dunn, 1976;Greenough, 1976).
Over a period of 20 years, Rosenzweig and co-workers have consistently demonstrated a relation between experiential enrichment and several chemical and morphological parameters of the rat cortex (Bennett, 1976;Rosenzweig & Bennett, 1977).Some factors that have been repeatedly and rather consistently shown to be influenced by the housing conditions of experimental animals include cortex weight and its content of macromolecules (protein, DNA, RNA).Certain enzymes related to brain function such as acetylcholinesterase have also been shown to be modified by the environment (Rosenzweig, Bennett, & Diamond, 1972).Many chemical and morphological changes have been delineated in some detail and supportive data has come from several laboratories (Altman & Das, 1964; Volkmar & Greenough, 1972; La Torre, 1968; Grouse, Schrier, Bennett, Rosenzweig, & Nelson, 1978).
The mechanisms underlying such changes are unknown.Various possible means by which the environment may modulate brain composition have been put forward.These include the concept of an endocrine basis, perhaps stress related, for such differences.This possibility has been largely excluded, as have other factors present in these experimental conditions such as differences in social stimulation, locomotor activity, handling, or earlier maturation (Rosenzweig & Bennett, 1977).Recent findings have supported the original hypothesis in these studies that the cerebral changes are caused by the increased opportunity for learning that animals have when they are maintained in the enriched situation (Bennett, Rosenzweig, Morimoto, & Hebert, 1979).The metabolic processes mediating learning and biochemical changes in the brain are also uncertain but may be related to dynamic physiological changes connected with the use or disuse of specific neuronal circuits.
While the causes of the dependence of brain chemistry upon the external environment are not well understood, such chemical changes have been shown to be concurrent with specific cerebral anatomical variations.Thus, rats exposed to the environmentally enriched (EC) paradigm delineated by Bennett, Diamond, Krech, and Rosenzweig (1964) have increased cortical depth relative to animals maintained in the environmentally impoverished (IC) housing situation (Bennett, 1976;Rosenzweig et al., 1972;Diamond, Lindner, Johnson, Bennett, & Rosenzweig, 1975).
These changes appear confined to certain cortical regions, as do differences in size of neuronal perikarya and nuclei (Rosenzweig et al., 1972) and glial cell number (Diamond, Law, Rhodes, Lindner, Rosenzweig, Krech, & Bennett, 1966).Other anatomical measures that seem in part environmentally regulated are the density of dendritic spines and extent of dendritic arborization in the rat cortex (Volkmar & Greenough, 1972;Globus, Rosenzweig, Bennett, & Diamond, 1973).The concept underlying the present work was the possibility that the increased number and density of dendritic spines and size of receptor areas observed in EC rats may reflect a difference in the number of cortical neurotransmitter binding sites.
Housing Conditions
Male rats of the Berkeley S 1 strain were used.This strain is derived from a Tryon maze bright stock.After weaning, paired littermates were housed in enriched conditions (EC) or in an improverished sensory condition (IC).Standard colony (SC) rats were reared in an intermediate environment (Bennett, 1976;Bennett & Rosenzweig, 1981).
In brief, EC animals were maintained in groups of 10 to 12 in a large cage provided with stimulus objects (ladders, wheels, etc.).IC animals were maintained singly in a dimly illuminated room in cages with solid side walls so that rats could not see each other.For the SC condition, three animals of the same sex were housed in each cage, measuring 21 x 34 x 20 cm, made of wire bars with shavings on the floor (Bennett & Rosenzweig, 1981).This housing condition resembles that used by most laboratories that maintain rats.Food and water were freely available to all groups.After 35 to 55 days of exposure to these conditions, animals were killed and the cerebral cortex and subcortical regions were dissected out, weighed, and labeled by a coding system (Bennett & Rosenzweig, 1981).Subcortex included thalamus, hippocampus, and hypothalamus but excluded pons, medulla, and cerebellum.Storage before biochemical processing was between -20°C and -60°C.
Membrane Preparation
A crude membrane fraction was prepared from tissue by homogenization in 19 volumes 0.32 M sucrose and centrifugation (50,000g for 10 min).The resulting precipitate was homogenized in the same volume of 40 mM Tris-HCl, pH 7.4, and recentrifuged (50,000g for 10 min).This procedure combined with the prior freezing of brain parts caused major lysis of cell components such as mitochondria and nerve endings.In the estimation of GABA and diazepam binding, two further washes with Tris buffer were necessary in order to remove an endogenous inhibitory material.
Binding Assay
The use of the binding technique in the assay of neurotransmitter receptors has been extensively described (Yamamura, Enna & Kuhar, 1978).Binding measurements were performed in a 1 ml incubation volume containing 40 mM Tris, pH 7.4, and appropriate radioactive and unlabeled pharmacological agents.Incubation was at 37°C for 10 min, except in the case of assay for GABA and diazepam receptors when a temperature of 2°C was used.The amount of membrane tissue in each incubation corresponded to 5 mg original wet weight and contained around 300-400 µg protein as determined by the method of Lowry, Rosenbrough, Farr, and Randall (1951).
A series of preliminary studies was carried out (Bondy, 1980) establishing that binding was at equilibrium within 10 min, was saturable and generally reversible, was between 75 and 95% specific, had an appropriate regional distribution, and was limited by and proportional to the amount of membrane present.
At the end of incubation, samples were filtered on glass fiber disks (25 mm diameter, 0.3-µm pore size, Gelman, Ann Arbor, Mich.) and washed twice rapidly with 10 ml Tris-HCI, pH 7.4, at 0°C.Filters were then dried and counted in 5 ml of a scintillation mixture (Aquasol, New England Nuclear Corp., Boston, Mass.) using a scintillation counter at an efficiency of 38 to 43%.
Statistics
All data presented are based on results from nine pairs of littermates, one of each pair being assigned to the EC or IC condition.Each region from each animal was individually assayed.Unpaired data were evaluated using Students' two-tailed t test.Paired data were subjected to the correlated t test for matched pairs.
RESULTS AND DISCUSSION
Cortical weights of EC animals were around 5% heavier (666 ± 11 mg) than those of IC animals (634 ± 14 mg). in 14 out of 15 cases, the EC cortical weight was greater than that of the paired IC littermate.The extent of binding of six ligands by membranes prepared from cortical tissue was measured (Table 1).No significant differences were found between receptor binding of EC and IC animals on a protein basis for any of the six receptors assayed.Homologous measurements were also made using membranes prepared from the cerebellum or subcortical regions.Very little difference between the binding capacity of homologous tissue derived from EC and IC rats was found for any ligand studied (Table 1), and none of these small differences was statistically significant.Four receptor sites were also measured in the cerebellum (the remaining two, dopamine and cx-norepinephrine, receptors being too low to detect).No significant differences were apparent between data from EC and IC animals (Table 1).However, since EC animals had heavier cortices containing more protein than the corresponding IC animals, it follows that the overall binding capacity of EC cortices was greater than that of the IC cortices.It is interesting that EC/IC values for almost all neurotransmitter receptors studied were above unity in cortical and below unity in subcortical tissues.This result is somewhat reminiscent of the finding of increased cortical weight relative to subcortex in EC-treated rats (Rosenzweig et al., 1972).
, Since certain subcortical regions may be sensitive to environmental modulation (Uphouse, 1980), receptor binding was studied in several more defined noncortical regions (Table 2).These regions were studied in more detail in order to increase the probability of detecting a regionspecific change in a receptor-ligand interaction.In this study, social control (SC) animals were also studied.Fewer isotopes were used in this study due to relatively small weights of tissue being available.This, combined with the lower N value may account for the somewhat greater variances in Table 2. Striatal spiroperidol and hypothalamic ergocryptine binding were chosen, as binding of these ligands has been reported to be altered by prolonged isolation (Guisado et al., 1980) or by handling (Uphouse, 1981), respectively.However, no significant differences were found between the three experimental groups (Table 2).
In conclusion, although gross sensory deprivation is known to cause very selective changes of certain synapse types in precise anatomical areas (Winfield, 1981), we have found no evidence for an especial vulnerability of any single class of neurotransmitter specific neuron.However, it is possible that real effects were masked in these studies for two reasons.First, the standard errors of the results reported are sufficiently large such that changes of 5 to 10% could remain undetected in several cases.Second, the anatomical area studied was relatively large, and differences between smaller submicroscopic regions could have been obscured.The generally nonselective nature of the differences between animals raised in differing environments suggests that regional nutrition may play a role in effecting these differences.Changes in cerebral blood flow have been found to follow altered sensory input (Bondy & Davis, 1972).These dynamic metabolic events that alter nutrient supply could underlie the differences in chemical composition of the brains of animals maintained in environments of varying sensory potential.
TABLE I Specific
Binding of Labeled Compounds to Membranes of Rats Maintained in an Enriched (EC) or Impoverished (IC) Environment" = 12 for each group.Data are expressed as pmoles ligand bound/g protein.Standard errors are presented.Experimental details are given in the text.Animals were maintained for 35 days in the two environmental conditions.Six to nine animals were used in each group.Data are expressed as pmoles ligand bound/g protein.Standard errors are presented.Experimental details are given in the text.Animals were maintained for 55 days in the three environmental conditions. | v3-fos-license |
2020-10-30T09:07:50.238Z | 2020-06-10T00:00:00.000 | 225712069 | {
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} | pes2o/s2orc | Functional in silico analysis of human tyrosinase and OCA1 associated mutations
Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. OCA1 exists in two forms: OCA1A and OCA1B. OCA1A is caused by a full loss of the human tyrosinase protein (Tyr), leading to an absence of pigment in skin, hair, and eyes, while OCA1B has reduced Tyr catalytic activity and pigment. The current understanding of the disease is hampered by the absence of information regarding the alterations of protein structure and the effects leading to either form of OCA1. Here, we used computational methods to find a general mechanism for establishing this link. Tyr and mutant variants were built through homology modeling, glycosylated in silico, minimized, and simulated using 100 ns molecular dynamics in water. For OCA1B mutants, cavity size is linked to ΔΔG values for mutants, suggesting that partial loss of Tyr is associated with the destabilizing effect of the EGF-like domain movement. In OCA1A, active site mutation simulations indicate that the absence of O2 leads to protein instability. OCA1B mutants are described in severity by the size of the cavity within the EGF–Tyr interface, while active site OCA1A mutants are unable to fully coordinate copper, leading to an absence of O2 and Tyr instability. In patients with known genotypes, free energy changes may help identify the severity of the disease by assessing either the allosteric effect of the EGF-Tyr cavity in OCA1B or the active site instability in OCA1A.
Introduction
Human tyrosinase is a type 1 trans-membrane and copper-containing glycoenzyme that catalyzes the rate-limiting step of melanin pigment production in melanosomes. 1,2 Mutations in the tyrosinase gene (TYR) can lead to oculocutaneous albinism Type 1 (OCA1), an autosomal recessive disease. Phenotypic signs of OCA1 include the absence (Type A) or reduction (Type B) of pigment in skin, hair, and eyes. OCA1A is associated with a complete loss of tyrosinase function, while OCA1B exhibits reduced tyrosinase enzymatic activity. 3 Nearly 350 TYR mutations have been identified as causes of OCA1 in patients with established diagnosis, 77% of which are missense mutations (OCA1A -67% and OCA1B -33%). 4 While recent attempts at crystallography of human tyrosinase have been unsuccessful, 5 the crystal structure of human tyrosinase-related protein 1 domain (residues 1-537) was determined at 2.3 Å resolution, and can be used as a reference for modeling intramelanosomal domains of mammalian tyrosinases due to their 40-50% sequence identity. 6,7 The active site contains four α-helices containing six His residues that coordinate two zinc ions involved in the catalytic mechanism of Tyrp1. These zinc ions are partially stabilized by one water molecule between them. Crystal structures of bacterial and fungal tyrosinase species are readily available; however, in crystallographic studies, the majority of protein structures did not demonstrate protein enzymatic activities, indicating possible differences from a protein native state. Also, a human tyrosinase structure is needed to answer OCA1 mutation-related questions.
Tyr is an N-linked glycoprotein that relies on glycosylation for its proper folding. 8,9 Glycosylation stabilizes human Tyr during the process of translocation from the endoplasmic reticulum (ER) to the cytoplasm of the melanocyte. 4 OCA1A mutants have been shown to destabilize the Tyr structure, resulting in a loss of enzymatic activity and proteolytic degradation. OCA1B also results in structural destabilization, but to a lesser degree, meaning the mutant appears to produce lower levels of melanin in melanocytes compared to the wild type. 4,10 The unfolding mutation screen (UMS) was developed in order to understand and evaluate the effect of missense mutations on protein folding and thermodynamic stability. 11 This program uses protein unfolding curves and thermodynamic changes in Gibbs free energy (ΔΔG) to calculate propensities of mutations in global mutagenesis. The results are then projected onto a protein model to highlight the critical residues and regions of structural importance to the protein.
While the difference in phenotype between OCA1A and OCA1B have been identified, the difference in the molecular mechanism leading to these results have yet to be discovered. Here, we used computational methods to find a general mechanism for establishing a link between mutation changes at the Tyr protein level and OCA1 disease phenotypes. Tyr and mutant variants were built through homology modeling, glycosylated in silico, minimized, and simulated using 100 ns molecular dynamics in water. For OCA1B mutants, cavity size is linked to free energy changes for mutants, suggesting that partial loss of Tyr in OCA1B is associated with the destabilizing effect of the EGF-like domain movement. In OCA1A, active site mutation simulations demonstrated that the absence of O 2 in an active site causes the inability to fully coordinate copper atoms and leads to Tyr protein instability. In patients with known phenotypes, ΔΔG values produced in our work may help identify the severity of OCA1 by assessing either the allosteric effect of the EGF-Tyr cavity in OCA1B or the active site instability in OCA1A.
Molecular modeling
A human homology model of the intra-melanosomal domain of human tyrosinase (TyrD, residues 19-469) was built, and refined using 2 ns molecular dynamics (MD). Ion concentration was added as a mass fraction with 0.9% NaCl used. Simulation temperature was set to 298 K with a water density of 0.997 at pH 7.4. The cell size extended to 10 Å beyond each side of the protein in the shape of a cube with dimensions 92.9 Å x 92.9 Å x 92.9 Å. Each mutation was run through molecular dynamics in YASARA using an AMBER03 forcefield, with a timestep of 5 fs. Tyr was run through the Unfolding Mutation Screen, and the atomic model was uploaded to the ocular proteome website (https://neicommons.nei.nih.gov/#/proteome). 11,12 Later, the structure of tyrosinase was glycosylated in-silico as follows. The OCA1B PDB files were glycosylated at five biologically likely Asn sites (N111, N161, N230, N337, and N371) using the online web tool Glycam-Web (http://glycam.org). The exact sugar composition in each N-glycosylation site is not known; the most biologically common eukaryotic core glycan was chosen and attached to the site: (N-acetylglucosamine, β-D-mannose, α-D-mannose). 13,14 Using the Build Structure function in UCSF Chimera, 15 bonds were added between N's Nδ2 atom and N-acetylglucosamine's C1 atom. The O1 and HO1 atoms on N-acetylglucosamine and HD21 atom on N were deleted.
Active site alterations
Tyrosinase and tyrosinase-related proteins each have coordinating molecules within the active site. 13 In order to define their role in tyrosinase function, the Tyr and OCA1A mutant structures were simulated without the dioxygen molecule. The dioxygen that binds copper within the active site were deleted from the PDB file. After deletion, each structure was energy-minimized in a water box using the AMBER14 force field, and subsequently run through molecular dynamics. The same parameters as the original mutants were used.
best-aligning pair of chains between reference and match structure was chosen for chain pairing. The Needleman-Wunsch algorithm using BLOSUM-62 was used for calculation. Matching was iterated by pruning long atom pairs until no pair exceeded 2.0 angstroms in order to remove far apart residues on the "match list" used to superimpose the structures.
Accessible area and cavity analysis
Tyr and all OCA1B mutant structure simulations were analyzed at 0 ns and 100 ns using MOLE 2.5 for cavities within the protein. Loading a protein structure leads to automatic detection of cavities based on an algorithm. 19 The default parameters for this algorithm are 3.00 Å for the probe radius, 1.25 Å for the interior threshold, and 5.00 Å for the minimum depth. These parameters were used in the calculation of cavities for the Tyr and OCA1B mutants. The three largest cavities of each structure were summed and used for analysis, assumed to be a reasonable approximation for the total cavity volume in each protein structure. For mutant variants, the effect of MD simulations was estimated as a difference between parameter changes in protein structure (mut) at 100 ns and 0 ns structure (wt). The change in solvent-accessible surface area (SA) over 100 ns between structures was described as ΔΔSA = ΔSA mut -ΔSA wt . The change in solvent-accessible volume (V) over 100 ns between structures was described as ΔΔV = ΔV mut -ΔV wt . The change in cavity volume (V cav ) over 100 ns between structures was described as ΔΔV cav = ΔV cav_mut -ΔV cav_wt .
Unfolding mutation screen
Global mutagenesis was conducted on Tyr, and each mutant was characterized by a thermodynamic change in Gibbs free energy (ΔΔG). These values were calculated using the semi-empirical method (FoldX) and standardized on a 0-1 scale, named the unfolding parameter, known as the fraction of protein in the unfolded state. 11,20 This parameter described the predicted effect of each mutation as weak (0-0.19), moderate (0.2-0.8), or severe (0.81-1). The Unfolding Mutation Screen (UMS) also outputted a foldability parameter to show critical residues in protein folding. This parameter is a sum of severityweighted unfolding propensities for the 20 mutations generated at a specific residue. Residues with the highest foldability were considered critical for protein folding. 11,14
Statistical analysis
Within the UMS pipeline, a procedure called "internal control" verifies the quality of the protein structure by mutating each residue from the protein sequence to itself. 11 The ΔΔG values of each identity mutation are calculated and then converted to unfolding propensities. The quality of the protein model is then determined by calculating the mean, standard deviation, p-value, and 95% confidence interval for those propensities. Each unfolding propensity should have a value of 0.5. Statistical significance of OCA1 structural parameters were assessed using Pearson's correlation coefficient and an adjusted R-squared value. The Pearson's correlation coefficient formula is: , where n is the number of points and k is the number of independent repressors.
Homology model of Tyr
Tyr homology model contains 12 alpha helices of at least one turn, four beta sheets, and seven disulfide bonds. The first five disulfide bonds are situated in the EGF-like domain, a repeat found in many extracellular and cell surface proteins ( Figure 1A), ( Figure 2). This domain is also found in homologous tyrosinases, Tyrp1 and Tyrp2. While the typical EGF-like repeat contains three disulfide bridges, the Tyr structure contains two more within the same region, providing more stability.
Structural changes caused by OCA1B mutations
In order to define key characteristics of OCA1 and molecular differences between OCA1A and OCA1B, eight OCA1B mutants and eleven active site His OCA1A mutants were analyzed. The ΔΔG value and unfolding parameter were calculated for each OCA1B mutant using UMS (Table 1).
Foldability parameters were listed for each residue of interest, highlighting each residue's importance to structural and thermodynamic stability ( Table 2).
For these residues, R77, M370, V393, and R402 were predicted to have the greatest destabilizing effect with ΔΔG = 2.1-5.7 kcal/mol. K142 is in α-helix H7, located away from the active site. S323 and T325 are residues in the coil between α-helix H14 and α-helix H15, located on the periphery of the protein, and do not contribute to the secondary/tertiary structure of Tyr. R77 is located within the beta hairpin of beta sheet B. M370 is located one residue after trans-membrane α-helix H16. V393 and R402 are both located within trans-membrane α-helix H19. W39 is located within the EGF-like motif ( Figure 2).
Mutant structures were overlaid onto the Tyr structure at 100 ns to identify visible structural changes ( Figure 4). No visible differences were found within the transmembrane α-helices, active site, EGF-like domain, or glycan-rich regions. The ΔΔG value of unfolding did not correlate with differences in secondary or tertiary structure in OCA1B mutants. K142M, a stable yet non-native mutant, and R77G, the most unstable mutant, exhibited similar minor differences in random loop fluctuations, but no difference in Tyr regions of importance (EGF-like domain, active site, and secondary structure Solvent-accessible volume and cavity volume within the protein were collected for each OCA1B mutant variant in MD simulations in order to identify a link between these parameters and Gibbs free energy changes. Solvent-accessible volume was calculated by YASARA. Calculated by Mole 2.5, total cavity volume was determined as the sum of the three largest cavities within the protein. Over 100 ns, the difference in solvent-accessible volume increased directly for each mutant with the ΔΔG value ( Table 1). The ΔΔSA value compared to ΔΔG had a Pearson's Coefficient of 0.55, indicating a low association between the severity of the mutation as described by ΔΔG and the total surface of the protein ( Figure 3B). The ΔΔV value compared to ΔΔG had a Pearson's Coefficient of 0.63, indicating that the total volume of the protein increases with the severity of the mutation ( Figure 5C).
While OCA1A is characterized as a complete loss of Tyr function, OCA1B exhibits reduced enzymatic activity. Tyr seems to complete the folding process; however, mutations within the protein result in a partial loss of activity. According to our data, once OCA1B mutants had completed the folding process, the protein structure began to slowly expand, creating cavities that channeled water molecules to the interior of the protein. The most severe mutations of Tyr resulted in large cavities of up to 6500 Å 3 of a difference compared to the native Tyr ( Figure 5C). Cavities were then analyzed for location commonality in regard to secondary structures, the EGF-like domain, and the active site.
Large Tyr cavities were found near the EGF-like domain, which is located within the first 100 residues of the protein. The EGF-like domain is a cysteine-rich region that contains five disulfide bridges ( Figure 6). 21 The hydrophobic cavity between the EGF-like domain and the rest of the Tyr domain (EGF-Tyr interface) contains many large aromatic residues (F98, F105, F176, W210, F429, Y433, and F438). This interface was monitored to assess destabilization in Tyr mutants in OCA1B (Table 3). The EGF-Tyr interface volume was 422 Å 3 at 100 ns for native Tyr. Mutants with ΔΔG<0 contained cavities with decreased size compared to Tyr within the cysteine-rich domain. Mutants with ΔΔG>0 contained significantly larger cavities within the EGF-Tyr interface, leading to destabilization of the protein ( Figure 5A). A strong correlation was found between the two parameters. Cavities within or near the active site show no correlation with ΔΔG values and, therefore, are not listed.
Large cavities within the EGF-Tyr interface suggest it to be a source for destabilization in OCA1B mutants. At 100 ns, the cavity encompassing the EGF-Tyr interface had perforated throughout the protein, ultimately affecting the active site in severe mutations. The native Tyr and weak mutants did not display these large cavities.
Human tyrosinase active site
Within the active site of tyrosinase there is a bundle of four alpha helices that contain six copper coordinating histidine residues. The two copper atoms are stabilized and tightly held together (2.7-2.9 Å) by two oxygen atoms ( Figure 1B). Mutations in copper coordinating histidine residues could cause a partial or complete loss of protein activity. 4 In order to find the mechanism leading to protein instability, we analyzed albinism causing genetic mutations localized in histidines coordinating copper within the active site of Tyr. The foldability parameter for each active site histidine residues is presented in Table 4. For these residues, mutations to H202 and H211 were predicted to have the greatest destabilizing effect. UMS calculations for individual mutations predicted significant changes causing a loss of protein stability or a stabilizing effect leading to a non-native stable structure ( Table 5).
The effect of mutations was simulated using MD. Each mutant variant was generated as described in the Methods section. After simulation of each OCA1A mutant was completed, the differences of the molecular-accessible volume and surface area between 0 ns and 100 ns were recorded in order to assess structural changes ( Table 5). No correlation was found between mutation severity and ΔΔV (Pearson's Coefficient = −0.51) or ΔΔSA (Pearson's Coefficient = −0.62), suggesting the correlation between ΔΔG and OCA1B mutant expansion might not apply to active site His mutants. No significant visual differences were found between Tyr and the OCA1A mutants, and there was no correlation with the ΔΔG values ( Figure 7). The structure comparison of Tyr and H363T, the most stable OCA1A mutant, produced an RMSD across pruned atom pairs of 1.1 Å and an RMSD across all atom pairs of 3.1 Å at 100 ns. The structure comparison of Tyr and H211T, the most severe OCA1A mutant, produced an RMSD across pruned atom pairs of 1.2 Å and an RMSD across all atom pairs of 3.1 Å at 100 ns.
Effect of O 2 in histidine active site mutant function in OCA1A
The glycosylated Tyr and all eight OCA1B mutant simulations were run with and without O 2 -binding copper within the active site to define its role in Tyr function. After 25 ns, the copper distance for Tyr expanded to 8.2 Å. None of the mutants were located within the active site or were able to affect copper stability, so there was no major difference with the OCA1B mutants. Although the stabilizing effect of O 2 was removed, three His residues still coordinated each copper and held them within the active site. The dioxygen molecule bound to both coppers, thought to be integral to protein stability, was removed from Tyr and the active site mutations. Simulations for all models without dioxygen were run for 25 ns, and the copper distance was measured at 0 ns, 4 ns, and 25 ns ( Table 6). For Tyr without O 2 , the copper atoms separated to 8.2 Å at 4 ns and were held stable for the rest of the simulation. For each active site His mutant, one of the two coppers lost coordination. The copper coordinated by two His residues was no longer held in place in the active site, and it either moved out and away from the two His residues or out of the active site entirely, indicating that the absence of O 2 leads to protein instability in active site His mutations ( Figures 5D & 8). In those mutants, the other copper was coordinated by three His residues and did not exhibit any signs of loss of coordination, keeping its relative position throughout the simulation. These results suggest that OCA1A mutant misfolding could be associated with protein instability within the active site.
Discussion
Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. OCA1 exists in two forms: OCA1A and OCA1B. OCA1A is caused by a full loss of the human tyrosinase protein (Tyr), leading to an absence of pigment in skin, hair, and eyes, while OCA1B has reduced Tyr catalytic activity and pigment. In this work, Tyr and mutant variants were built through homology modeling, glycosylated in silico, minimized, and simulated using 100 ns molecular dynamics in water. The effect of cavity formation due to mutation changes, in part, could be interpreted at the level of interatomic interactions. Indeed, mutation K142M results in the removal of two hydrogen bonds. The first is between hydrogen 1HZ and oxygen OD2 atoms of K142-1HZ and D174-OD2, and is 2.0 Å. The second is between K142-2HZ and D174-OD1, and is 2.7 Å. These bonds are located on the surface of the protein and, in the K142M mutation, M142 rotates towards the interior. W39 is located between R52 and R43, and the W39R mutation can result in repulsive interactions between three consecutive arginine residues. W39R is 4.1 Å away from R52 and 4.4 Å away from R43, leading to greater destabilization in the EGF-like region and a greater ΔG value. The effect of the M370Vmutation is less definitive. Arginine R77 forms two hydrogen bonds with aspartic acid D75. The first is between R77-1HH1 and D75-OD1, and its length is 1.7 Å. The second is between R77-HE and D75-OD2, and is 1.8 Å. The R77G mutation removes both hydrogen bondsand replaces Arg with a glycine residue. S323R and T325A do not experience much of a change in environment. R402 is located on the surface of the protein at the end of α-helix H19. The R402G mutation may affect the stability of the α-helix, though it is not shown in the simulation. V393D introduces a polar residue within the hydrophobic core and forms the hydrophobic bond D393:HD1-H389:OD1 of length 1.7 Å. The simulation does not show a change in the shape of α-helix H19; however, the D393 changes orientation from V393, which could destabilize an α-helix containing His-coordinating copper. While mutations might have an allosteric effect on the EGF-Tyr interface, large ΔΔG values can also be attributed to changes within those mutations' environments for most cases.
There are six total His-Cu coordinating bonds in Tyr. When a His residue is mutated within the active site, five other His-Cu coordinating bonds are still able to keep the protein intact. After 100 ns of His active site OCA1A mutant simulations, there was no evidence of denaturation or significant structural differences between the mutants and Tyr. This suggests that His active site mutations are destabilized prior to protein folding, since OCA1A mutants are known to be denatured. Parameters derived from the UMS calculation indicate that OCA1A active site histidine residues are not necessarily critical for protein folding; however, this parameter does not account for the loss of copper coordination ( Table 4). Lack of significant change in free energy suggests that active site mutations are not able to coordinate the copper before the protein folding process is completed.
The OCA1A mutant folding process may hinge on oxygen-binding copper bringing together two halves of the protein, which could build the protein catalytic site by the formation of native 4-helix bundle motif. To test protein structure stability without the oxygen, the O 2 molecule was removed from the protein and simulated for 100 ns. Without the oxygen molecules the copper atoms are no longer held tightly together, and their separation gradually increases, thereby affecting the substrate binding site located between the α-helices and destabilizing the protein structure. For almost every mutant, the solvent-accessible surface area and solvent-accessible volume increased compared to the Tyr structure. Additionally, when the histidine residues are no longer completely able to coordinate copper with the help of dioxygen, the surface area and volume of the protein accessible to solvent begins to increase greatly in comparison to the surface area and volume of the tyrosine wild type (Tables 5, 6).
In patients with known OCA1 genotypes, computer simulations performed in our study may help identify the severity of OCA1. By assessing either the allosteric effect of the EGF-Tyr cavity in OCA1B or the active site instability in OCA1A, we can guide treatment tailored to individual patients with OCA1. In disease, this can help identify the Tyr molecular mechanism associated with partial or complete protein misfolding.
Conclusions
From our analysis of human tyrosinase structure, we demonstrated an association between changes in mutant variants and OCA1 phenotypes. Indeed, OCA1B mutants are described in severity by the size of the cavity within the EGF-Tyr interface, while active site OCA1A mutants are unable to fully coordinate copper, leading to an absence of O 2 and Tyr instability. In patients with known genotypes, free energy changes may help identify the severity of the disease by assessing either the allosteric effect of the EGF-Tyr cavity in OCA1B or the active site instability in OCA1A. Sequence of the intra-melanosomal domain of human Tyr with respective secondary structure elements labeled. Obtained using the pdbsum generate server (https:// www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html). Patel Histidine residues required for copper coordination are shown in stick form with copper atoms in bronze for the Tyr and cyan for the H202Q mutant. The distance between the two copper atoms is 5.7 Å for the WT and 6.4 Å for the mutant. (B) The ribbon structures of the Tyr (bronze) and the H202Q mutant (cyan) are overlaid onto one another at 25 ns. Histidine residues required for copper coordination are shown in stick form with copper atoms in bronze for Tyr and cyan for the H202Q mutant. The distance between the two coppers is 8.2 Å for Tyr and 12.4 Å for the mutant. Q202 is not able to coordinate the copper ion, increasing the separation between the atoms. The active site of H202Q is larger than the WT, due to a slight unfolding of the protein. Patel For mutants with ΔΔG values below 0, we assume the structures to have a more stable but still non-native structure. Therefore, they are labeled as severe | v3-fos-license |
2017-09-24T23:49:46.360Z | 2017-09-08T00:00:00.000 | 34656390 | {
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} | pes2o/s2orc | Biodiesel Production from Bombacopsis glabra Oil by Methyl Transesterification Method
The objective of this work was to produce methyl biodiesel from Bombacopis glabra (B. glabra) oil degummed with H3PO4. The methyl biodiesel was prepared in an alkaline medium, and characterized by physico-chemical parameters, thin-layer chrmatograghy (TLC), gas chromatograph (GC), (Nuclear magnetic resonance of hydrogen (H-NMR), thermogravimetry and infrared analysis. The physico-chemical parameters of biodiesel were in accordance with the limits established by National Agency of Petroleum, Natural Gas and Biofuels (ANP) Resolution 45/2014, except oxidation stability, where it was corrected with the addition of antioxidants such as TBHQ and BHT.
Introduction
The research in biodiesel area has intensified in recent years, particularly due to the environmental advantages it presents compared to petroleum diesel. Since it is free from sulfur and aromatic compounds, it emits lower particulate content, such as hydrocarbons (HC), carbon monoxide (CO) and carbon dioxide (CO 2 ). It is not toxic, being biodegradable, it comes from renewable sources, and therefore, the world will benefit from the lower emission of gases contributing to the greenhouse effect [1,2].
Brazil, a tropical country, has a great potential for biodiesel production, possessing large areas of productive land and a large variety of oilseed to obtain vegetable oils [3]. In some regions of the country, there are plantations with a high yield potential. In other regions, climatic conditions provide specific and almost exclusive crops, and there are regions where only the extraction would be enough to boost production on an industrial scale [3]. Thus, the study of native plants of each region is essential to evaluate their productive potential, to aggregate value to these species, and contribute to generate employment and income to the rural population.
The B. glabra is a woody ornamental plant and can measure from 4 to 6 meters tall. The seeds of this species are the main means of propagation, with 100% germination, occurring five to ten days after sowing [6,8]. The flowering occurs from September to November, with fruit ripening at the beginning of the year. B. glabra can be used in the recovery of degraded areas, with a good development of seedlings in full sun and it tolerates 30 to 50% shade. Moreover, it is used as a living fence post (hedge), by "açourianas" communities of Santa Catarina Island, Brazil [8].
The fruit of B. glabra contains on average 18 seeds rich in oil. It has a pleasant taste, and is quite often consumed by humans and wild animals, yet however is poorly investigated for its economic exploitation [9]. The annual production is about 63 fruits per plant corresponding to an estimate of 570 kg of seeds per hectare containing a total of 400 plants [10].
Species belonging to the family Malvaceae have as a common chemical characteristic the presence of triglycerides of cyclopropenoid fatty acids (CPFA) in the oil of their almonds [5]. There are reports in the literature that compounds containing a cyclopropene ring are associated with multiple biological effects on animals including carcinogenic activities and co-carcinogenic [11][12][13]. Although B. glabra seeds are consumed by humans, their ingestion is not recommended due to the presence of CPFA. Due to the presence of this substance (CPFA), this oil does not compete with the edible oils for biodiesel production.
In some studies, reported in the literature about the chemical composition of B. glabra oil, the percentage of CPFA ranged from 24.5% to 34%, and, in other studies there was no mention about the existence of these substances [7,10]. The divergence in the results of the analyses is because the CPFA are unstable and easily decompose under heating in acidic medium, depending, therefore, on the form of extraction, the conditions of derivatization and chromatographic analysis [12,13].
Brazil has invested in research to diversify the raw material used in the production of biodiesel. The Sterculia striata oil which contains about 15% CPFA [13] was investigated for the production of ethyl biodiesel [15], however, its density was above the value specified by the Brazilian Petroleum Agency.
In some studies, reported in the literature about the chemical composition of B. glabra oil, the percentage of CPFA ranged from 24.5% to 34%, and, in other studies there was no mention about the existence of these substances [7,10]. The divergence in the results of the analyses is because the CPFA are unstable and easily decompose under heating in acidic medium, depending, therefore, on the form of extraction, the conditions of derivatization and chromatographic analysis [12,13].
Brazil has invested in research to diversify the raw material used in the production of biodiesel. The Sterculia striata oil which contains about 15% CPFA [13] was investigated for the production of ethyl biodiesel [15], however, its density was above the value specified by the Brazilian Petroleum Agency.
This study aimed to extract the oil of almonds of B. glabra, to synthesize and to characterize methyl biodiesel obtained from the degummed oil with H 3 PO 4 . Moreover, methyl biodiesel was evaluated with synthetic antioxidants, butylated hydroxyquinone (TBHQ), and butylated hydroxytoluene (BHT), and another of natural origin, the cashew nut shell liquid (CNSL).
Physicochemical Parameters of Degummed Oil
The 1 H-NMR spectrum of the oil degummed with H 3 PO 4 did not present singlet in δ 0.77, corresponding to the methylene hydrogens of the cyclopropene ring showing that under these conditions there was a degradation of these groups (Figure 2), in accordance with that which was observed for oil of Sterculia striata (Malvaceae) [12].
Physicochemical Parameters of Degummed Oil
The 1 H-NMR spectrum of the oil degummed with H3PO4 did not present singlet in δ 0.77, corresponding to the methylene hydrogens of the cyclopropene ring showing that under these conditions there was a degradation of these groups (Figure 2), in accordance with that which was observed for oil of Sterculia striata (Malvaceae) [12]. According to Table 1, the degummed oil presented an acidity level within the limits observed for refined oils, whose value is less than 0.6 mg KOH/g of oil [12], being therefore suitable for conversion into biodiesel. The value of the index of saponification degummed oil (Table 1) was lower than that reported in the literature (196-211 mg KOH/g oil) [10,16] and closer to palm oil (190 mg KOH/g oil) [17]. According to Table 1, the degummed oil presented an acidity level within the limits observed for refined oils, whose value is less than 0.6 mg KOH/g of oil [12], being therefore suitable for conversion into biodiesel. The value of the index of saponification degummed oil (Table 1) was lower than that reported in the literature (196-211 mg KOH/g oil) [10,16] and closer to palm oil (190 mg KOH/g oil) [17]. The B. glabra oil had a viscosity higher than that of coconut (27.4 mm 2 /s), palm (36.0 mm 2 /s) [18], soybean (32.6 mm 2 /s), rapeseed (37.0 mm 2 /s), and cotton oil (33.5 mm 2 /s) [19], and below castor (239.4 mm 2 /s) [20] and crambe (Crambe abyssinica) oil (53.2 mm 2 /s) [21].
Biodiesel Analysis by TLC, 1 H-NMR and GC
In biodiesel synthesis, sodium hydroxide (NaOH) in concentrations of 0.5%, 0.75%, and 1.0% in relation to the oil mass was used. It was observed that a low NaOH concentration (0.5%) led to an incomplete reaction. On the other hand, although an excess of NaOH (1.0%) accelerates the trasesterification, it also increases the amount of water in the reaction medium in the formation of sodium methoxide step, favoring the undesired hydrolysis of biodiesel [2]. In this work, the best biodiesel yield was obtained when the NaOH concentration was 0.75%.
The methyl biodiesel of degummed oil of B. glabra prepared with 0.75% NaOH produced 90% yield, however this value is influenced in part by the formation of stable emulsions, as caused by undesirable reactions such as saponification of the methyl esters and triacylglycerols [2].
The TLC analysis of the methyl biodiesel, as produced from the degummed oil with H 3 PO 4 , showed that practically all the oil was transesterified ( Figure 3). 1 H-NMR spectrum ( Figure 4) did not show signs characteristic of triacylglycerols between δ 4.10-4.40. Moreover, the spectrum confirmed the occurrence of the transesterification reaction of the oil because it showed a singlet at δ 3.61 assigned to methoxy hydrogens of ester [22]. The conversion percentage of triacylglycerols to methyl ester as determined by analysis of the 1 H-NMR spectrum was 95%. However, in the gas chromatograph (GC) analysis, the conversion percentage was 91.3%, below the minimum value (96.5%) established by the ANP Resolution 45/2014 [23]. The B. glabra oil had a viscosity higher than that of coconut (27.4 mm 2 /s), palm (36.0 mm 2 /s) [18], soybean (32.6 mm 2 /s), rapeseed (37.0 mm 2 /s), and cotton oil (33.5 mm 2 /s) [19], and below castor (239.4 mm 2 /s) [20] and crambe (Crambe abyssinica) oil (53.2 mm 2 /s) [21].
Biodiesel Analysis by TLC, 1 H-NMR and GC
In biodiesel synthesis, sodium hydroxide (NaOH) in concentrations of 0.5%, 0.75%, and 1.0% in relation to the oil mass was used. It was observed that a low NaOH concentration (0.5%) led to an incomplete reaction. On the other hand, although an excess of NaOH (1.0%) accelerates the trasesterification, it also increases the amount of water in the reaction medium in the formation of sodium methoxide step, favoring the undesired hydrolysis of biodiesel [2]. In this work, the best biodiesel yield was obtained when the NaOH concentration was 0.75%.
The methyl biodiesel of degummed oil of B. glabra prepared with 0.75% NaOH produced 90% yield, however this value is influenced in part by the formation of stable emulsions, as caused by undesirable reactions such as saponification of the methyl esters and triacylglycerols [2].
The TLC analysis of the methyl biodiesel, as produced from the degummed oil with H3PO4, showed that practically all the oil was transesterified ( Figure 3). 1 H-NMR spectrum ( Figure 4) did not show signs characteristic of triacylglycerols between δ 4.10-4.40. Moreover, the spectrum confirmed the occurrence of the transesterification reaction of the oil because it showed a singlet at δ 3.61 assigned to methoxy hydrogens of ester [22]. The conversion percentage of triacylglycerols to methyl ester as determined by analysis of the 1 H-NMR spectrum was 95%. However, in the gas chromatograph (GC) analysis, the conversion percentage was 91.3%, below the minimum value (96.5%) established by the ANP Resolution 45/2014 [23].
Analysis in Infrared Region
The infrared spectra of degummed oil and methyl biodiesel of B. glabra are illustrated in Figure 5. The lack of absorption bands among 2500-3300 cm −1 indicated the absence of moisture in the samples [24]. In the biodiesel spectrum, small displacements of the absorption bands were found as compared to those in the spectrum of oil: 1743 cm −1 , related to the stretching vibration of C=O of methyl esters, 1242, 1172, and 1112 cm −1 of bond C-O [24].
Analysis in Infrared Region
The infrared spectra of degummed oil and methyl biodiesel of B. glabra are illustrated in Figure 5. The lack of absorption bands among 2500-3300 cm −1 indicated the absence of moisture in the samples [24]. In the biodiesel spectrum, small displacements of the absorption bands were found as compared to those in the spectrum of oil: 1743 cm −1 , related to the stretching vibration of C=O of methyl esters, 1242, 1172, and 1112 cm −1 of bond C-O [24].
Thermogravimetric Analysis
Because biodiesel is a mixture of alkyl esters, it has similar physical properties to the pure esters. Therefore, it tends to show volatility and boiling point dependency on fatty acid composition, especially the chain length and number of double bonds. Thus, to a certain biodiesel, the boiling point will be the value average of types and amounts of esters of fatty acids present [25,26].
Thermogravimetric Analysis
Because biodiesel is a mixture of alkyl esters, it has similar physical properties to the pure esters. Therefore, it tends to show volatility and boiling point dependency on fatty acid composition, especially the chain length and number of double bonds. Thus, to a certain biodiesel, the boiling point will be the value average of types and amounts of esters of fatty acids present [25,26].
The thermogravimetric analysis has been shown as a rapid technique for measuring the boiling point and vapor pressure of many organic compounds, including vegetable oils or alkyl esters of vegetable oils. According Goodrum [25], this method does not show visible evidence that the samples of esters suffer breakdown before or during boiling. The thermogravimetry and derived thermogravimetry (TG/DTG) curve of crude oil of B. glabra, extracted with hexane at room temperature ( Figure 6a) showed a weight loss of 88.23% in the temperature range 255.73 to 451.74 • C, with the "onset" temperature (T onset ) of 381.83 • C concerning the boiling point of triacylglycerols. The second mass loss of 9.81% occurred among 451.74 to 505.47 • C, attributed to volatilization from longer chain triglycerides and/or minority constituents present in the oil [1,20]. It is noteworthy that the TG/DTG curve of B. glabra oil extracted at room temperature was very similar to that for other vegetable oils, and, despite the existence of 28.82 ± 1.98% of CPFA as determined by 1 H-NMR [7], thermogravimetric analysis did not show changes that could be attributed to such substances.
The degummed oil with acid showed a TG/DTG curve similar to that of crude oil (Figure 6b). The volatilization of triglycerides occurred between 261.03 to 447.20 • C (88.17% mass loss) and a boiling point of 373.37 • C. The loss of mass which occurred in the range of 447.20 to 503.20 • C (8.82%) was also attributed to volatilization of triglycerides with a higher molecular mass and/or minority constituents present in oil, once this process of degumming removes only partially the polar residues from oil [27]. There was still a small decay at 106.64 to 261.03 • C, with loss of mass of 2.10%, which can be attributed to degradation products of CPFA.
The The TG/DTG curve of methyl biodiesel of Jatropha curcas L. oil degummed with phosphoric acid exhibits two events, being that the first occurred between 9.40 to 246.65 • C (loss of mass of 79.47%; T onset = 175.81 • C) and the other from 246.65 to 301.14 • C (loss of mass 18.32% and T onset = 287.52 • C) [28].
Methyl biodiesel of "babaçu" (Orbignya phalerata) and soy showed a main thermal event with T onset corresponding to 222.11 • C [1] and 347.65 • C [25], respectively, which were attributed to the volatilization of the methyl esters. The presence of possible CPFA decomposition products likely contributed to the reduction of this value in the main event of the TG/DTG curve of B. glabra biodiesel.
The temperature interval from 310.97 • C to 429.03 • C and 429.03 • C to 502.44 • C, with a mass loss of 10.70% and 1.28%, and T onset of 387.91% and 463.36%, respectively, were associated with the waste monoglycerides, once verified the absence of di-and triacylglycerols in the gas chromatography analysis ( Table 2). It may also be associated with minority constituents in the oil, which were only removed partially in the processing biodiesel, therefore, observed with a lower content. This finding is supported by the TLC analysis, since part of the spots relative to constituents polar, observed for the degummed oil did not appear in biodiesel (Figure 3). Methyl biodiesel of "babaçu" (Orbignya phalerata) and soy showed a main thermal event with Tonset corresponding to 222.11 °C [1] and 347.65 °C [25], respectively, which were attributed to the volatilization of the methyl esters. The presence of possible CPFA decomposition products likely contributed to the reduction of this value in the main event of the TG/DTG curve of B. glabra biodiesel.
Physico-Chemical Parameters
The physico-chemical parameters of B. glabra biodiesel ( Table 2) are within the limits established in the ANP Resolution 45/2014 [23], with the exception of the oxidation stability at 110 • C (0.76 h) that may be corrected with the addition of antioxidants to meet specifications. The low oxidation stability of biodiesel was in agreement with the low thermal stability in the thermogravimetric analysis. This behavior must be due to the presence of degradation products of CPFA in the degummed oil with H 3 PO 4 and hence the biodiesel.
The percentage of monoacylglycerol and the absence of di and triacylglycerol in GC analysis, associated with low levels of total glycerin, showed that there was a high rate of conversion of oil in methyl ester.
Addition Effect of Antioxidants in Stability from Methyl Biodiesel of B. glabra
Aiming to increase the oxidation stability of the B. glabra, biodiesel tests were carried out using synthetic antioxidants, butylated hydroxyquinone (TBHQ), and butylated hydroxytoluene (BHT), and another of natural origin, the cashew nut shell liquid (CNSL), as shown in Figure 7. lubricant [35]. According to literature reports, an improvement of the thermal stability of the oil and castor biodiesel is observed when using CNSL as an additive [35]. Anacardic acid TBHQ and BHT were effective in an increase of induction period of the methyl biodiesel ( Figure 8). An addition of 2500 ppm of both antioxidants was necessary to reach the specification of this parameter, as required by ANP Resolution 45/2014, given that the biodiesel present with stability only 0.76 h.
It was observed that in the concentrations studied of TBHQ and BHT, the induction period was significantly similar. However, research showed that in the soybean biodiesel the induction period was only 0.16 h, and TBHQ presented a greater potential of stability. TBHQ was utilized in higher concentrations and showed differences in stabilization, when compared to BHT [29]. TBHQ and BHT are primary antioxidants that interrupt oxidative reactions in the chain, by the donation of hydrogens atoms from phenolic hydroxyl to the free radicals of esters, later becoming free Energies 2017, 10, 1360 9 of 14 radical, which stabilized by resonance without starting or spreading the oxidation [29,30]. The potential of these antioxidants in the stabilization of soy, palm, and jatropha biodiesel has been verified by several researchers [29,[31][32][33][34].
CNSL is comprised of meta-alkylphenols (anacardic acid, cardanol and cardol) (Figure 7), which act as free radical scavengers and sometimes as metal chelators, in steps of initiation and the spread of the oxidation process (Figure 8) [29], and may be used as an antioxidant of fuel and lubricant [35]. According to literature reports, an improvement of the thermal stability of the oil and castor biodiesel is observed when using CNSL as an additive [35].
TBHQ and BHT were effective in an increase of induction period of the methyl biodiesel ( Figure 8). An addition of 2500 ppm of both antioxidants was necessary to reach the specification of this parameter, as required by ANP Resolution 45/2014, given that the biodiesel present with stability only 0.76 h.
It was observed that in the concentrations studied of TBHQ and BHT, the induction period was significantly similar. However, research showed that in the soybean biodiesel the induction period was only 0.16 h, and TBHQ presented a greater potential of stability. TBHQ was utilized in higher concentrations and showed differences in stabilization, when compared to BHT [29]. In Figure 9 and Table 3, the induction period gradually increases with the use of high concentrations of CNSL. In addition, this increase remained practically constant above 2% of antioxidant. In these concentrations, the physico-chemical characteristics of biodiesel may be TBHQ and BHT were effective in an increase of induction period of the methyl biodiesel ( Figure 8). An addition of 2500 ppm of both antioxidants was necessary to reach the specification of this parameter, as required by ANP Resolution 45/2014, given that the biodiesel present with stability only 0.76 h.
It was observed that in the concentrations studied of TBHQ and BHT, the induction period was significantly similar. However, research showed that in the soybean biodiesel the induction period was only 0.16 h, and TBHQ presented a greater potential of stability. TBHQ was utilized in higher concentrations and showed differences in stabilization, when compared to BHT [29].
In Figure 9 and Table 3, the induction period gradually increases with the use of high concentrations of CNSL. In addition, this increase remained practically constant above 2% of antioxidant. In these concentrations, the physico-chemical characteristics of biodiesel may be compromised. The results show that CNSL is not appropriate to be used as an antioxidant additive of biodiesel of B. glabra.
Instrumentation
Automatic The methyl esters were analyzed in GC-FID, GC17A model, equipment Shimadzu with SELECT FAME column, Varian, and on a gas chromatograph GC17A model, with mass detector (GC-MS) QP5000, Shimadzu, column: SP 2560, SUPELCO. The NMR analysis were obtained in the spectrometer Varian Inova 500 (Center for NMR Spectroscopy, Pullman, WA, USA); the infrared analysis in Nicolet, Impact 400 (Nicolet Impact listings on LabX, Markham, Ontario, CA, USA), and thermogravimetric analysis in thermogravimetric balance.
Plant Material
Almonds of Bombacopsis glabra (Pasq.) Robyns were collected in in June 2003 in Brasilia-DF and stored in paper bags at room temperature. Dr. Carolyn Elinore Barnes of the University of Brasilia-UnB performed the botanical identification and the voucher specimen was deposited in the Herbarium of UnB in the UB 76691 number.
Extraction and Degumming of Oil for Obtaining Biodiesel
Samples of almonds of B. glabra, after being crushed, were submitted to extraction with hexane by 24 h at room temperature [7]. The oil obtained was degummed with H 3 PO 4 [27]. The oil was preheated to below 60 • C, where 0.1% phosphoric acid (based on the mass of oil used) and NaOH solution (8%), sufficient to neutralize 70-90% of the acid added. Then, 2% water was added, with subsequent heating and formation/decanting of the first gums, which were separated. The latter procedure was repeated, now with 3% water, to remove the remaining gums. The oil was dried with anhydrous sodium sulfate.
Synthesis of Biodiesel, TLC and 1 H-NMR Analysis
The degummed oil with H 3 PO 4 was converted into biodiesel using the alkaline transesterification reaction. Sodium hydroxide was dissolved in methanol and added to the oil in weight ratios of 100:20 (oil:MeOH) [1,20]. Values NaOH used in this process were 0.5, 0.75, and 1.0% in relation to the oil. The mixture was kept under agitation at room temperature for 1 h [36][37][38], posteriorly it was transferred to a separating funnel and the methyl esters were separated from glycerol, washed with water, and dehumidified at 100 • C. The biodiesel obtained was analyzed by TLC of silica gel, eluting with hexane-AcOEt (95:5), and iodine vapor as a dyeing reagent. The reaction yield was calculated for the expression: Yield (%) = (mass of biodiesel/oil mass) × 100.
The conversion percentage of triacylglycerols into methyl esters was determined by GC, as well as by the analysis of the 1 H-NMR spectrum from biodiesel dissolved in CDCl 3 using the following expression: C = 100 × 2A CH 3 /3A CH 2 , where C = % conversion, A CH 3 = integration area of the signal at δ 3.7 of methoxyl hydrogens and A CH 2 = integration area of the signal at δ 2.3 from methylene hydrogens α-carbonyl of methyl esters and triacylglycerol [39].
Physicochemical Characterization, Infrared and Thermogravimetric Analysis of Biodiesel
The physicochemical analysis of biodiesel included: specific mass, kinematic viscosity, water and sediments, bright point, corrosiveness to copper, cold filter plugging point (CFPP), total and free glycerin, mono, di and triacylglycerol, methanol, and oxidation stability were realized according to the standards of the American Society of Testing and Materials (ASTM) and Européen de Normalisation Committee (CEN) as indicated by Resolution 45/2014 of the National Agency of Petroleum, Natural Gas and Biofuels (ANP) [23]. The acid value of biodiesel was determined according to the Analytical standards of the Institute Adolfo Lutz [36]. The viscosity of the biodiesel was performed by the method (ASTM D445).
The infrared spectra of the degummed oil and biodiesel were obtained in the region of 4000-400 cm −1 , KBr shares.
The TG/DTG curves of oil extracted with hexane at room temperature, of degummed oil, and biodiesel were obtained in a temperature range of 30 • C to 600 • C at a heating rate of 10 • C/min, in a nitrogen atmosphere with a flow rate of 50 mL/min using an aluminum pan with a hole approximately 0.5 mm in the cap in diameter. The loss of sample mass was determined as the difference between the initial and final mass. The boiling point (temperature "onset") was considered as the point of intersection of the tangent of mass loss slope with the initial baseline. The product software was used to draw the tangent lines and record the boiling temperature [25,26].
Tests for Oxidation Stability
The oxidation stability was measured according to EN 14112, where samples of 3 g were analyzed under heating at 110 • C and a constant airflow of 10 L h −1 . In Rancimat, the airflow passes through the sample and subsequently bubbled into a flask containing deionized water, dragging the volatile carboxylic acids (degradation product) which solubilize and increase the conductivity of the water. The response elicited is a curve of electrical conductivity vs. time, in which two tangents intersect at a point, corresponding in time scale, the induction period, or oxidation stability [40].
Statistical Analysis
With the exception of some physicochemical parameters, other analyzes were performed in triplicate, and presented the mean and standard deviation of the measurements.
Conclusions
In this study, Bombacopsis glabra oil, which was extracted at room temperature and degummed with H 3 PO 4 , presented an acidity level suitable for conversion into biodiesel.
Methyl biodiesel was obtained for the first time from B. glabra oil through the transesterification process, using the base catalyst (NaOH) with the superb conversion efficiency of triacylglycerols to methyl ester (more than 90% was obtained).
The physico-chemical parameters of biodiesel were in accordance with the limits established by ANP Resolution 45/2014, except for oxidation stability, which was corrected with the addition of antioxidants such as TBHQ and BHT.
The oil of B. glabra is a important oil for biodiesel production because it is not competes with edible oils due the presence of cyclopropenoid fatty acid, although some people eat their almonds. | v3-fos-license |
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} | pes2o/s2orc | Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo)
Introduction Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. Methods We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. Results DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. Conclusion Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.
Introduction
Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ.
Methods
We have assessed receptor binding profile of DeNo and compared with dermorphin and N/ OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca 2+ in cells coexpressing recombinant receptors and a the chimeric protein Gα qi5 , (ii) stimulate the binding of GTPγ[ 35 S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats.
Results
DeNo bound to Mu (pK i ; 9.55) and NOP (pK i ; 10.22) and with reasonable selectivity. This translated to increased Ca 2+ in Gα qi5 expressing cells (pEC 50 Mu 7.17; NOP 9.69), increased binding of GTPγ[ 35 S] (pEC 50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC 50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC 50 Mu 6.36;NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu Introduction While the majority of clinical opioids mainly target the Mu (μ) receptor, work in cell and animal models would suggest targeting two or more opioid receptors simultaneously may produce drugs with reduced harmful effects. A large body of this work has concentrated on simultaneous targeting of the Delta (δ) receptor to produce drugs with attenuated tolerance profiles [1][2][3]. Work in in vitro cell based systems has demonstrated that antagonism of Delta, or disruption of the Mu-Delta heterodimer, leads to recycling of the Mu receptor, rather than ubiquitination after activation and internalisation [4][5][6]. These findings were confirmed in animal studies with Delta knockout mice showing a complete attenuation of morphine tolerance, as did preproenkephalin knockout mice [7]. Despite this compelling evidence no Mu-Delta mixed ligands have yet reached the clinic.
The nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) is a comparatively new member of the opioid family and is often referred to as a 'non-opioid branch" due to little or no affinity for the non-selective opioid antagonist, naloxone [8]. The NOP receptor is located throughout the pain pathways producing anti-opioid effects supraspinally and analgesic effects spinally [8][9][10][11]. NOP has been shown to co-localise in the pain pathways with Mu [9]. Activation of the NOP receptor has demonstrated several advantages over the classical opioid receptors. For instance, NOP agonists are able to efficiently treat neuropathic pain, a condition which classical opioid do not adequately treat [8,9]. Of particular note, intrathecal co-administration of N/OFQ and morphine in non-human primates led to a potentiation of morphineinduced antinociception, without the associated morphine-induced side effects (itch) [12]. From a cellular perspective, Mu and NOP have been demonstrated to co-express in close proximity and display differential signalling activity in vitro, suggesting the formation of a heterodimer [13][14][15].
Results from both in vitro and in vivo models have subsequently led to the development of a number of mixed molecules targeted to Mu and NOP. One of the first examples of a mixed NOP-opioid ligand was [Dmt 1 ]N/OFQ(1-13)-NH 2 , a non-selective opioid agonist acting at both the classical opioid receptors and NOP. [Dmt 1 ]N/OFQ(1-13)-NH 2 demonstrated potent and sustained activity in vivo in the monkey tail withdraw assay, with a 30-fold increase in potency over N/OFQ [16]. The most recently developed Mu-NOP bifunctional pharmacophore is cebranopadol. Cebranopadol is a full Mu agonist and a high efficacy partial agonist at NOP [17]. In rat models, cebranopadol demonstrated a long duration of action and did not disrupt motor coordination or respiration. Furthermore, cebranopadol produced tolerance by 26 days compared to only 11 days for morphine [17,18]. The results demonstrated by cebranopadol would suggest that targeting of Mu and NOP could provide substantial improvement in the treatment of pain. To further understand the interactions between Mu and NOP, a detailed examination of the interactions of a full agonist dual-targeted drug needs to be further made. In order to further explore the interactions between Mu and NOP we have synthesized a mixed Mu-NOP agonist named DeNo (Fig 1). The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs [19]. The NOP component is the endogenous agonist N/OFQ. We have assessed receptor binding, upstream and downstream signalling in cells and tissues and assessed in vivo spinal anti-nociceptive effects in rats.
Membrane preparation
In the radioligand displacement binding assays, homogenization/wash buffer consisting of 50 mM Tris-HCl pH to 7.4 with KOH, for CHO hMu , CHO hDelta and CHO hKappa or additional 5 mM MgSO 4 for CHO hNOP was used. Homogenisation buffer (50 mM Tris and 0.2 mM EGTA pH 7.4 with NaOH) was used in GTPγ[ 35 S] assays. Membranes were centrifuged at 20374 g for 10 min at 4°C. This process was repeated at least three times. The resulting pellet was resuspended in an appropriate amount of the necessary buffer and the protein concentration was determined by Lowry assay [29].
Displacement Binding Assay
Membrane protein (40 μg) was incubated in 0.5ml of 50 mM Tris, 0.5% BSA and~0.8nM [ 3 H]-DPN (for CHO hMu , CHO hDelta and CHO hKappa ) or~0.8nM [ 3 H]-UFP-101 (for CHO NOP cells), as well as varying concentrations (10 μM-1pM) of the reference ligand DeNo. Non-specific binding was determined in the presence of 10 μM naloxone for CHO hMu , CHO hDelta and CHO hKappa or 1μM of N/OFQ for CHO NOP cells. Samples were incubated for 1 hr at room temperature and reactions were terminated by vacuum filtration, onto PEI-soaked Whatman GF/B filters, using a Brandel harvester. The concentration of displacing ligand producing 50% displacement was corrected for the competing mass of radioligand to yield pK i , a measure of its affinity [3].
Calcium mobilisation assay
When confluence was reached (3-4 days), cells were seeded at a density of 50,000 cells/well into 96-well black, clear bottom plates. After 24 h incubation, the cells were loaded with medium supplemented with 2.5 mM probenecid, 3 μM of the calcium sensitive fluorescent dye Fluo-4 AM and 0.01% pluronic acid, for 30 min at 37°C. Afterwards, the loading solution was aspirated and 100 μl/well of assay buffer (Hank's balanced salt solution supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 μM Brilliant Black (Aldrich)) was added. Serial dilutions of ligands were made in Hank's balanced salt solution / HEPES (20 mM) buffer (containing 0.02% BSA fraction V). After placing both plates (cell culture and compound plate) into the FlexStation II (Molecular Device, Union City, CA 94587, US), fluorescence changes were measured at 37°C. Online additions were carried out in a volume of 50μl/well. Maximum change in fluorescence, expressed in percent of baseline fluorescence, was used to determine agonist response [27].
BRET Assay
Membrane extracts taken from HEK-293 and SH-SY5Y cells stably expressing NOP-RLuc and Mu-RLuc respectively together with Gβ 1 -RGFP were used to assess the effects of drugs on receptor/G protein interaction in concentration response curve experiments. For G-protein experiments, enriched plasma membrane aliquots from transfected cells were prepared by differential centrifugation; cells were detached with PBS/EDTA solution (1 mM, pH 7.4 NaOH) then, after 5 min 500 g centrifugation, Dounce-homogenized (30 strokes) in cold homogenization buffer (TRIS 5 mM, EGTA 1 mM, DTT 1 mM, pH 7.4 HCl) in the presence of sucrose 0.32 M. Three further centrifugations were performed at 1000 g (4°C) and the supernatants kept. Two 25,000 g (4°C) subsequent centrifugations (the second in the absence of sucrose) were performed for separating enriched membranes that, after discarding the supernatant, were kept in ultrapure water at -80°C [32]. The protein concentration in membrane preparations was determined using the QPRO-BCA kit (Cyanagen Srl, Bologna, IT) and a Beckman DU 520 spectrophotometer (Brea, CA, USA). Luminescence in membranes was recorded in 96-well untreated white opaque microplates (PerkinElmer, Waltham, MA, USA) using the Victor 2030 luminometer (PerkinElmer, Waltham, MA, USA). For the determination of receptor/ G-protein interaction, membranes (3 μg of protein) prepared from cells co-expressing NOP or Mu-RLuc and Gβ1-RGFP were added to wells in Dulbecco's PBS. For the determination of receptor/β-arrestin 2 interaction, cells co-expressing NOP-RLuc or Mu-RLuc and β-arrestin 2-RGFP were plated 24 h before the experiment in poly-D-Lysine treated plates (100,000 cells/ well), while for Mu expressing cells untreated plates were used. The cells were prepared for the experiment substituting the medium with DPBS supplemented with MgCl 2 (0.5 mM) and CaCl 2 (0.9 mM). Coelenterazine, at a final concentration of 5 μM, was injected 15 minutes prior to reading the cell plate. Receptor/G protein interaction was measured in cell membranes to exclude the participation of other cellular processes (i.e. arrestin recruitment, internalization). Different concentrations of ligands in 20 μL of PBS-BSA 0.03% were added and incubated for an additional 5 min before reading luminescence. All experiments were performed at room temperature.
Guinea pig ileum bioassay
With approval of Animal Subjects Review Board of the University of Ferrara and from the Italian Ministry of Health (PROT-186) ileum tissues were taken from male albino guinea pigs of 350-400 g (Pampaloni, Pisa, Italy). The animals were treated in accordance with European guidelines (86/609/ECC) and national regulations (DL 116/92). They were housed in 560 x 320 x 180 mm cages (Techinplast), three per cage, under standard conditions (22°C, 55% humidity, 12 h light/dark cycle, light on at 7:00 h) with food (complete feed for guinea pig, Mucedola, Milano, Italy) and water ad libitum. The day of the experiment the animals were killed with an isofluorane overdose. Bioassay experiments were performed as previously described [33]. The tissues were suspended in 5 ml organ baths containing Krebs solution (composition in mM: NaCl 118.5, KCl 4.7, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 25, CaCl 2 1.8, glucose 10), hexamethonium bromide 22 μM, benadril 0.34 μM and oxygenated with 95% O 2 and 5% CO 2 . The temperature was set at 37°C. At resting tension, 1 g was applied to the tissues. Tissues were stimulated through two platinum electrodes with a supramaximal rectangular pulse of 1 ms duration, 0.05 Hz frequency, 80V amplitude. Electrically evoked contractions were measured isotonically by means of Basile strain gauge transducers (Basile 7006; srl Ugo Basile, Varese, Italy) and recorded with a computer-based acquisition system (Power Lab 8, ADInstruments, USA). After an equilibration period of about 60 min, the contractions induced by electrical field stimulation were stable. At this time, cumulative concentration response curves to agonists were constructed (0.5 log unit steps). Antagonists were injected into the baths 15 minutes before constructing agonist concentration response curves.
In Vivo Studies
For all the experiments described below, male Sprague-Dawley rats (Harlan, Varese, Italy), weighing approximately 280-300g at the beginning of the experimental procedure, were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least one week after their arrival. One rat was housed per cage (size 26x41 cm); animals were fed with standard laboratory diet and tap water ad libitum, and kept at 23±1°C with a 12 h light/dark cycle, light at 7 a.m. All animal manipulations were carried out according to the European Community guidelines for animal care (DL 116/92, application of the European Communities Council Directive of 24 November 1986 (86/609/EEC). The ethical policy of the University of Florence complies with the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (NIH Publication No. 85-23, revised 1996; University of Florence assurance number: A5278-01). Formal approval to conduct the experiments described was obtained from the Animal Subjects Review Board of the University of Florence and from the Italian Ministry of Health (N°54/2014-B). Experiments involving animals have been reported according to ARRIVE guidelines [34]. All efforts were made to minimize animal suffering and to reduce the number of animals used. All animals were monitored daily using a scoring system (based on: Appearance, Food and Water Intake, Clinical Signs, Natural Behaviour and Provoked Behaviour [35]. Maximum score is 20 and animals reaching 10 are euthanized. In the experiments reported here no animals reached this score and none died before the end of the experiment) and at the end of the experiment were euthanized by CO 2 overdose.
Intrathecal catheterization
Rats were anaesthetised with 2% isoflurane and an intrathecal catheter was surgically implanted according to the method of Yaksh & Rudy (1976) [36]. Rats were shaved on the back of the neck and placed in the stereotaxic frame with the head securely held between ear bars. The skin over the nape of the neck was cleaned with ethyl alcohol and incised for 1 cm. The muscle on either side of the external occipital crest was detached and retracted to expose about 3-4 mm 2 of the atlanto-occipital membrane. The membrane was incised by a needle, which led to the escape of cerebrospinal fluid. The caudal edge of the cut was lifted and about 7.0 cm of 28G polyurethane catheter (0.36 mm outer diameter; 0.18 mm inner diameter; Alzet, USA) was gently inserted into the intrathecal space in the midline, dorsal to the spinal cord until the lumbar enlargement. The exit end of the catheter was connected to 4.0 cm polyurethane tube (0.84 mm outer diameter; 0.36 mm inner diameter) and was taken out through the skin, flushed with saline solution, sealed and securely fixed on the back of the head with a silk wire. Animals were placed in individual cages until recovery. All animals used during behavioural tests did not show surgery induced motor impairment as evaluated by Rota rod test. Animals presenting any kind of motor disability were excluded from the behavioural measurements. Behavioral measurements were performed on 5 rats for each treatment.
Intrathecal drug treatments
Dermorphin and DeNo were dissolved in sterile saline solution. Acute measures were performed after the intrathecal (i.t.) infusion of 0.1-1 nmol dermorphin and DeNo. All compounds were administered in a final volume of 10 μl. Behavioral tests were carried out after 15, 30, 45, 60, 90, 120 and 180 min.
Paw Pressure test
Nociceptive threshold was determined with an analgesimeter (Ugo Basile, Varese, Italy), according to the method described by Leighton et al. (1988) [37]. Briefly, a constantly increasing pressure was applied to a small area of the dorsal surface of the paw using a blunt conical probe by a mechanical device. Mechanical pressure was increased until vocalization or a withdrawal reflex occurred while rats were lightly restrained. Vocalization or withdrawal reflex thresholds were expressed in grams. Rats scoring below 40 g or over 75 g during the test before drug administration were rejected. For analgesia measures, mechanical pressure application was stopped at 150 g. All experiments were performed by a researcher blind to drug treatment.
Rota-rod test
Rota-rod apparatus (Ugo Basile, Varese, Italy) consisted of a base platform and a rotating rod with a diameter of 6 cm and a non-slippery surface. The rod was placed at a height of 25 cm from the base. The rod, 36 cm in length, was divided into 4 equal sections by 5 disks. Thus, up to 4 rats were tested simultaneously on the apparatus, with a rod-rotating speed of 10 rpm. The integrity of motor coordination was assessed on the basis of the number of falls from the rod in 60s measured 15, 30, 45, 60, 90, 120 and 180 min after treatments.
Data analysis
Data are expressed as mean±SEM or with confidence intervals as appropriate. For more than 2 groups, data are analysed using ANOVA with post-hoc testing using Dunnett's or Bonferroni's test as appropriate. Where there are only 2 groups paired or unpaired t-tests were used. P values of less than 0.05 were considered significant. All curve fitting was accomplished using Graphpad-Prism (V6). The concentration of drug producing 50% of the maximum response (pEC 50 ) and the maximum response (E max ) are quoted. In gpI experiments the antagonist potency (pK b ) is calculated from the rightward shift of the agonist concentration response curve by a fixed antagonist concentration.
Displacement Binding assay
In displacement binding studies at CHO hMu , dermorphin and DeNo displaced the binding of [ 3 H]-DPN in a concentration dependent and saturable manner (Fig 2). DeNo (pK i 9.55) demonstrated a significant increase in affinity at Mu, when compared to the parent compound dermorphin (8.69). At CHO hNOP , DeNo displaced [ 3 H]-UFP-101 in a concentration dependent and saturable manner (Fig 2). DeNo (10.22) displayed a similar pK i value, for NOP, to its parent compounds N/OFQ (10.69) (Fig 2, Table 1). At CHO hDelta , DeNo (8.12) demonstrated an increase in affinity compared to its parent compounds. Dermorphin displayed an affinity of 7.17, while N/OFQ failed to displace [ 3 H]-DPN at the Delta receptor. Furthermore, DeNo (7.34) showed affinity for the Kappa receptor, whereas the parent compounds (dermorphin and N/OFQ) failed to displace [ 3 H]-DPN at this receptor (Fig 2, Table 1).
GTPγ[ 35 S] Assay
Dermorphin and DeNo stimulated the binding of GTPγ[ 35 S] in a concentration dependent and saturable manner at the Mu receptor (Fig 4). DeNo (E max 2.68) demonstrated a similar maximal response to that of dermorphin (2.63). The pEC 50 values for DeNo (7.77) showed no significant difference to that of the parent compound, dermorphin (7.83) (Fig 4, Table 3). At CHO hNOP , N/OFQ and DeNo stimulated the binding of GTPγ[ 35 S] in a concentration dependent and saturable manner (Fig 4). DeNo produced a maximal response (E max 2.49) similar to that of its parent compound, N/OFQ (2.57). The pEC 50 value of 9.50 achieved by DeNo was similar to that of N/OFQ (9.05). (Table 3). Leu-enkephalin and DeNo stimulated the binding of GTPγ[ 35 S] in a concentration dependent and saturable manner in membranes expressing Delta receptors (Fig 4). DeNo (E max 1.84) produced a maximal response similar to that of the endogenous Delta peptide, Leu-enkephalin (1.90). However, the pEC 50 value for DeNo (6.78) was significantly lower than that of Leu-enkephalin (8.50) ( Table 3). At CHO h-Kappa , dynorphin-A and DeNo stimulated the binding of GTPγ[ 35 S] in a concentration dependent and saturable manner (Fig 4). DeNo (E max 2.36) produced a maximal response similar to that of dynorphin-A (2.33). The pEC 50 value of DeNo (5.91) was significantly lower than that of dynorphin-A (9.36) ( Table 3).
Cyclic AMP assay
To further assess functional activity of DeNo, inhibition of cyclic AMP (cAMP) formation was assessed. Since DeNo demonstrated a higher affinity and potency at Mu and NOP, assays were performed using CHO hMu and CHO hNOP cells only. Addition of forskolin in CHO hMu cells lead to a 24.3 (±1.79) fold increase in cAMP production, when compared to basal activity ( Fig 3). Co-incubation of 1μM dermorphin, or 1μM DeNo reverses the effects of forskolin, returning cAMP levels to basal. In CHO hNOP cells, forskolin stimulation leads to a 21.23 (±3.86) fold increase in cAMP production, when compared to basal activity. The addition of 1μM N/OFQ reverses forskolin stimulated cAMP production. The addition of 1μM DeNo has a similar effect, returning cAMP levels to basal.
BRET Assays
In HEK-293 membranes, N/OFQ promoted NOP-RLuc/G-protein-RGFP interaction in a concentration-dependent manner with high potency (pEC 50 9.22) and a maximal effect corresponding to 0.42±0.04 stimulated BRET ratio. Dermorphin was very weak only increasing BRET ratio at micromolar concentrations (Fig 6, Table 4). In SH-SY5Y membranes, dermorphin produced a concentration dependent stimulation of G-protein interaction, also with high potency (pEC 50 8.13). In this cell type, the maximal effect was larger at 1.39±0.14 stimulated BRET ratio. N/OFQ was very weak increasing BRET ratio only at micromolar concentrations. Under the same experimental conditions, DeNo was tested in both cells lines. In HEK-293 membranes, DeNo mimicked the stimulatory effect of N/OFQ with similar potency (9.02) and maximal effect (α 1.01) (Fig 6). In SH-SY5Y membranes, DeNo concentration dependently increased BRET ratio with similar potency and maximal effect (pEC 50 8.03 and α 0.98) to dermorphin (Fig 6, Table 4). HEK-293 and SH-SY5Y cells stably expressing the human NOP or Mu (NOP/Mu-RLuc) receptors and β-arrestin 2-RGFP were used to evaluate NOP and Mu interaction with β-arrestin 2.
Guinea pig ileum bioassay
DeNo was assessed in the electrically stimulated guinea pig ileum. In this preparation, concentration response curves to DeNo were assessed in comparison with N/OFQ and dermorphin (Fig 8). N/OFQ inhibits contractions induced by electrical field stimulation in a concentration dependent manner, with a pEC 50 of 8.03 (7.84-8.23) and maximal effect of 71±4%. Dermorphin mimicked the effects of N/OFQ with higher potency and maximal effects (pEC 50 9.55 (9.33-9.77) E max 86 ± 2%). The new compound, DeNo, inhibited electrically induced twitch, with a potency of 8.63 and maximal effects similar to those of dermorphin (E max 90 ± 1%). In order to determine the site of action of DeNo in the guinea pig ileum, antagonist assays were undertaken. The standard non-selective opioid antagonist naloxone (100 nM) does not affect the inhibitory action of N/OFQ, while 100 nM SB-612111 was able to shift to the right the concentration response curve to N/OFQ with a pK b of 8.36. In contrast, the effects of dermorphin were sensitive to naloxone (pK b 8.57) but not to SB-612111 (Fig 8). Finally, the effects of DeNo were challenged with naloxone, SB-612111, and the cocktail of the two antagonists. Naloxone antagonized the inhibitory effect of DeNo producing a rightward shift of the concentration response curve and no modification of maximal effects. A pK b value of 7.54 was derived from these experiments. SB-612111 was also able to counteract DeNo effects by producing a displacement to the right of the concentration response curve; a pK b value of 7.07 was derived from these experiments. When the two antagonists were assayed together they displayed a clear additive effect.
Paw pressure Test
Time-courses were produced for both dermorphin and DeNo given intrathecally (i.t.) in rats subjected to the plantar test. Dermorphin produced significant antinociceptive effects (Fig 9A). Similar effects were measured in response to i.t. DeNo which was less potent producing statistically significant effects only at the top dose of 1 nmol (Fig 9B). However it should be ubderscored that in the same range of doses both compounds impaired animal performance in the rotarod test (data not shown).
Discussion
In this study we have characterised a novel peptide bivalent Mu-NOP ligand, DeNo; created by combining dermorphin and N/OFQ. DeNo bound to Mu and NOP receptors and was~5fold A) The activity of phosphorylated p38 compared to total p38 at CHO hMu caused by dermorphin (1μM) and DeNo (1μM). B) The activity of phosphorylated ERK1/2 compared to total ERK1/2 at CHO hMu caused by dermorphin (1μM) and DeNo (1μM). C) The activity of phosphorylated ERK1/2 at CHO hNOP caused by N/OFQ (1μM) and DeNo (1μM). All data are after a 15 minute incubation period. Data are mean (±SEM) for n = 5. *p<0.05; according to ANOVA followed by Dunnett's test for multiple comparison. Representative blots are shown above mean data.
doi:10.1371/journal.pone.0156897.g005 NOP selective. There was between 1 and 2 logs selectivity over Delta and Kappa receptors. At conventional upstream (GTPγ[ 35 S]) and downstream (cAMP) assays and in Ca 2+ mobilisation experiments using chimeric G-protein constructs DeNo was a full agonist at both Mu and NOP. In BRET assays to assess receptor G-protein interaction and arrestin recruitment, DeNo also behaved as a full agonist. Moreover, DeNo activated ERK1/2 as a full agonist at Mu and NOP; there was no activation of p38 at NOP. In a more intact preparation DeNo inhibited contraction of the electrically stimulated gpI via simultaneous activation of NOP and Opioid receptors and was antinociceptive via the i.t. route in rats.
At the receptor, the most striking difference was a log increase in DeNo binding affinity at Mu compared to dermorphin and this may result from the structure of the linker between the two 'pharmacophores'. Comparing CHO data and taking into account different assays/buffer systems there was a marked difference in pK i / pEC 50 for Mu falling 60 fold at GTPγ[ 35 S] (upstream) and 331 fold at the chimeric G-protein (downstream). In contrast at NOP there was a 7 and 3 fold difference in affinity compared to potency. As the point of interrogation (assay) moved further down the signal transduction cascade the difference in potency at Mu protein assay suffers from hemi-equilibrium problems and we have discussed this in detail previously [38].
Using BRET based assays, we have been able to assess receptor/G protein interaction and arrestin recruitment. These end points were examined in HEK cells for NOP and SH-SY5Y cells for Mu; this is a minor drawback in comparison with the other assays reported but because we have used dermorphin and N/OFQ then we can cross compare. In all assays, the parent compounds and DeNo behaved in essentially the same manner (potency) and as full agonists. A possible confounder is that SH-SY5Y cells have been shown to express both the Delta receptor and, more relevantly, the NOP receptor; at very low expression levels [39]. Previous work with Mu/NOP heterodimers has demonstrated that activation of NOP often leads to a reduction in the potency of Mu agonists throughout the cell signalling cascade [15]. It is possible in this cell line that DeNo is engaging both Mu and NOP. It has been known for some time that opioid receptors couple to MAPK with some variation in coupling [40,41]. In CHO cells expressing recombinant Mu and NOP there were marked differences. ERK1/2 was ubiquitously activated but p38 was only activated by Mu. ERK1/2 is typically involved in proliferative and differentiation responses but there is good evidence for a role in more chronic opioid receptor activation and possibly withdrawal [42][43][44]. This kinase along with p38 also plays a role in apoptosis [45]; the distinction of roles is not clear cut. In terms of activation, two pathways have been suggested-the first involving a typical G-protein mediated event and the second via an arrestin-MAPK protein scaffold. That arrestin is activated is confirmed by the BRET assay but without full concentration response curves and pertussis toxin sensitivity experiments it is not possible to delineate the relative importance of these pathways. The fact that dermorphin and N/OFQ behaved as full agonists with no striking differences in potency suggests no ligand bias as expected for naturally occurring agonists. We have used the gpI as a more intact bioassay that (1) endogenously expresses both Mu and NOP [33] and (2) links the in vitro recombinant data set to the in vivo behavioural experiments. In this preparation, N/ OFQ was sensitive to the non-peptide antagonist SB-612111 but not naloxone; dermorphin was sensitive to naloxone but not SB-612111. These antagonists displayed lower potency when tested against DeNo whose concentration response curve was additively shifted when a cocktail of antagonists was used. Collectively, these results demonstrated that the biological action of DeNo in this preparation is due to the simultaneous activation of NOP and Mu receptors despite the greater loss of potency at Mu than at NOP found in recombinant systems. In vivo, after spinal administration in rats dermorphin (present results) and N/OFQ [46] elicited similar antinociceptive actions. Under the same experimental conditions, DeNo mimicked the effects of dermorphin and N/OFQ being neither more potent nor more effective. This result contrasts with the extensive literature evidence suggesting that the simultaneous activation of Mu and NOP receptor elicits synergistic antinociceptive effects both in rodents [47][48][49] and in non-human primates [12,16,50,51]. In vitro studies clearly demonstrated that DeNo is able to bind and activate both Mu and NOP receptor in the same range of concentrations; this may not be the case in vivo. A similar situation has been previously well documented in the studies of the MDAN series of compounds [52]. Under the present experimental conditions there were almost no differences between doses inducing antinociceptive effects and those disrupting animal performance on the rotarod. Further studies are needed to assess in detail any potential spinal antinociceptive actions of DeNo.
In conclusion we have created a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP); this molecule behaves essentially as the parent compounds. Despite this promising profile, analgesic actions of DeNo are poor in the model employed here. As a mixed molecule, this ligand represents a useful addition to the non-peptides BU08028 [53,54], the SRI-International compounds exemplified by SR16435 [54,55] cebranopadol and the peptide [Dmt 1 ]N/OFQ(1-13)-NH 2 . DeNo may be a useful tool, particularly in vitro for investigating the consequences of the simultaneous activation of NOP and mu receptors. | v3-fos-license |
2019-04-06T13:10:55.993Z | 2014-06-27T00:00:00.000 | 97707140 | {
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} | pes2o/s2orc | Mass Transfer During Osmotic Dehydration of Chub Mackerel Cylinders in Ternary Solution
In the analysis, design and optimization of an osmotic dehydration process is important to know the kinetic of water loss and solutes gain. In this study, the mass transfer during osmotic dehydration of chub mackerel (Scomber japonicus) cylinders in ternary solution glycerol/salt/water was analyzed. The models of Zugarramurdi & Lupín and Azuara were used to describe mass transfer and to estimate equilibrium values. The radial effective diffusion coefficient was estimated using the analytical solution of Fick's second law. Diffusion coefficients were determined for a finite cylinder, for an infinite cylinder considering only the first term of the series and considering higher order terms of the series. The profiles of water and solutes during the osmotic dehydration were calculated by using the estimated water and solutes diffusivities. According to the results obtained, using three terms in the analytical solution of the Fick's second law is appropriate to determine the diffusion coefficients. The diffusion coefficient for infinite cylinder were 2.63×10, 4.11×10 and 4.25×10 cm/s for water loss, salt and glycerol gain respectively. For a finite cylinder these values were 2.30×10, 3.67×10 and 3.78×10 cm/s respectively. All the models proposed were in agreement with experimental data for solutes gain ((0.967<Radj<0.986); (0.0016<RMSE<0.039) and (4.17<P<10.0)). The model based on the solution of Fick’s Law for an infinite cylinder with higher order terms was the best fit for water loss and solutes gain. The equilibrium values estimated with Azuara model agree with the experimental (0<relative error<9.8). Water and solute distributions as a function of time and location in the radial direction were plotted.
Introduction
Osmotic dehydration (OD) is a technique used for the partial removal of water when foods are placed in a hypertonic solution.During the process, two counter-current flows take place: water flows out from the food to the solution and the solute is transferred from the solution to the food.Mass transfer rate during osmotic dehydration depends on many factors such as concentration, temperature, composition of the osmotic solution, immersion time, nature of the food and their geometry, solution agitation, etc. (Li & Ramaswamy, 2006;Uribe et al., 2011;Abbasi Souraki, Ghaffari, & Bayat, 2012).For developing food-processing technology based on OD process, it is important to understand OD kinetics so as to set the desired dehydration level during the process.The kinetic of mass transfer can be predicted using an unsteady-state diffusion model (Fick's second law).Analytical solutions of the equation are available for idealized geometric media: spheres, infinite cylinders, infinite slabs, and semi-infinite media (Li & Ramaswamy, 2006) and the diffusion coefficients for both water loss and solids gain individually or simultaneously can be estimated.However, only limited research has been carried out on finite cylinders under typical osmotic dehydration conditions that describe mass transfer phenomena (Rastogi, Raghavarao, & Niranjan, 1997;Li & Ramaswamy, 2006;Abbasi Souraki et al., 2012).For fish products there is no information available on cylinder geometry.
Since diffusion equations have analytical solutions only for classical geometries, for non-classical geometries, numerical methods are necessary for their solution.Because of these limitations the use of empirical models is of interest, since these simple models have no geometric restrictions for their application.Water loss and solute gain kinetics during OD of fish products has been modeled by mean of empiric relations like Peleg and Zugarramurdi & Lupín models (Zugarramurdi & Lupín, 1980;Turhan, Sayar, & Gunasekara, 2002;Corzo & Bracho, 2005, 2006;Schmidt, Carciofi, & Laurindo, 2009;Czerner & Yeannes, 2010) and probabilistic models like Weibull (Corzo & Bracho, 2008).The information in the literature about the application of such empirical and probabilistic models for describing OD of fish products in ternary solutions containing glycerol is very scarce (Checmarev, Casales, Yeannes, & Bevilacqua, 2013;Checmarev, Casales, & Yeannes, 2013).
Chub mackerel (Scomber japonicus) is found along the Atlantic coast of South America.It is bounded on north by latitude 23°S (Rio de Janeiro) and on the south by latitude 39°S (Bahía Blanca) (Angelescu, 1980).In Argentine, during 2010 and 2011 the fishing fleet reached 27.558 and 28.253 tons respectively (MAGYP, 2011).The usual preservation of chub mackerel consists mainly in the traditional process of frozen, canned and smoked.Considering the aforementioned, it is important to develop new processing alternatives for this species.Osmotic dehydration represents a good alternative for the development of new products of chub mackerel.
In this work the kinetics of water loss and solid gain for chub mackerel cylinders during osmotic dehydration in water/salt/glycerol were studied.The empirical models of Zugarramurdi and Lupín and Azuara and a model based on the solution of Fick's Law for cylinder geometry were used.The water and solutes distributions during dehydration were predicted.
Raw Materials
Chub mackerel (Scomber japonicus) caught in Mar del Plata, Argentine, in October 2012 and stored at -18 °C was used in this study.
Sampling
Experimental works were carried out with long cylindrical samples of mackerel, so that the process could be considered as one-dimensional diffusion in the radial direction.
To determine the mass transfer kinetic and the diffusion coefficients, cylinders (cooked mackerel loin) of 16.5 cm long and 1.9 cm in diameter were immersed in ternary solution containing salt, water and glycerol and were kept under refrigeration temperature (7±1 °C).At different times, three cylinders were removed from the solution and the ends of the cylinders were discarded in order to prevent edge effects, only the central area of each cylinder (9 cm in length) were analyzed (Graiver, Pinotti, Califano, & Zaritzky, 2006).The contents of water, salt and glycerol in the samples were determined at 0.5, 1, 1. 5, 2, 2.5, 3, 4, 7, 10.5, 15, 17, 19 and 24 h.The cylinders were drained, superficially rinsed with distilled water, dried with absorbent paper and weighed.Two experiments were carried out.
To validate the models were carried out two experiments in which immersion in the ternary solution was performed at 10 °C.
Physicochemical Analyses
The water content was determined at 105 °C until constant weight (AOAC, 1990) using a drying oven (Marne, 644, Córdoba, Argentine).The ashes were determined by calcinations at 550 °C as described by AOAC (1990) using an electric oven (Indef, 332, Córdoba, Argentine) and the sodium chloride content using the Mohr method adapted for food (Kirk, Sawyer, & Egan, 1996).The glycerol content was determined using an enzymatic UV method (Boehringer Mannheim/R-Biopharm) with a spectrophotometer (Shimadzu® UV-1601 PC, Kyoto, Japan).All analyses were done in triplicate.
Mass Transfer Model Based on Fick's Second Law
The unsteady state Fickian diffusion model was applied to describe the water and solute diffusion into a cylinder (Crank, 1975).
(1) Analytical solution of Equation ( 1) is obtained by using the following initial and boundary conditions: Uniform initial moisture and salt concentrations: , 0 0 (2) Symmetry of concentration at the center: It is assumed that the shape and the diffusion coefficient are constant during dehydration.Constant equilibrium moisture and salt concentrations at the surface (negligible external resistance to mass transfer) are considered: C(r, t) = C eq for r R (4) Constant solution concentration (high solution to fish mass ratio).
Equation ( 1) and boundary conditions can be rewritten in dimensionless form: φ= φ(x,τ); 0 1; 0 , 0 x=0, τ> 0 (8) Being ; ; (9) The solution of the Equation (5) for the average ratio of water loss and solute gain is given by: ∑ Where x t is the water loss or solute gain (expressed as g on a non-salt and non-glycerol dry matter basis, g/gdm) at time t, x eq the equilibrium values and x 0 the initial values.The δ n are the roots of the Bessel equation of the first kind of zero order: Jo(δ n )=0.
Model Based on Fick's Second Law for an Infinite Cylinder
The effective diffusion coefficient was calculated using 1, 3 and 5 terms of the Equation (10).
Equation ( 10) can be simplified by considering only the first term in their series expansion and written in logarithmic form as: ln The water and solute effective diffusivities are derived from the slope of the plot of versus t.
2.6.1.2Model Based on Fick's Second Law for a Finite Cylinder Prediction of mass transfer in a finite cylinder requires the use of analytical solutions obtained for both infinite cylinder and infinite slab.The solution of the Equation ( 5) for the average ratio of water loss and solute gain is given by: The effective diffusion coefficient was calculated using 5 terms of each series.
Water and Solute Profiles Into Mackerel Cylinder During Osmotic Dehydration
The concentration distribution of water and solutes is considered only along its radius r as a function of time t since the mackerel cylinder length l is many times larger than its diameter 2R.The water and solutes distributions along the radius will be of the form (Crank, 1975): or in the following dimensionless form:
Empirical Models
Simple empirical models that have no geometric restrictions for their application have been reported for describing mass transfer in food subjected to OD and to predict water loss and solid gain at equilibrium condition (Azuara, Beristain, & García, 1992;Corzo & Bracho, 2006;Schmidt et al., 2009, among others).
Azuara Model
Azuara et al. (1992) developed a two parameters model with the following expression: (15) where x t and x eq are water loss, salt or glycerol gain (expressed as g on a non-salt and non-glycerol dry matter basis, g/gdm) at time t and at equilibrium, respectively, and S (h -1 ) is the model constant (rate constant).
Zugarrramurdi and Lupin Model (Z&L Model)
Zugarramurdi and Lupín (1980) proposed a mathematical model, with an exponential approach to the salt and water equilibrium values: (16) where x t and x eq are the water loss, salt or glycerol gain (g/gdm) at a given time t (h) and at the equilibrium respectively and k is the specific rate constant (h -1 ).Integrating Equation ( 16) with the initial
Statistical Analysis
The fitting of the models to the experimental data was performed using OriginPro 8.The goodness of fit was determined using the adjusted determination coefficient (R 2 adj ), the root mean square error (RMSE, Equation ( 18)) and the average relative deviation (P, Equation ( 19)).
where x i is the experimental value, x pi is the predicted value and n the number of data pairs.Values of P less than or equal to 10% are considered to fit the experimental data satisfactorily (Azoubel & Murr, 2003).
Effective Diffusivities of Water and Solutes in Mackerel Cylinders
Effective diffusivities were calculated by fitting the unsteady Fick´s second law solutions (Equations ( 10), ( 11) and ( 12)) to the experimental data.The predicted values of water, salt and glycerol effective diffusivities for an infinite cylinder with 1, 3 and 5 terms of the series and for a finite cylinder are shown in Table 1.The values of R 2 adj , RMSE and P are shown in Table 2.
Table 1. Diffusion coefficients of water and solutes
Values are mean±standard deviation of two experiments.
Table 2. Statistical parameters of models
In all cases, R 2 adj was higher than 0.924 and RMSE was lower than 0.18 indicating the acceptability of the model proposed.Values of P were less than or equal to 10%, excepting for diffusion coefficient of water loss estimated by the linearized equation.These results indicated that the models fit the experimental data.There were no significant differences to the 95% of confidence level between using three and five terms in the analytical solutions of the Fick second law to determine the diffusion coefficient.Therefore, an increase in the number of terms in the series did not generate changes in the diffusion coefficient as these terms were very small and negligible for the calculation.Ramallo, Schvezov, & Mascheroni (2004) studied the osmotic dehydration of pineapple in sucrose solution and reported that there were no significant differences between using three and four terms in the analytical solutions of Fick second law.When the diffusion coefficients were estimated by the linearized equation the RMSE was higher (0.10<RMSE<0.18) and also the P value for water loss was greater than 10%.Validation of models is shown in Figure 1, as can be seen the model followed the general trend of the experimental osmotic dehydration curves.The values of water loss, salt and glycerol gain predicted by the Fick model for infinite cylinder and for finite cylinder were very close to the experimental values.The model based on the solution of Fick's Law for an infinite cylinder was the best fit for water loss and salt gain.For glycerol gain there was no difference in the R 2 adj, RMSE and P values of the model based on the solution of Fick´s Law for infinite and finite cylinder respectively.
Water and Solutes Profiles During Osmotic Dehydration
Since the predicted values of average water loss and solutes gain were found to fit well to the experimental data (Figure 1), the water, salt and glycerol distributions into mackerel cylinder were predicted.Equation ( 14) was solved using the predicted effective water, salt and glycerol diffusivities (Table 1).Dimensionless water, salt and glycerol contents vs. dimensionless time (τ = Dt/R 2 ) were plotted as a function of dimensionless radius (x = r/R) in Figure 2.This Figure shows that the central point has the maximum water content and minimum salt and glycerol content (for water C o >C eq , for salt and glycerol C 0 =0 and C 0 <C eq ).Therefore, water is transferred from interior to the surface and salt and glycerol are transferred into the sample in the reverse direction.As it can be seen in this figure, the decrease of water content and the increase of salt and glycerol contents is mainly carried out in the region of surface of the mackerel cylinder and slowly progresses to the interior.At the end of dehydration, the water, salt and glycerol contents in mackerel cylinder reached the equilibrium with osmotic solution.
Figure 2. Dimensionless water, salt and glycerol distribution inside mackerel cylinder during osmotic dehydration
Kinetics of Osmotic Dehydration and Equilibrium Water Loss and Solutes Gain
Empirical models can be used to represent mass transfer during osmotic treatment, when the diffusive model is difficult to use or when experimental determination of the equilibrium values requires long immersion times that can lead to food tissue changes.The models developed by Z&L and Azuara (Equations ( 15) and ( 17)) were used to predict the kinetics of osmotic dehydration and to determine the final water loss and solutes gain at equilibrium.The main advantage of these models is its capacity to predict the equilibrium values.Table 3 shows the parameters of the models as well as the R 2 adj , the root mean square error (RMSE) and P value.For solutes gain, in all cases the R 2 adj was higher than 0.93, RMSE was lower than 0.016 and P was less than or equal to 10 indicating the acceptability of the Z&L and Azuara models.For water loss, P value was greater than 10.
Conclusions
In all the cases, Fick's Model for finite and infinite cylinder and empirical models of Z&L and Azuara better predicted the salt and glycerol gain than the water loss, during the osmotic dehydration of mackerel cylinders in hypertonic water/salt/glycerol solution.
Three terms in the analytical solution of the Fick equation for infinite cylinder is appropriate to determine the diffusion coefficients.
The model based on the solution of Fick's Law for an infinite cylinder was the best fit for water loss and solutes gain.The equilibrium values estimated with Azuara model agree with that determined experimentally.
Using calculated effective diffusivities of water, salt and glycerol, the water and solutes distributions into mackerel cylinder samples were predicted.The changes of water, glycerol and salt were mainly confined in the region of the surface and slowly progresses to the interior.At the end of the osmotic dehydration, the water, glycerol and salt in the mackerel cylinder reached the equilibrium with osmotic solution.
Figure 1 .
Figure 1.Fit of experimental data to Fick´s Models error, x i experimental value and x pi predicted value.
Figure 3 .
Figure 3. Fit of experimental data to Azuara and Z&L Models
Table 3 .
Parameters of Z&L and Azuara modelsValidation of models is shown in Figure3, as it can be seen, the water loss as well as solutes gain increased with time and the predicted values are very close to the experimental values.Comparison of experimental and predicted equilibrium values is shown in Table4.As can be seen in this table, Z&L model underestimates the equilibrium of water, salt and glycerol contents.On the other hand, the equilibrium values predicted by the Azuara model agree with the experimental ones according with the lower values of E. | v3-fos-license |
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} | pes2o/s2orc | Bioprinting Perfusion-Enabled Liver Equivalents for Advanced Organ-on-a-Chip Applications
Many tissue models have been developed to mimic liver-specific functions for metabolic and toxin conversion in in vitro assays. Most models represent a 2D environment rather than a complex 3D structure similar to native tissue. To overcome this issue, spheroid cultures have become the gold standard in tissue engineering. Unfortunately, spheroids are limited in size due to diffusion barriers in their dense structures, limiting nutrient and oxygen supply. Recent developments in bioprinting techniques have enabled us to engineer complex 3D structures with perfusion-enabled channel systems to ensure nutritional supply within larger, densely-populated tissue models. In this study, we present a proof-of-concept for the feasibility of bioprinting a liver organoid by combining HepaRG and human stellate cells in a stereolithographic printing approach, and show basic characterization under static cultivation conditions. Using standard tissue engineering analytics, such as immunohistology and qPCR, we found higher albumin and cytochrome P450 3A4 (CYP3A4) expression in bioprinted liver tissues compared to monolayer controls over a two-week cultivation period. In addition, the expression of tight junctions, liver-specific bile transporter multidrug resistance-associated protein 2 (MRP2), and overall metabolism (glucose, lactate, lactate dehydrogenase (LDH)) were found to be stable. Furthermore, we provide evidence for the perfusability of the organoids’ intrinsic channel system. These results motivate new approaches and further development in liver tissue engineering for advanced organ-on-a-chip applications and pharmaceutical developments.
Introduction
Engineering tissues for in vitro organ models has always been a challenge. The creation of complex 3D tissues is motivated by the fact that 3D cell cultures commonly result in biology closely representing native tissues [1]. Nevertheless, complex tissues require cultivation conditions of elevated complexity, such as perfusion of the organoid in bioreactors like multi-organ-chips [2]. In order to empower their full potential, organoids can, not only be cultivated in monocultures resembling specific cell niches [3], but can also be combined in multi-organ cultures for metabolic and systemic studies [4]. Recent advances in bioprinting technology have enabled us to push the development of complex 3D tissues for in vitro applications even further, by setting up microfluidic channels within the printed organ equivalents for perfusion and the possibility of vascularization.
There are many different bioprinting techniques that have introduced additive manufacturing technology to the field of tissue engineering [5]. Among laser-induced-forward-transfer (LIFT) [6] and extrusion-based bioprinting [7], stereolithographic bioprinting combines many advantages and features the preparation of high-resolution hydrogels [8] with detailed architectures and embedded vascularization, supplying cell-laden tissues for long-term cultivation [9][10][11][12]. Current research pushes the trends and limitations of bioprinting technology [13], implementing solid-freeform manufacturing, formerly referred to as a tool for rapid-prototyping [14,15], into cell culture laboratories for tissue manufacture [16,17]. For the successful printing of tissue models, not only progress in 3D printing technology, but also the development of suitable bioinks, are necessary. Gelatin-based bioinks show great promise for embedding cells and creating suitable conditions for the proliferation and differentiation of many cell types [18][19][20]. By customizing bioink properties, specific cell-biomatrix interactions, such as cell migration [21] or directional matrix degradation [22], are controllable.
In view of these developments, many tissue models have already been engineered for in vitro cell culture applications [23,24]. Ma et al. showed higher liver-specific gene expression levels and increased metabolic product secretion in a 3D bioprinted liver model consisting of human induced pluripotent stem cell (hiPSC)-derived hepatic progenitor cells, human umbilical vein endothelial cells, and adipose-derived stem cells. Furthermore, their model found both phenotypic and functional enhancements in comparison to 2D monolayer culture [25]. Tsang et al. reported the fabrication of 3D hepatic tissues by additive photopatterning of modified polyethylenglycol (PEG) hydrogels, whereas bulk and patterned hydrogels were compared, showing favorable performance in a continuous flow bioreactor over a 12 day cultivation period [26]. In addition, Lewis et al. reported architecture-dependent hepatic quality in a 3D printed gelatin scaffold. They found increased albumin secretion, CYP activity (CYP3A4 and CYP2C9), and bile transport in interconnected scaffolds compared to different geometries and 2D controls [27]. Other liver tissue models involve polyelectrolyte multilayer templates to combine primary hepatocytes and fibroblasts in a patterned co-culture. This approach results in controllable cell-cell and cell-surface interactions, as reported by Kidambi et al. [28]. In a similar approach, Puttaswamy et al. arranged HepG2 cells in a hexagonal liver lobule like structure, in which cells remained viable despite the application of an electric field for cell manipulation and positioning [29]. Using an enhanced field-induced dielectrophoresis trap, Ho et al. combined hepatic and endothelial cells, mimicking the morphology of liver lobule tissue with about 95% cell viability. Furthermore, an 80% enhancement of CYP1A1 activity was reported compared to non-patterned pure HepG2 cells [30]. Khetani et al. showed that tissue function depends on hierarchical structures extending from single cells to functional subunits that coordinate organ functions. In their multiwell culture system for human liver cells with optimized microscale architectures, phenotypic functions were maintained for several weeks. Their results emphasized the combination of different technologies to advance tissue engineering for 'human-on-a-chip' applications [31].
Here, we present a bioprinted tissue equivalent, representing the smallest functional unit of the liver. The printed lobule consists of a hexagonal structure possessing twelve channels running from the model edges to the central port. Channels are open at both sides, so that fluids can perfuse the complete construct.
In order to provide basic characterization of the printed liver equivalents, we chose standard tissue engineering analyses, including quantitative PCR, immunohistochemistry (IHC) staining and metabolic activity assays. Different protein targets were defined to classify the quality of the printed tissue compared to HepaRG cells cultivated in monolayer. We focused on albumin, cytochrome P450 3A4 (CYP3A4), cytokeratin 8/18 (Ck8/18), vimentin, multidrug resistance-associated protein 2 (MRP2) and zonula occludens-1 (ZO-1) expression. Albumin indicates the maturity of hepatocytes, whereby it is crucial to verify the presence of the protein using IHC [32]. Cytokeratin 8/18 is expressed by hepatocytes with proliferative capacity, which contribute to the tissues' regenerative potential [33]. For toxicity screenings, the activity of the Cytochrome P450 enzymes is fundamental. Since CYP3A4 is one of the most important enzymes of that family, analyses were focused on its expression in order to provide a proof-of-concept for cytochrome activity in the printed liver equivalent [34,35]. One of the liver's main functions is the conversion and transportation of substances. Thereby, the liver secretes bile, which is transported inter-and intracellularly by specific transporters. We, therefore, also investigated the expression of the MRP2, actively controlling bile in-and out-fluxes [36]. Functional hepatocytes require close cell-cell interactions through tight junctions, which are represented by peripheral membrane proteins, such as ZO-1 [37]. As the printed liver equivalents, not only contain HepaRGs, but also stellate cells, we chose vimentin, a protein of mesenchymal origin [38], to visualize cell distribution within the tissue.
Cell Culture
Cell culture components were purchased from Corning and cultures were incubated in HepaRG medium at 37 • C and 5% CO 2 , unless otherwise stated. HepaRG cells were obtained from Biopredic International (Rennes, France) and maintained as described by Gripon et al. [39]. Briefly, cells were cultured in HepaRG medium, consisting of William's Medium E supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units mL −1 penicillin, 100 µg mL −1 streptomycin, 5 µg mL −1 human insulin, 2 mM l-glutamine, and 5 × 10 −5 M hydrocortisone hemisuccinate (Sigma-Aldrich, St. Louis, MO, USA). Undifferentiated cells were maintained in 75 cm 2 tissue culture flasks (Greiner Bio One, Solingen, Germany) at a seeding density of 2 × 10 4 cells cm −2 for two weeks. Induction of differentiation was initiated by allowing the cells to reach confluence by maintaining the cells in a growth medium for two weeks. Differentiation medium containing 2% (v/v) dimethyl sulfoxide (DMSO; Carl Roth GmbH, Karlsruhe, Germany) was added for another two weeks. Human hepatic stellate cells (SteCs) and their culture supplements were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). The cells were seeded at 5 × 10 3 cells cm −2 in 75 cm 2 tissue culture flasks in stellate cell medium for maintenance, according to the manufacturer's instructions. Medium was exchanged every three days. Cells were harvested for further use at 90% confluence.
Bioink Preparation
For stereolithographic printing, two bioinks based on gelatin and PEG were used. Both bioinks were synthesized, as previously shown [40,41]. In short, 10 wt % gelatin (porcine skin Type B, Sigma) was dissolved in phosphate buffered saline (PBS) at 50 • C. After, 20-fold molar excess methacrylic anhydride (Sigma) was added and the reaction continued for 3 h. After reaction, the product (GelMA) was dialyzed against distilled water. Products were freeze dried and lyophilized for precise bioink preparation. Degradable PEG-bis-(acryloyloxy acetate) was synthesized in a two-step reaction. First, PEG-bis-chloroacetate was synthesized by reacting PEG (Sigma) with chloroaceryl chloride (Sigma). In the second step, acrylic groups were added by reacting the product with sodium acrylate (Sigma). Products were recovered by precipitation in cold ethylether (Sigma), dialyzed against distilled water and freeze-dried for long-term storage. The photoinitiator lithium phenyl-2,4,6-trimethylbenzoy phosphinate was used at 0.1 wt % in all bioinks. For the bioprinting process, cells were mixed with bioink-solutions containing the photoinitiator to form a bioink cell suspension ready for photopolymerization.
Tissue Model and Printing Process
The liver equivalents are designed with hollow channels to allow for perfusion of the organoid. Within the hexagonal construct, there were twelve channels running from the model edges to a central port. The channels were open at both sides ( Figure 1a). The printed liver model consisted of two materials. Channels were printed with degradable PEG at 7 wt %. For the cell-containing structures (Figure 1a, in grey), HepaRGs were harvested, mixed with SteCs 24:1 and resuspended in GelMA, thereby forming the main bioink at 7% (w/v), possessing a cell density of 10 × 10 7 cells/mL. A model mimicking the sinusoidal structure of the liver lobule was designed with a diameter of 4 mm, using computer-aided design (CAD) software (Rhinoceros 5, McNeel Europe, Barcelona, Spain). The CAD file was processed using a Cellbricks Bioprinter and was printed, layer-by-layer. as shown in Figure 1b. During printing, the bioink was changed automatically according to the material and cells used for the current structure.
Genes 2018, 9, x FOR PEER REVIEW 4 of 15 CAD file was processed using a Cellbricks Bioprinter and was printed, layer-by-layer. as shown in Figure 1b. During printing, the bioink was changed automatically according to the material and cells used for the current structure. During the printing process, each layer of the tissue construct was photopolymerized directly onto the print head using blue light illumination for 30 s per layer. Stereolithographic printing technology enabled us to manufacture six tissue models, in parallel, during one printing process. Each model contained a total of 10 6 cells. After printing, the constructs were detached from the bioprinter and placed in a 24-well plate filled with 1 mL cell culture medium for cultivation. Tissue constructs were incubated at 37 °C and 5% CO2 over 14 days of cultivation time. Two time points, directly after printing and 14 days after printing, were chosen for analysis. Medium changes were performed every day.
qPCR
Gene expression of albumin, CYP3A4, ZO-1 and MRP2 were analyzed by qPCR, which was performed directly after printing, on day zero, as a control, and after 14 days of cultivation. RNA isolation was performed using the NucleoSpin ® RNA isolation Kit (Macherey-Nagel, Düren, Germany) by harvesting the tissue model in RA1 buffer containing 1% β-mercaptoethanol according to the manufacturer's protocols. A total of 150 ng of RNA was reverse transcribed into cDNA with the TaqMan ® kit (Applied Biosystem, Foster City, CA, USA), for each sample. Quantitative RT-PCR experiments were conducted according to the manufacturer's protocols using the Stratagene system During the printing process, each layer of the tissue construct was photopolymerized directly onto the print head using blue light illumination for 30 s per layer. Stereolithographic printing technology enabled us to manufacture six tissue models, in parallel, during one printing process. Each model contained a total of 10 6 cells. After printing, the constructs were detached from the bioprinter and placed in a 24-well plate filled with 1 mL cell culture medium for cultivation. Tissue constructs were incubated at 37 • C and 5% CO 2 over 14 days of cultivation time. Two time points, directly after printing and 14 days after printing, were chosen for analysis. Medium changes were performed every day.
qPCR
Gene expression of albumin, CYP3A4, ZO-1 and MRP2 were analyzed by qPCR, which was performed directly after printing, on day zero, as a control, and after 14 days of cultivation. RNA isolation was performed using the NucleoSpin ® RNA isolation Kit (Macherey-Nagel, Düren, Germany) by harvesting the tissue model in RA1 buffer containing 1% β-mercaptoethanol according to the manufacturer's protocols. A total of 150 ng of RNA was reverse transcribed into cDNA with the TaqMan ® kit (Applied Biosystem, Foster City, CA, USA), for each sample. Quantitative RT-PCR experiments were conducted according to the manufacturer's protocols using the Stratagene system (Agilent Technologies, Waldbronn, Germany) with a SensiFast SYBR No-ROX One-Step kit (Bioline, Luckenwalde, Germany). Used primers and their sequences are presented in Table 1. Cycle threshold and melting curves were determined using LightCycler software and results were processed using the 2-∆∆Ct method for relative gene expression analysis [42,43]. Changes in gene expression were normalized using the TATA-Box binding protein (TBP) as a housekeeping gene. For each time point, six tissue samples were taken. Monolayer cultures were measured with n = 10. Statistical analyses, such as the unpaired t-test, were performed in Prism 7 (Graphpad Software, La Jolla, CA, USA).
Immunohistochemistry
For immunohistological analysis, constructs were embedded in Tissue-Tek ® (Sakura, The Netherlands), frozen in liquid nitrogen and cryosectioned at a thickness of 10 µm. Sections were fixed in acetone at −20 • C for 10 min. After washing in PBS and blocking in 10% goat serum for 20 min, immunostaining was performed using primary antibodies (ThermoFisher, Waltham, MA, USA) targeting albumin, CYP3A4, ZO-1, MRP2, vimentin and cytokeratin 8/18 at 4 • C overnight. Secondary antibodies (goat-anti-mouse and goat-anti-rabbit CF594, Biotium, Fremont, CA, USA) were incubated for 45 min at room temperature with 1:5000 DAPI for cell nuclei staining. Afterwards, coverslips with mounting solution were added to seal the staining. Fluorescent microscopy was performed using a Biorevo BZ-9000 (Keyence, Osaka, Japan). Viability was determined using TUNEL/Ki67 double staining, using the Apo-Direct Apoptosis Detection Kit (eBioscience, San Diego, CA, USA), according to the manufacturer's protocols, in combination with Ki67 antibody (eBioscience, San Diego, CA, USA).
Metabolic Analysis
Daily medium samples were taken for the determination of glucose-, lactate-and lactate dehydrogenase (LDH)-content. Absorbance-related measurements were performed in 384-well plates (Greiner Bio-One, Solingen, Germany) in a microplate-reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). LDH activity in the medium was measured using a Cytotoxicity Detection Kit PLUS (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocols. The average absorbance per minute (∆A/min), at 450 nm, was determined over three minutes using medium as a reference. As a positive control, samples were treated with 0.1% Triton X-100 for two hours and supernatants were analyzed. Daily glucose consumption was measured with the Glucose Kit Glu 142 (Diaglobal, Berlin, Germany) according to manufacturer's protocols, using medium as standard. Lactate concentration was screened in daily medium supernatants using a Lactate Kit Lac 142 (Diaglobal), according to the manufacturer's protocols. Absorbance was measured at 520 nm using standards (10 mM/mL) as a reference.
Morphological Analysis after Printing
The printed liver equivalents had a diameter of 4 mm from edge to edge. This size was verified in all printed constructs. A mean of 4053.6 µm with a standard deviation of 67.2 µm was determined (based on 30 samples). Multiple tissue constructs printed at the same time displayed high equality in morphology and cell distribution. Cells were found within the printed matrix at a high density and with a homogenous distribution (Figure 2a). The channel-structure was defined by the sacrificial matrix, as shown in Figure 2a. The channels had a diameter of approx. 200 µm, with a standard deviation of under 10 µm, as shown in Figure 2a,b. Immediately after printing, some cells were loosely attached to the model edges. After washing, these cells were removed and only cells that were truly incorporated within the bioink remained. After three days in culture, the PEG was fully degraded, as seen in Figure 2, by focusing on the edges (Figure 2b) and the bottom (Figure 2c) of the channel. Furthermore, we were able to flush the complete channel system, thus demonstrating the organoids' suitability for cultivation under perfusion (Figure 2d,e and Supplementary Video S1: Perfusion of 3D bioprinted liver equivalent.).
Genes 2018, 9, x FOR PEER REVIEW 6 of 15 (based on 30 samples). Multiple tissue constructs printed at the same time displayed high equality in morphology and cell distribution. Cells were found within the printed matrix at a high density and with a homogenous distribution (Figure 2a). The channel-structure was defined by the sacrificial matrix, as shown in Figure 2a. The channels had a diameter of approx. 200 µm, with a standard deviation of under 10 µm, as shown in Figure 2a,b. Immediately after printing, some cells were loosely attached to the model edges. After washing, these cells were removed and only cells that were truly incorporated within the bioink remained. After three days in culture, the PEG was fully degraded, as seen in Figure 2, by focusing on the edges (Figure 2b) and the bottom (Figure 2c) of the channel. Furthermore, we were able to flush the complete channel system, thus demonstrating the organoids' suitability for cultivation under perfusion (Figure 2d,e and Supplementary Video S1: Perfusion of 3D bioprinted liver equivalent.).
Viability after Bioprinting
Viability was confirmed by TUNEL/Ki67 staining (Figure 3). Printed liver equivalents showed high viability throughout the cultivation time of 14 days. Ki67-positive, proliferative cells were observed at day zero and day 14, whereas TUNEL-positive, apoptotic cells could only be observed in the positive control treated with Triton X-100. Less proliferative cells were observed at day 14 compared to day zero, right after printing.
Viability after Bioprinting
Viability was confirmed by TUNEL/Ki67 staining (Figure 3). Printed liver equivalents showed high viability throughout the cultivation time of 14 days. Ki67-positive, proliferative cells were observed at day zero and day 14, whereas TUNEL-positive, apoptotic cells could only be observed in the positive control treated with Triton X-100. Less proliferative cells were observed at day 14 compared to day zero, right after printing.
Glucose, Lactate and LDH Metabolics
Glucose consumption was observed to be higher at the beginning of cultivation, measuring around 0.15 mg to 0.3 mg glucose consumption per day (Figure 4a). Complete medium changes with a maximal glucose concentration of 2 mg per culture were performed every day. Cells treated with Triton X-100 consumed more than 1.95 g/L glucose per day. LDH levels were observed to be far below levels of the positive control, indicating viable cells throughout the 14 day of cultivation. LDH levels started at about 100 mU/mL in the first two days of cultivation, but declined and stabilized around 70 mU/mL in the following days ( Figure 4). Tissue constructs treated with Triton X-100 represented the positive control for apoptotic cells. In these samples, LDH levels rose to 830 mU/mL. Lactate content was found to be between 3.7 and 6.4 mmol/mL. Similar to observations made for LDH, these levels were elevated in the first two days of cultivation, dropping down to an average lactate concentration of 4.5 mmol/L (Figure 4). Lactate content in pure culture medium without cells was found to be 3 mmol/L (data not shown).
Glucose, Lactate and LDH Metabolics
Glucose consumption was observed to be higher at the beginning of cultivation, measuring around 0.15 mg to 0.3 mg glucose consumption per day (Figure 4a). Complete medium changes with a maximal glucose concentration of 2 mg per culture were performed every day. Cells treated with Triton X-100 consumed more than 1.95 g/L glucose per day. LDH levels were observed to be far below levels of the positive control, indicating viable cells throughout the 14 day of cultivation. LDH levels started at about 100 mU/mL in the first two days of cultivation, but declined and stabilized around 70 mU/mL in the following days ( Figure 4). Tissue constructs treated with Triton X-100 represented the positive control for apoptotic cells. In these samples, LDH levels rose to 830 mU/mL. Lactate content was found to be between 3.7 and 6.4 mmol/mL. Similar to observations made for LDH, these levels were elevated in the first two days of cultivation, dropping down to an average lactate concentration of 4.5 mmol/L (Figure 4). Lactate content in pure culture medium without cells was found to be 3 mmol/L (data not shown).
qPCR Marker Expression
The following gene expression results for 3D printed liver equivalents and for HepaRG monolayer cultures are summarized in Figure 5. In printed liver equivalents, albumin expression was found to rise significantly from day zero to day 14 (Figure 5a). Monolayers showed the opposite effect; a significant drop in albumin expression from day zero to day 14 was detected. Overall, albumin expression levels were higher in 3D bioprinted liver equivalents than in HepaRG monolayers. Similar results were observed in terms of CYP3A4 expression. Gene expression in the bioprinted tissue constructs appeared to be, on average, 150-fold higher on day 14 compared to day zero. Monolayers showed a significant drop in CYP3A4 expression from day zero, right after maturation, to day 14, two weeks in culture after maturation. Overall CYP3A4 expression appeared to be more than seven times higher in 3D bioprinted tissues compared to monolayer cultures at their highest expression levels (monolayer: Day zero; prints: Day 14) (Figure 5b). The tight junction protein ZO-1 was expressed in both 3D tissues and monolayer cultures throughout the experiments with a significant increase of expression in monolayers. Overall expression levels appeared to be similar, but were slightly higher in monolayers than they were in the printed liver equivalents at day 14 ( Figure 5c). MRP2 expression dropped slightly in bioprinted liver equivalents and expression remained stable over the two week cultivation. In contrast, the monolayer demonstrated a significant increase in MRP2 expression levels from day 0 to day 14 (Figure 5d).
qPCR Marker Expression
The following gene expression results for 3D printed liver equivalents and for HepaRG monolayer cultures are summarized in Figure 5. In printed liver equivalents, albumin expression was found to rise significantly from day zero to day 14 (Figure 5a). Monolayers showed the opposite effect; a significant drop in albumin expression from day zero to day 14 was detected. Overall, albumin expression levels were higher in 3D bioprinted liver equivalents than in HepaRG monolayers. Similar results were observed in terms of CYP3A4 expression. Gene expression in the bioprinted tissue constructs appeared to be, on average, 150-fold higher on day 14 compared to day zero. Monolayers showed a significant drop in CYP3A4 expression from day zero, right after maturation, to day 14, two weeks in culture after maturation. Overall CYP3A4 expression appeared to be more than seven times higher in 3D bioprinted tissues compared to monolayer cultures at their highest expression levels (monolayer: Day zero; prints: Day 14) (Figure 5b). The tight junction protein ZO-1 was expressed in both 3D tissues and monolayer cultures throughout the experiments with a significant increase of expression in monolayers. Overall expression levels appeared to be similar, but were slightly higher in monolayers than they were in the printed liver equivalents at day 14 ( Figure 5c). MRP2 expression dropped slightly in bioprinted liver equivalents and expression remained stable over the two week cultivation. In contrast, the monolayer demonstrated a significant increase in MRP2 expression levels from day 0 to day 14 (Figure 5d).
Immunohistochemistry
Negative controls for both the double and single staining showed no unspecific antibody interactions (Figure 6e). Immunohistochemistry is shown at day 14 for vimentin, cytokeratin 8/18, albumin (Alb), CYP3A4 and ZO-1. Vimentin and cytokeratin 8/18 were co-stained visualizing the cell distribution. Stellate cells visualized by positive staining of vimentin (Figure 6a, in green) were found to be distributed homogenously throughout the printed tissue. Some accumulation due to cell proliferation was observed at the bottom edge. Cytokeratin 8/18 was predominantly stained within the center of the organoid. Only a few cytokeratin 8/18-positive cells were found on the edges, within a dense cell layer surrounding the whole organoid. Both albumin and CYP3A4 appeared to be highly expressed ( Figure 6, albumin (b) and CYP3A4 (c)). Their expressions were found to be stronger at day 14 compared to day zero (data not shown). ZO-1 expression was observed in densely-populated areas throughout the printed tissue (Figure 6f). In each graph, the qPCR results of albumin (a), CYP3A4 (b), ZO-1 (c) and MRP2 (d) of monolayer cultures and printed liver constructs at day 14 are compared to day 0, respectively. Results showed statistically significant differences. (*** p < 0.0005; **** p < 0.0001).
Immunohistochemistry
Negative controls for both the double and single staining showed no unspecific antibody interactions (Figure 6e). Immunohistochemistry is shown at day 14 for vimentin, cytokeratin 8/18, albumin (Alb), CYP3A4 and ZO-1. Vimentin and cytokeratin 8/18 were co-stained visualizing the cell distribution. Stellate cells visualized by positive staining of vimentin (Figure 6a, in green) were found to be distributed homogenously throughout the printed tissue. Some accumulation due to cell proliferation was observed at the bottom edge. Cytokeratin 8/18 was predominantly stained within the center of the organoid. Only a few cytokeratin 8/18-positive cells were found on the edges, within a dense cell layer surrounding the whole organoid. Both albumin and CYP3A4 appeared to be highly expressed ( Figure 6, albumin (b) and CYP3A4 (c)). Their expressions were found to be stronger at day 14 compared to day zero (data not shown). ZO-1 expression was observed in densely-populated areas throughout the printed tissue (Figure 6f).
Discussion
In this study, we successfully demonstrated the stereolithographic printing principle of a complex hepatic tissue construct with an intrinsic hollow channel system. The results build a foundation for future detailed characterization and development of a functional liver model with possible applications in metabolic and toxicologic assays. The translation of a 3D digital drawing into a highly-structured, cell-laden hydrogel-based organoid was successfully performed (Figure 2). Right after printing, some cells were loosely attached to the printed model due to the high cell density within the bioinks. These cells can easily be washed away, and only the designed structure with the incorporated cells remains. Multiple tissue constructs, printed at the same time, showed a high equality in morphology and cell distribution constituting an accurate representation of the intended design for the tissue model, thus reproducible printing of organoids was demonstrated. Furthermore, apoptotic cells could only be found in the positive control after two weeks of cultivation (Figure 3). Proliferating cells were found both at day zero and after two weeks in culture demonstrating a high viability for cultivation over at least 14 days. More Ki67 positive cells were observed at day 0 compared to day 14, which indicates a loss of proliferative capacity due to cell differentiation. In this study, we compared HepaRG cells cultivated in monolayer with the same cells incorporated in the printed lobular constructs. For both cultivation methods, cells were matured over two weeks in monolayer. Afterwards one half of the cell culture was printed and cultivated in 3D, the other half remained in the monolayer culture. Unfortunately, we had to exclude stellate cells from the monolayer controls, as these cultures appeared unstable over 14-day cultivation, contracting into an elliptic random construct, whereas monolayers without stellate cells remained stable over the two week cultivation period. Both monolayer and printed tissue cultivations were performed over 14
Discussion
In this study, we successfully demonstrated the stereolithographic printing principle of a complex hepatic tissue construct with an intrinsic hollow channel system. The results build a foundation for future detailed characterization and development of a functional liver model with possible applications in metabolic and toxicologic assays. The translation of a 3D digital drawing into a highly-structured, cell-laden hydrogel-based organoid was successfully performed (Figure 2). Right after printing, some cells were loosely attached to the printed model due to the high cell density within the bioinks. These cells can easily be washed away, and only the designed structure with the incorporated cells remains. Multiple tissue constructs, printed at the same time, showed a high equality in morphology and cell distribution constituting an accurate representation of the intended design for the tissue model, thus reproducible printing of organoids was demonstrated. Furthermore, apoptotic cells could only be found in the positive control after two weeks of cultivation ( Figure 3). Proliferating cells were found both at day zero and after two weeks in culture demonstrating a high viability for cultivation over at least 14 days. More Ki67 positive cells were observed at day 0 compared to day 14, which indicates a loss of proliferative capacity due to cell differentiation. In this study, we compared HepaRG cells cultivated in monolayer with the same cells incorporated in the printed lobular constructs. For both cultivation methods, cells were matured over two weeks in monolayer. Afterwards one half of the cell culture was printed and cultivated in 3D, the other half remained in the monolayer culture. Unfortunately, we had to exclude stellate cells from the monolayer controls, as these cultures appeared unstable over 14-day cultivation, contracting into an elliptic random construct, whereas monolayers without stellate cells remained stable over the two week cultivation period. Both monolayer and printed tissue cultivations were performed over 14 days. Overall gene expression of selected markers was found to be higher in the printed tissues compared to the monolayer cultures, confirming the direct effect of the three-dimensional cultivation on the cells' biology ( Figure 5). In the printed liver equivalents, the tight junction protein ZO-1 was found to be stably expressed over 14 days of cultivation. At day 14, its standard deviation dropped to a minimum. As cells expressed tight junctions when in contact with each other, and more organoids showed a higher ZO-1 expression at day 14 compared to day 0, these results suggest some cell proliferation within the hydrogel. One of the models already showed maximal expression on day zero, maintaining this level over the entire cultivation time. Since the cells are pre-differentiated over 14 days in monolayers, detached, suspended in the bioink and then printed in high cell density, tight junction protein expression might remain high from the first day on, but this assumption requires further experiments. MRP2 expression was found to increase in monolayers, whereas there was a slight decrease in expression levels found in printed tissues ( Figure 5). In monolayer cultures, the HepaRGs proliferate until complete confluency, differentiating and forming bile ducts with transporter expression. In the printed tissues, cells are incorporated in a hydrogel at a given concentration. Thus, the cells are surrounded by the printed matrix, but not all cells interact with each other. Cells that expressed MRP2 in the confluent monolayer prior to printing might not have any cell-cell contact after printing within the gelatin hydrogel. This might explain the decrease in MRP2 expression in our printed tissues over 14 days of cultivation. An increase in bioink cell concentration in future organoids might support the expression of MRP2 as more cells stay in contact with each other, supporting the formation of bile ducts and the main bile acid transporters [36]. There was a high difference in protein expression at day zero, comparing printed tissue and monolayers, although the cells were pre-differentiated equally in both experiments. Albumin expression was found to be two-fold higher and CYP3A4 expression was found to be even 20-fold higher in monolayers, than in printed tissues, at day zero ( Figure 5). This difference can be explained by the procedure used to take the RNA samples, as different cell handling results in metabolic alterations [44]. For the monolayers, RNA samples were taken by lysing the attached cells directly from the tissue culture flask so that no changes in expression were expected. For printed tissues, cells were detached, mixed with bioink, resulting in a single-cell-suspension, and printed within the hydrogels. Right after the printing process, the tissue constructs were lysed to extract the RNA. The cells remained in suspension before the bioprinting process, thus downregulating liver-specific gene expression (albumin, CYP3A4, MRP2). Only ZO-1 was not affected by this phenomenon in all printed tissues. Nevertheless, this procedure was chosen to investigate the actual changes in gene expression from day zero to day 14. Monolayer controls lose albumin and CYP3A4 expression over the two weeks cultivation period. Usually, HepaRG functionality in monolayers is maintained by adding DMSO after the pre-differentiation. As the printed tissues are cultivated without DMSO, monolayer cultures were also kept without DMSO, resulting in a loss of hepatic function. The printed tissues, however, maintained hepatic functionality under these native conditions. Protein expression was verified by immunohistology, being in accordance with the results from the qPCR experiments ( Figure 6). Some morphological deformations were observed in cryosections due to the freezing and cutting procedure of the tissues. As some sections appeared to be squeezed in one direction (e.g., y-direction, Figure 6a) these deformations were most likely introduced by the cutting procedure. In Figure 6a,e, a part of the channel structure is visible (black linear area within the staining). Not all channels are visible at once due to the cutting angle. An accumulation of vimentin positive cells (stellate cells) was observed at the bottom edge of the tissue section ( Figure 6a). As HepaRGs and SteCs are homogenously mixed in the bioinks, and SteCs were found homogenously distributed throughout the printed constructs at day zero, this accumulation suggests the proliferation or migration of stellate cells at this spot. As cells at the edges of the printed tissues might not be fully incorporated in the printed matrix, they have more space to proliferate and populate the construct surface. Cytokeratin 8/18-positive cells within the same staining suggest some proliferative hepatocytes, representing an active tissue with regenerative capabilities.
Metabolic analyses revealed the production of small amounts of lactate (Figure 4c) in accordance with glucose consumption (Figure 4a). These results suggest oxygen limitation so that glucose is converted to lactate. The oxygen limitation might be a result of the medium amount used for cultivation. In this study, the printed tissues were cultivated in a 24-well format with 1 mL of medium. Manufacturers like Greiner Bio-one suggest using only 0.5 mL of medium in this format. The higher liquid level limits the amount of oxygen at the bottom of the culture well, where the organoid can be found [45]. In future experiments, medium levels will need to be adapted and cultivation under perfusion will help to alleviate this problem. As LDH levels appeared to stay stable after the first two days (Figure 4, center), the printed tissues stay viable. Metabolic data suggest homeostasis, as no significant increases or drops in neither glucose consumption nor lactate concentration were visible.
In future experiments, the printed liver organoid will be cultivated in a multi-organ-chip platform, which facilitates the in-and efflux of oxygen, carbon dioxide, nutrients and metabolites [46]. Co-cultivation with other organ models, such as pancreatic islets, neural tissue or skin, might pose a promising strategy in testing and optimizing the printed liver equivalents at their current state [4,47,48]. As the channel system allows for perfusion (Figure 2d,e and the supplementary video), adding endothelial cells to form tight homogenous channel walls is a crucial extension to the development of this organ model for physiologic perfusion experiments. In native tissue, substances need to pass endothelial barriers before reaching biological active hepatocytes [49], thus, the addition of endothelial cells will support hepatic polarization, leading to a higher biological activity and a more detailed physiology [50,51].
Conclusions
In this study, a complex liver organoid was precisely printed using a stereolithographic bioprinting approach. We were able to print a hollow channel system within the cell-laden hydrogel. The printed liver tissue equivalents were found to have higher albumin and CYP3A4 expression over a two week cultivation period, when compared to monolayer controls. Tight junction protein ZO-1 and MRP2 expression remained stable in the printed tissue. However, monolayer controls showed an increase in the expression of these genes, so there still is potential to adapt cell densities within the printed organoids. In its current state, we found that the printed liver organoid has great potential for future lab-on-a-chip and organ-on-a-chip applications, as medium can flow through the channel system within the tissue model, preparing it for cultivation under perfusion. We successfully established the stereolithographic printing technology, thus enabling development of the model. Now that we demonstrated the feasibility of the printing principle, detailed analyses of the major enzymes of the cytochrome P450 family are crucial to fully characterize the model for applications in metabolic and toxicology assays. Furthermore, the application of potential toxic substances will give insight into the enzyme kinetics and overall organoid performance. As our printing technology is constantly being developed, we aim to incorporate endothelialized channels in the future to support physiologic hepatic polarization for long-term toxicity screenings. The organoid was established using HepaRG cells, but, as the presented bioprinting technique it is not limited to cell lines, we suggest the integration of iPSCs or a primary material for potential personalized medicine applications.
Acknowledgments: This work was partially funded by the Federal Ministry for Economic Affairs and Energy funding program EXIST Transfer of Research funding 03EFEBE077. T.G., A.T. are employees of Cellbricks GmbH engineering human tissue models via bioprinting technology. Lutz Kloke and Roland Lauster declare no conflict of interest.
Author Contributions: T.G. and L.K. conceived and designed the experiments. T.G. and A.R. performed and analyzed the data supported by A.T., A.-K.A. checking experimental design and implementation. B.P.N. contributed reagents and materials used in this study. R.L. provided laboratories and access to research equipment. T.G. wrote the paper under the supervision of his co-authors. | v3-fos-license |
2020-04-23T09:07:53.543Z | 2020-04-01T00:00:00.000 | 216075623 | {
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} | pes2o/s2orc | Cytotoxicity of the Sesquiterpene Lactone Spiciformin and Its Acetyl Derivative against the Human Leukemia Cell Lines U-937 and HL-60.
Spiciformin (1) is a sesquiterpene lactone with a germacrane skeleton that is found in two Tanacetum species endemic to the Canary Islands. In this study, the cytotoxicities of 1 and its acetyl derivative (2) were evaluated against human tumor cells. These sesquiterpene lactones were cytotoxic against human acute myeloid leukemia (U-937 and HL-60) cells, even in cells over-expressing the pro-survival protein Bcl-2, but melanoma (SK-MEL-1) and human mononuclear cells isolated from blood of healthy donors were more resistant. Both compounds are apoptotic inducers in human leukemia U-937 cells. Cell death was mediated by the processing and activation of initiator and effector caspases and the cleavage of poly(ADP-ribose) polymerase, and it was blocked by a broad-spectrum caspase inhibitor and (in the case of sesquiterpene lactone 2) by the selective caspase-3/7, -8, and -9 inhibitors. In addition, certainly in the case of compound 2, this was found to be associated with a decrease in mitochondrial membrane potential, downregulation of the anti-apoptotic protein Bcl-2, activation of the mitogen-activated protein kinases signaling pathway, and generation of reactive oxygen species. It will, therefore, be relevant to continue characterization of this class of compounds.
Introduction
The discovery of new anticancer agents is of great interest since the currently available drugs against cancer exhibit several critical problems, including serious adverse effects, insufficient effectiveness, and the development of multidrug resistance [1]. Acute myeloid leukemia is a lethal form of hematologic malignancy and is the most usual type of acute leukemia in adults and accounts for 15%-20% of all tumors diagnosed in children aged 1 to 14 years [2]. Despite recent advances to treat and cure acute myeloid leukemia, mortality rates are still very high [3].
Many small molecules approved as anticancer drugs are based on natural products [4]. Sesquiterpene lactones are naturally occurring compounds formed from the condensation of three isoprene units and contain one or more lactone rings. A large number of sesquiterpene lactones exhibit cancer cell cytotoxicity, which depends on the presence of the α-methylene-γ-lactone moiety. This functional group acts as an alkylating agent in a Michael-type reaction with biomolecules involved in survival and cell death. It has been shown that sesquiterpene lactones inhibit nuclear factor κB, phosphatidylinositol-3-kinase, Janus kinase/signal transducer, and activator of transcription and mitogen-activated protein kinase signaling, as well as epigenetic factors such as DNA methyltransferase 1 and histone deacetylase 1, together with activation of c-Jun N-terminal kinases and reactive oxygen species generation (reviewed in Reference [6]). The effect of sesquiterpene lactones on multiple targets triggers the blocking of cancer development and progression and might sensitize cancer cells to conventional chemotherapy. Interest in this kind of natural product has emerged mainly because they are selective toward tumor and cancer stem cells [7].
Several sesquiterpene lactones have been evaluated in cancer clinical trials [8]. Specific sesquiterpene lactones are apoptotic inducers in different types of cancer cells [9]. This type of regulated cell death is characterized by cytoplasmic shrinkage, plasma membrane blebbing, phosphatidylserine exposure at the cell surface, loss of mitochondrial membrane potential, chromatin condensation, nuclear fragmentation, and formation of apoptotic vesicles [10,11]. It can occur with or without the activation of a class of aspartate-specific cysteine proteases known as caspases [12][13][14]. Two main apoptotic pathways have been described, namely the extrinsic and intrinsic pathways [15]. The intrinsic or mitochondrial pathway is controlled by pro-apoptotic and anti-apoptotic proteins of the B-cell lymphoma 2 (Bcl-2) family and involves mitochondrial outer membrane permeabilization, which promotes the cytosolic release of apoptogenic factors including cytochrome c [16]. Cytosolic cytochrome c is involved in the assembly of apoptosome and pro-caspase-9 stimulation, which activates the executioner caspase-3 and -7. The death receptor or extrinsic pathway is mediated by plasma membrane receptors and induces activation of caspase-8, which mainly activates caspase-3 [17,18]. In addition, most of the anticancer drugs trigger apoptosis induction in malignant cells and reactivation of this pathway is critical for more effective treatments [19].
Two Tanacetum species endemic of the Canary Islands, Tanacetum ptarmiciflorum and T. ferulaceum var. latipinnum contain spiciformin (1) (Figure 1), a sesquiterpene lactone with a germacrane skeleton [20]. Its potential antiproliferative activity against human cancer cells has not been investigated to date. The justification for studying the cytotoxic effects of spiciformin against human cancer cells is two-fold: (1) it has the same skeleton as parthenolide, a sesquiterpene lactone with potent anti-cancer and anti-inflammatory activity, and (2) its chemical structure allows the addition of new substituents to improve its cytotoxicity. The present study explores the potential cytotoxicity of spiciformin (1) and its acetyl derivative (2) against human tumor cells and their underlying mechanisms of cell death, including the disruption of mitochondrial membrane potential, the activation of the caspase cascade and the mitogen-activated protein kinase pathway, the changes in the Bcl-2 family proteins expression, and the generation of reactive oxygen species.
Spiciformin Acetate (2) is More Cytotoxic Than Spiciformin (1) Against Human Tumor Cells
In the present study, the effects of the naturally occurring spiciformin (1), an epoxylated germacranolide, and its acetyl derivative (2) on the growth of human acute myeloid leukemia (U-937 and HL-60) and melanoma (SK-MEL-1) cells were evaluated. Spiciformin acetate (2) was synthesized and evaluated for cytotoxicity against human tumor cells since its higher hydrophobicity can facilitate diffusion through the plasma membrane and, therefore, enhance cytotoxicity in vitro. No
Spiciformin Acetate (2) is More Cytotoxic Than Spiciformin (1) Against Human Tumor Cells
In the present study, the effects of the naturally occurring spiciformin (1), an epoxylated germacranolide, and its acetyl derivative (2) on the growth of human acute myeloid leukemia (U-937 and HL-60) and melanoma (SK-MEL-1) cells were evaluated. Spiciformin acetate (2) was synthesized and evaluated for cytotoxicity against human tumor cells since its higher hydrophobicity can facilitate diffusion through the plasma membrane and, therefore, enhance cytotoxicity in vitro. No studies have yet addressed the cytotoxicity of these germacranolides against human acute myeloid leukemia and melanoma cell lines.
To evaluate the potential antiproliferative effect of both sesquiterpene lactones, cells were treated with increasing concentrations and viability was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay. These compounds were found to inhibit the cell viability in a concentration-dependent manner and displayed cytotoxic effects against the human leukemia cell lines (Figure 2a). The IC 50 (concentrations inducing a 50% inhibition of cell growth) values for spiciformin (1) were similar in both leukemia cells (IC 50 values of ca. 5 µM), while in SK-MEL-1 the IC 50 value was approximately four-fold higher ( Table 1). The human tumor U-937 cells were more sensitive than SK-MEL-1 melanoma cells to the cytotoxic effects of spiciformin (1). The IC 50 values for spiciformin acetate (2) were lower than spiciformin (1) in all cell lines assayed, except in HL-60 cells. In these experiments, etoposide, which is an agent commonly used for the treatment of acute leukemia, was included as a positive control. The IC 50 values of etoposide were 1.2 ± 0.4 µM, 0.4 ± 0.1 µM, and 10 ± 4 µM, for U-937, HL-60, and SK-MEL-1, respectively. Over-expression of the pro-survival protein Bcl-2 did not suppress the cytotoxicity induced by sesquiterpene lactones 1 and 2 since the IC 50 values were similar for U-937 and U-937/Bcl-2 cells ( Figure 2b, Table 1). This result is very interesting since Bcl-2 protein confers resistance to apoptosis by antagonizing the mitochondrial outer membrane permeability and exerts anti-apoptotic functions through the binding of pro-apoptotic members of the Bcl-2 family [21].
Treatment with both sesquiterpene lactones induced profound morphological changes as well as a marked reduction in the number of cells. Representative images obtained with an inverted phase-contrast microscope of U-937 cells treated with increasing concentrations of spiciformin (1) and spiciformin acetate (2) for 24 h are shown in Figure 2c. It was also investigated whether both sesquiterpene lactones were likewise cytotoxic for human mononuclear cells isolated from peripheral blood of healthy donors (PBMC). As shown in Figure 2d, a pronounced reduction was detected in the proliferation of U-937 cells while quiescent PBMC showed higher resistance than leukemia cells, even at 30 µM sesquiterpene lactones. The IC 50 values determined at 24 h for 1 and 2 were 30.4 ± 3.6 µM and 41.0 ± 3.8 µM against PBMC, while the IC 50 values against U-937 cells were 10 ± 4.0 µM and 12.2 ± 2.6 µM, respectively. Interestingly, the IC 50 values in U-937 cells for both compounds were similar at 24 h. These results suggest that both sesquiterpene lactones affected viability of peripheral blood mononuclear cells (PBMC) to a lesser extent than U-937 cells. Control experiments with the fibroblast-like Vero cell line showed no appreciable toxicity even at 100 µM spiciformin or spiciformin acetate for 24 h. As a positive control, doxorubicin (3 µM) was included in the experiment and there was a 50% decrease in the viability of these cells (Figure 2e). Future studies addressing the selectivity and efficacy of in vivo concentrations of these compounds are necessary to determine their potential for human health.
percentage of hypodiploid cells (i.e., sub-G1 fraction) increased about 10-fold and 14-fold in cells treated with compounds 1 and 2, respectively, compared with control cells after 24 h exposure at a concentration of 30 µM. Representative histograms obtained after treatment with both sesquiterpene lactones and subjected to flow cytometric analysis of nuclei stained with propidium iodide are shown ( Figure 2h). These results indicate that sesquiterpene lactones 1 and 2 induce apoptosis in human U-937 cells.
Spiciformin (1) and Spiciformin Acetate (2) Induce Apoptosis in Human Acute Myeloid Leukemia Cells
To elucidate whether growth inhibition mediated by sesquiterpene lactones was caused by apoptosis, fluorescence microscopy using Hoechst 33258 staining and flow cytometry experiments were performed in U-937 cells. To this end, cells were treated with 30 µM of compounds for 24 h, and morphological aspects were visualized by fluorescent microscopy. As shown in Figure 2f, control cells showed round nuclei with uncondensed and dispersed chromatin, while treated cells with sesquiterpene lactones showed fragmented and condensed nuclei. The evaluation of the number of annexin V-fluoresceine isothiocyanate (FITC) positive cells by flow cytometry revealed that the percentage of apoptosis increased about five and nine times in cells treated with 10 µM sesquiterpene lactones 1 and 2 for 24 h, respectively ( Figure 2g). In accordance with the annexin V-FITC studies, the percentage of hypodiploid cells (i.e., sub-G 1 fraction) increased about 10-fold and 14-fold in cells treated with compounds 1 and 2, respectively, compared with control cells after 24 h exposure at a concentration of 30 µM. Representative histograms obtained after treatment with both sesquiterpene lactones and subjected to flow cytometric analysis of nuclei stained with propidium iodide are shown ( Figure 2h). These results indicate that sesquiterpene lactones 1 and 2 induce apoptosis in human U-937 cells.
Spiciformin (1) and Spiciformin Acetate (2) Induce Cell Death by a Caspase-Dependent Pathway
To determine whether compounds 1 and 2 activate the caspase cascade, U-937 cells were incubated in the absence or presence of both sesquiterpene lactones and poly(ADP-ribose) polymerase (PARP), as well as the initiator (caspase-8 and -9) and executioner (caspase-3 and -7) caspases were determined by immunoblotting using specific antibodies. Dose-response and time-course experiments revealed that both compounds induce PARP cleavage and the processing of pro-caspases-3, -7, and -9 ( Figure 3a,b). The processing of pro-caspase-8 (detected as a reduction of this zymogen) was evident even with the lower concentration of compound 2 (10 µM) and PARP cleavage was detected at concentrations as low as 10 µM of each sesquiterpene lactone (Figure 3a). Caspase-4, which is involved in the endoplasmic reticulum stress, was also cleaved in U-937 cells treated with 1 and 2. In these experiments, Ponceau S staining prior to antibody detection was used as an alternative loading control.
Enzymatic assays were also performed to confirm caspase activation. To this end, U-937 cells were incubated in the presence of 30 µM sesquiterpene lactones 1 and 2 for 24 h and cell lysates were analyzed for cleavage of the tetrapeptide substrates DEVD-pNA, IETD-pNA, and LEHD-pNA as specific substrates of caspase-3/7, -8 and -9, respectively. The results revealed that both sesquiterpene lactones stimulate the activity of the initiator caspases, caspase-8 and -9, as well as the effector caspases-3/7. In addition, compound 2 was a more potent caspase activator than compound 1 (Figure 3c). To determine whether caspases are involved in sesquiterpene lactones-induced cell death, it was investigated the effects of the pancaspase inhibitor Z-VAD-FMK, as well as the following selective caspase inhibitors: the caspase-3/7 inhibitor Z-DEVD-FMK, the caspase-4 inhibitor Ac-LEVD-CHO, the caspase-8 inhibitor Z-IETD-FMK, and the caspase-9 inhibitor Z-LEHD-FMK. Pretreatment of cells with Z-VAD-FMK completely blocked the increase in the percentage of sub-G 1 cells (Figure 3d Taken together, these results demonstrate that both sesquiterpene lactones induce apoptosis by a mechanism dependent on caspase since cell death was completely inhibited by the broad-spectrum caspase inhibitor Z-VAD-FMK. In addition, the selective caspase inhibitors for caspase-3 and -7, -8, and -9 were effective at blocking the increase in the percentage of annexin V positive cells and the increase in the percentage of hypodiploid cells induced by these sesquiterpene lactones. This result suggests that the mechanism of cytotoxicity is dependent on caspase-8 and caspase-9 activation. Although pro-caspase-4 was also processed by these sesquiterpene lactones, the selective caspase-4 inhibitor did not block cell death, suggesting a minor role of the endoplasmic reticulum stress signaling pathway in the mechanism of cell death. The mechanism of cytotoxicity exhibited by spiciformin (1) and spiciformin acetate (2) is different from the previously described sesquiterpene lactones depending on the cell types. For example, parthenolide, a sesquiterpene lactone isolated from the plant Tanacetum parthenium, induces cell death that is not dependent on caspases in human osteosarcoma MG63 and melanoma SK-MEL-28, because cell death was not blocked by the pan-caspase inhibitor Z-VAD-FMK [22]. Parthenolide cytotoxicity has also been described to be partly caspase-dependent, as the broad-spectrum caspase inhibitor Z-VAD-FMK could partially protect multiple myeloma cells [23].
Since the intrinsic pathway of apoptosis involves release of mitochondrial proteins such as cytochrome c into the cytosol, it was explored whether this crucial protein is involved in the cell death induced by both sesquiterpene lactones. Dose-response experiments revealed an increase in cytochrome c in the cytosol after 24 h of treatment with 10 µM of compound 2 (Figure 4a). This was accompanied by a decrease in cytochrome c in the mitochondria-enriched fraction. However, results at earlier time points (6 h or 12 h; Figure 4b) remain largely elusive for both compounds. Control determinations using anti-cytochrome c oxidase (COX IV) antibody revealed that the supernatants of the cytosolic fraction were free of mitochondrial contamination. Therefore, sesquiterpene lactones have reasonably been shown to induce late release of cytochrome c as well as activation of multiple caspases, including caspase-3, -4, -7, -8, and -9, emphasizing that both the extrinsic and the intrinsic pathways play a role in the observed cell death. In addition, there were no changes in Bax levels (whole cell lysates) after treatment with both sesquiterpene lactones (Figure 4a), but there was a decrease in Bax levels in the cytosolic fraction after treatment with 30 µM sesquiterpene lactone 2 for 24 h. This was associated with the increase of Bax in mitochondria-enriched fraction. This Bcl-2 family protein is characterized by its ability to form pores across the outer mitochondrial membrane [16]. An interesting result of these experiments was that both sesquiterpene lactones downregulate the Bcl-2 levels (Figure 4a) and this could be the mechanism by which these germacranolides block the protection conferred by Bcl-2. This anti-apoptotic protein plays a crucial role in apoptosis induction by antagonizing the mitochondrial outer membrane permeability. In addition, the inhibition of Bcl-2 in acute myeloid leukemia may overcome chemoresistance without affecting normal hematopoietic stem cells. to apoptosis induced by this sesquiterpene lactone. In these experiments, the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control. Activation of caspase-8 may result in the proteolytic cleavage of Bid, a Bcl-2 family protein, which translocates to mitochondria to release cytochrome c [24]. A significant decrease in full-length Bid after treatment was observed for both sesquiterpene lactones, in accordance with the processing and activation of caspase-8.
Spiciformin (1) and Spiciformin Acetate (2) Activate Mitogen-Activated Protein Kinases (MAPKs) and Induce Reactive Oxygen Species (ROS) Generation
To determine whether cell death was associated with significant changes in mitochondrial membrane potential (∆Ψm), U-937 cells were treated with these compounds for 4 h or 24 h and analyzed by flow cytometry after staining with JC-1. The results indicated an important loss of ∆Ψm at 4 h of treatment with compound 2 (Figure 4c), suggesting that the alteration of ∆Ψm contributed to apoptosis induced by this sesquiterpene lactone. In these experiments, the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control.
Spiciformin (1) and Spiciformin Acetate (2) Activate Mitogen-Activated Protein Kinases (MAPKs) and Induce Reactive Oxygen Species (ROS) Generation
The effects of sesquiterpene lactones 1 and 2 on the activation of the mitogen-activated protein kinases (MAPKs) were investigated because these enzymes play a crucial role in survival and cell death. To this end, U-937 cells were treated for different time periods and phosphorylation of the three major protein kinases of this signal transduction pathway were determined by Western blot. In an attempt to identify the primary targets and early mechanism of action of sesquiterpene lactones, we interrogated the MAPK pathway activation by using high concentrations of the drugs (> antiproliferative IC 50 values). The results showed that sesquiterpene lactones induce fast phosphorylation (0.5 h) of JNK/SAPK (c-Jun N-terminal kinases/stress-activated protein kinases) and p38 MAPK (p38 MAPKs) and the activation of ERK (extracellular signal-regulated kinases) 1/2 took place after 4-6 h under identical experimental conditions (Figure 5a). These results indicate that both sesquiterpene lactones induce activation of the three main mitogen-activated protein kinases following different kinetics. Fast activation of JNK/SAPK and p38 MAPK has been reported to trigger cell death in response to several cellular stressors including oxidative stress [25,26]. The effects of sesquiterpene lactones 1 and 2 on the activation of the mitogen-activated protein kinases (MAPKs) were investigated because these enzymes play a crucial role in survival and cell death. To this end, U-937 cells were treated for different time periods and phosphorylation of the three major protein kinases of this signal transduction pathway were determined by Western blot. In an attempt to identify the primary targets and early mechanism of action of sesquiterpene lactones, we interrogated the MAPK pathway activation by using high concentrations of the drugs (> antiproliferative IC50 values). The results showed that sesquiterpene lactones induce fast phosphorylation (0.5 h) of JNK/SAPK (c-Jun N-terminal kinases/stress-activated protein kinases) and p38 MAPK (p38 MAPKs) and the activation of ERK (extracellular signal-regulated kinases) 1/2 took place after 4-6 h under identical experimental conditions (Figure 5a). These results indicate that both sesquiterpene lactones induce activation of the three main mitogen-activated protein kinases following different kinetics. Fast activation of JNK/SAPK and p38 MAPK has been reported to trigger cell death in response to several cellular stressors including oxidative stress [25,26].
The involvement of reactive oxygen species (ROS) in the apoptosis induced by germacranolides 1 and 2 was investigated because they are considered important mediators and are generated by many apoptosis-inducing agents. To this end, U-937 cells treated with both sesquiterpene lactones were stained with 2',7'-dichlorodihydrofluorescein diacetate (H2-DCF-DA) and analyzed by flow cytometry. The results revealed a fast increase (1 h) in ROS levels in sesquiterpene lactones-treated cells (Figure 5b,c). In these experiments, the antioxidant N-acetyl-L-cysteine (NAC, 5 mM) and hydrogen peroxide (200 µM) were used as negative and positive controls. The involvement of reactive oxygen species (ROS) in the apoptosis induced by germacranolides 1 and 2 was investigated because they are considered important mediators and are generated by many apoptosis-inducing agents. To this end, U-937 cells treated with both sesquiterpene lactones were stained with 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 -DCF-DA) and analyzed by flow cytometry. The results revealed a fast increase (1 h) in ROS levels in sesquiterpene lactones-treated cells (Figure 5b,c). In these experiments, the antioxidant N-acetyl-l-cysteine (NAC, 5 mM) and hydrogen peroxide (200 µM) were used as negative and positive controls.
Sesquiterpene Lactones Evaluated
The extraction and isolation of spiciformin (1) from the aerial parts of Tanacetum ptarmiciflorum and Tanacetum ferulaceum var. latipinnum was performed as previously described [20]. The acetyl derivative of spiciformin (2) was obtained by treatment with acetic anhydride and pyridine and purified by column chromatography over silica gel using n-hexane-ethyl acetate as eluent. Known compounds spiciformin and spiciformin acetate were identified by HR-MS (high resolution mass spectrometry) as well as 1 H-and 13 C-NMR spectrometry. The previously unreported 13 C-NMR data of spiciformin acetate were obtained on a Bruker model AMX-500 spectrometer with standard pulse sequences operating at 126 MHz. 13
The cytotoxicity of sesquiterpene lactones on human tumor, human mononuclear cells, and Vero cells was determined by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay [30]. Cells (5000) were seeded in 96-well microculture plates with increasing concentrations of germacranolides for 24 or 72 h at 37 • C. The IC 50 values were determined graphically for each experiment as described previously [27]. Values are means ± standard errors of the means S.E.M. from at least three independent experiments, with three determinations each.
Evaluation of Apoptosis
Fluorescent microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained nuclei were performed as described [28]. Briefly, for fluorescent microscopy, cells were washed with phosphate buffered saline PBS, fixed in 3% paraformaldehyde, and stained with 20 µg/mL Hoechst 33258. For flow cytometry of propidium iodide-stained cells, cells were fixed in 70% ethanol overnight at −20 • C and then washed in PBS, incubated in the dark with 1 U/mL RNase A (DNase free) and 10 µg/mL of propidium iodide at room temperature. Double staining with annexin V-fluoresceine isothiocyanate (FITC) and propidium iodide was performed using an Annexin V-FITC apoptosis detection kit (BD PharMingen, San Diego, CA, USA) according to the manufacturer's protocol. All flow cytometry assays were performed using a BD FACSVerse cytometer (BD Biosciences, San Jose, CA, USA).
Western Blot Analysis and Subcellular Fractionation
Western blot analyses of caspases, PARP, Bcl-2 family members, and MAPKs and cytochrome c release from mitochondria were performed as described previously [28]. Briefly, for whole-cell lysates cells were resuspended in lysis buffer (20 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, 20 mM sodium β-glycerophosphate, 10 mM NaF, 2 mM EDTA, 2 mM tetrasodium pyrophosphate, 10% glycerol, 137 mM NaCl, 2 mM sodium orthovanadate, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 1 µg/mL leupeptin and aprotinin)), sonicated, and centrifuged. For the subcellular fractionation to determine cytochrome c release, cells were resuspended in ice-cold buffer [20 mM HEPES (pH 7.5), 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 1 µg/mL aprotinin, leupeptin, and pepstatin A], lysed by being pushed them several times through a 22-gauge needle, and the lysate was centrifuged at 1000× g for 5 min at 4 • C to eliminate nuclei and unbroken cells. The resulting supernatant was centrifuged to 10,000× g at 4 • C for 20 min to obtain the mitochondrial fraction. The supernatant was centrifuged again at 105,000× g for 45 min at 4 • C and the resulting supernatant was used as the soluble cytosolic fraction.
Statistical Analysis
Statistical differences between means of control and treated samples were tested using Student's t-test (two samples) or one-way ANOVA (3 or more samples) with Tukey's test used for a posteriori pairwise comparisons of means. P-values below 0.05 were considered as statistically significant.
Conclusions
In conclusion, spiciformin (1) and spiciformin acetate (2) are cytotoxic against the human acute myeloid leukemia cells, including cells that overexpress Bcl-2, and display less cytotoxicity against the melanoma cell line SK-MEL-1 and mononuclear cells isolated from healthy volunteers. Human U-937 cells responding to germacranolides 1 and 2 manifested a great reduction in the levels of the anti-apoptotic protein Bcl-2. The mechanism of cytotoxicity triggered by these sesquiterpene lactones was due to caspase-dependent apoptosis and associated with mitogen-activated protein kinase pathway activation and reactive oxygen species generation. The results support that these sesquiterpene lactones have an impact in at least two cellular hallmarks of cancer, such as resistance to apoptosis and sustained proliferative signaling. However, the early mode of action of these compounds and relative signaling cascade remains to be elucidated in more detail. | v3-fos-license |
2018-12-02T19:39:54.678Z | 2018-11-28T00:00:00.000 | 54124831 | {
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} | pes2o/s2orc | The Role of PEG-40-stearate in the Production, Morphology, and Stability of Microbubbles Microbubbles: Exploring Gas-Liquid Interfaces for Biomedical Applications
: Phospholipid coated microbubbles are currently in widespread clinical use as ultrasound contrast agents and under investigation for therapeutic applications. Previous studies have demonstrated the importance of the coating nanostructure in determining microbubble stability and its dependence upon both composition and processing method. While the in fl uence of di ff erent phospholipids has been widely investigated, the role of other constituents such as emulsi fi ers has received comparatively little attention. Herein, we present an examination of the impact of polyethylene glycol (PEG) derivatives upon microbubble structure and properties. We present data using both pegylated phospholipids and a fl uorescent PEG-40-stearate analogue synthesized in-house to directly observe its distribution in the microbubble coating. We examined microbubbles of clinically relevant sizes, investigating both their surface properties and population size distribution and stability. Domain formation was observed only on the surface of larger microbubbles, which were found to contain a higher concentration of PEG-40-stearate. Lipid analogue dyes were also found to in fl uence domain formation compared with PEG-40-stearate alone. “ Squeezing out ” of PEG-40-stearate was not observed from any of the microbubble sizes investigated. At ambient temperature, microbubbles formulated with DSPE-PEG(2000) were found to be more stable than those containing PEG-40-stearate. At 37 ° C, however, the stability in serum was found to be the same for both formulations, and no di ff erence in acoustic backscatter was detected. This could potentially reduce the cost of PEGylated microbubbles and facilitate simpler attachment of targeting or therapeutic species. However, whether PEG-40-stearate su ffi ciently shields microbubbles to inhibit physiological clearance mechanisms still requires investigation. for 15 min (the typical half-life of microbubbles in the human body being less than 5 min). 25 This was performed at both room temperature and 37 ° C. Measurement of Backscatter Acoustic Intensity of Microbubbles. Imaging of the microbubbles and measurement of their response to ultrasound was carried out using a tissue-mimicking agarose fl ow phantom model with an embedded 1.2 mm channel. 26 A peristaltic pump (Minipuls Evolution, Gilson Scienti fi c UK, Dunstable, Bedfordshire) was used to fl ow the freshly prepared microbubble solution (500 μ L) through the channel at a fl ow rate of 2 mL/min. An L12-5 probe of the iU-22 diagnostic ultrasound scanner (Philips Medical Systems, Bothell, WA) was used to interrogate and image the microbubbles at contrast mode and a low nondestructive mechanical index (MI = 0.04). The frame rate was set at 1 Hz to ensure replenishment of the channel with fresh microbubbles, and video loops of at least 10 images were captured. Quanti fi cation of the average backscatter intensity was done in Matlab (Mathworks, Natick MA, United States). The procedure was repeated for three separate solutions for each type of microbubble. Experiments were carried out in a degassed water tank at 37 ° C. Statistical analysis was performed using a one-tailed Student ’ s t -test.
■ INTRODUCTION
Ultrasound offers a safe, convenient, and cost-effective method of medical diagnostic imaging. Image contrast between blood vessels and the surrounding tissue, however, is typically lower than in other modalities such as magnetic resonance imaging (MRI). 1 To overcome this limitation, ultrasound contrast agents consisting of gas microbubbles stabilized by a surfactant or polymer coating 2 are injected into the patient's bloodstream. Due to their high compressibility, microbubbles are able to scatter ultrasound much more efficiently than red blood cells. Moreover, they exhibit a nonlinear response enabling the scattered signal to be distinguished from that due to neighboring tissues. 1 Therapeutically, the oscillations of microbubbles under ultrasound can increase the permeability of both cell and tissue membranes for delivery of bioactive compounds. 3 Microbubbles are also being explored for use as oxygen carriers for the treatment of ischemia and tumor hypoxia. 4−6 The key component of the microbubble for imparting stability and preventing coalescence is the coating. Microbubbles formulated with cross-linked serum albumin, such as Albunex and Optison, have relatively high coating stiffness and the propensity to rupture beyond a critical strain/expansion ratio, which limits the duration of contrast enhancement. To overcome this, Levovist used a surfactant (palmitic acid) to provide a more flexible coating and hence improve the acoustic response, but this resulted in a reduction in stability. Subsequently, however, it was found that other surfactants such as phospholipids could simultaneously provide stability and a strong acoustic response. This has led to multiple phospholipid formulations reaching the clinic, in particular Sonovue (Bracco Imaging), Sonazoid (GE Healthcare) and Definity (Bristol Myers-Squibb). In addition to phospholipids, smaller quantities of an emulsifier, typically polyethylene glycol derivatives are used in microbubble formulations to promote bubble formation, inhibit coalescence, and reduce nonspecific adsorption of blood plasma proteins. 7 Only a limited number of studies have been performed that examine the composition and structural properties of the phospholipid microbubble coating. For instance, Kim et al. analyzed the mechanical properties of phospholipid monolayers using micropipette aspiration. They also employed fluorescent phospholipids and freeze fracture electron microscopy 8 to examine domain formation in the coating. This study utilized microbubbles composed of 10:1 lipid:PEG-40-stearate molar ratio and varied both lipid chain length and cooling time during bubble fabrication. The results indicated that the monolayer microstructure was dependent on the cooling rate with larger domains formed at slower cooling rates. Bubbles with a coarse domain structure exhibited a resistance to shear deformation higher than those with a fine structure.
Borden et al. examined the surface phase behavior and microstructure of lipid/PEG-emulsifier coated microbubbles again with lipids of different chain length and a fluorescent dye (NBD-PC) via fluorescence microscopy and using lipid films in a Langmuir trough. 9 These results confirmed the polycrystalline nature of the two-component coating and that the surface morphology was affected by the bubble fabrication process. NMR and FTIR spectroscopy were then employed to investigate lateral phase separation. These results indicated that, at least in microbubbles greater than 10 μm in diameter, the coating comprised ordered domains consisting of lipid and interdomain regions enriched by PEG-40-stearate. 10 It was also suggested that below a certain size (<20 μm), the bubble surface pressure would be too high for the emulsifier rich regions to coexist with the lipid domains and would only remain attached through surface associated aggregates.
Lozano and Longo 11 employed fluorescence microscopy to study dissolving microbubbles coated with either a phosphatidylcholine (C16, C18, or C20) and PEG-40-stearate mixture or a phosphatidylcholine (C16, C18, or C20) and a PEG conjugated phospholipid (DSPE-PEG (2000)). Dissolution times for microbubbles containing DSPE-PEG(2000) were four times longer than those containing PEG-40-stearate. This is because the DSPE-PEG(2000) remains in the condensed phase with the lipid, whereas with PEG-40-stearate, it separates into the expanded phase, which is more gas permeable. Unlike the studies of Kim et al. 8 and Borden et al. 9 , these experiments were performed with microbubbles in degassed water, using the lipid analogue DiIC18 fluorophore to examine domain formation and/or surface deformation. Moreover, these experiments were performed with air-filled bubbles (rather than the clinically used perfluorocarbons which have been shown to alter the properties of lipid membranes 12−14 ) and at room temperature with bubbles of approximately 20 μm.
From these previous experiments, the behavior of the emulsifier was thus inferred from the observed lipid behavior. Moreover, the fluorescence-based technique for the observation of lipid domains was applied on microbubbles above 5 μm only to enable resolution of surface structures.
Kooiman et al. 15 investigated the impact of lipid chain length on the distribution of ligands around the microbubble surface, using super-resolution microscopy. Two lipid chain lengths, DPPC (16 carbons) and DSPC (18 carbons), were used to make microbubbles with DSPE-PEG(2000)-biotin. Fluorescent streptavidin was attached to biotinylated microbubbles, and a more even distribution and larger targeting area was found with DPPC relative to DSPC. 15 This was attributed to the fact that DSPE-PEG(2000)-biotin was more heterogeneously distributed on the microbubble surface in DSPC relative to DPPC, reducing the targeting area.
Abou-Saleh et al. examined the impact of PEGylated lipids on microbubbles created by microfluidics and observed changes in microbubble stability and mechanical properties with increasing concentration of PEGylated lipid. Above a certain threshold, the lifetime and concentration of the microbubbles were seen to decrease. 16 Shih et al. performed similar experiments and found bubble lifetime to be related to stable size, and that bubble size reduced with increasing concentration of emulsifier, down to a threshold of 30% beyond which stability was compromised. 17 Segers et al. also found that during insonation shedding of the pegylated lipid from the microbubble can occur potentially altering the coating stiffness. 18 Recent results from Carugo et al. have shown transfer of material from the microbubble coating to nearby cell membranes and that polyethylene glycol has a major impact on lipid ordering and the extent of this lipid transfer. 19 Hosny et al. used molecular rotor fluorescence lifetime imaging to quantify the spatial distribution of viscosity in a microbubble coating. Varying composition and production methodologies indicated that there can be a large variation in viscosity across a microbubble population independent of the individual microbubble size. 20 The aim of this study is to build on the results of previous research examining the properties of microbubbles formulated with PEG-40-stearate. A fluorescent PEG-40-stearate analogue was synthesized to visualize the location of PEG-40-stearate in the microbubble coating. The impact of PEG-40-stearate concentration on the lipid packing was also examined via fluorescence spectral imaging of an environmentally sensitive fluorophore (C-laurdan). The concentration, size, and stability of microbubbles formulated with both PEG-40-stearate and DSPE-PEG(2000) were then measured. Only one chain length of 18 carbons (DSPC) was used throughout the experiments for consistency. Finally, the acoustic responses of the different microbubbles were compared. Microbubble and Vesicle Preparation. The relevant mixtures of lipids and emulsifier, dissolved in chloroform, were mixed in a glass vial at a selected molar ratio (9:1) lipid to emulsifier. 9,11,20 This was heated to 50°C and left for 12 h. The lipid film was suspended in aqueous solution (2 mL) for over 1 h at 75°C under constant stirring. The stir bar was removed, and the solution was then sonicated for 90 Langmuir Article s using an ultrasonic cell disruptor (XL2000, probe diameter 3 mm; Misonix Inc., Farmingdale, NY, United States) operating at 22.5 kHz and level 4 corresponding to 8 W RMS output power. Sonication at the gas−water interface in an SF 6 gas environment was then performed for 20 s at sonication level 19 (38 W RMS ). The microbubble suspension was then placed in an ice bath for cooling for approximately 10 min. The same procedure was followed for vesicles, but the last sonication step was omitted.
For those experiments involving fluorescent labeling of microbubbles, a stock solution of the lipophilic dye DiI at a concentration of 2.5 mg/mL in dimethyl sulfoxide (DMSO, Sigma-Aldrich, UK) was prepared. Seven microliters of the stock solution was added to the chloroform lipid solution, and the protocols outlined previously were followed for microbubble production. To remove excess DiI from the formed microbubble suspension, the microbubble sample was centrifuged once at 1000g for 10 min (400R Heraeus Labofuge, Thermo Fisher Scientific Inc., United States), and microbubbles were resuspended in PBS after each centrifugation cycle.
To an ice cold solution (5°C) of fluorescein-PEG-NH 2 (50 mg, 0.02 mmol) in anhydrous dichloromethane (5 mL) under N 2 atmosphere was added a catalytic amount of triethylamine. The solution was stirred for 30 min at 5°C, and a solution of stearoyl chloride (7 mg, 0.02 mmol) in anhydrous dichloromethane (5 mL) was added dropwise, maintaining the temperature between 0 and 5°C . The reaction was allowed to proceed at room temperature for 24 h and kept in the dark. The product fluorescein-PEG-stearate was recovered by dissolution in dichloromethane and precipitation in ether and finally purified by preparative thin layer chromatography using methanol−chloroform (20%) as the eluant.
Measurement of Lipid Order and Lipid Structure in the Microbubble Coating. C-Laurdan Staining. The lateral order of the lipids within the microbubble coating was quantified using Claurdan, an environmentally sensitive fluorescent probe, the emissions from which shift according to the degree of hydration in the membrane. This technique has been widely used to characterize the surface properties of lipid structures, including investigation of the interactions between microbubbles and cells. 19 To reduce fluorescence background due to lipids in suspension, microbubbles were washed once by centrifugation. Approximately 3 mL of freshly prepared microbubbles were loaded into a disposable 10 mL Luer-Lok syringe (BD, MediSupplies, Unit 3, 2 Lansdowne Crescent, Bournemouth, UK) adapted to fit upright within a 50 mL centrifuge tube (Corning, Fogostraat 12, 1060 LJ Amsterdam, The Netherlands). They were then centrifuged at 1000g relative centrifugal force (RCF) at 10°C for 10 min in a swing bucket rotor (Heraeus Labofuge 400R, Thermo Fisher Scientific, Waltham, MA, United States) to form a bubble "cake" against the plunger. The subnatant was discarded, and the bubbles were resuspended within the syringe using 2 mL of PBS. This was then transferred to a glass vial for future use. To stain with C-laurdan, 1.5−5 μL of C-laurdan (400 nM) was added to 30 μL of washed microbubbles diluted in 70 μL of Milli-Q water (total sample volume = 100 μL). This was incubated for up to 30 min at 4°C before use.
Spectral Fluorescence Imaging. Spectral imaging was performed on a LSM 780 Inverted Confocal Microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using a previously reported methodology. 21 Briefly, 10 μL of stained microbubbles was loaded onto a 170 μmthick glass slide coverslip (Logitech Ltd., Scotland) and covered with another coverslip. Individual microbubbles were then located and imaged using an oil immersed 63× objective with the microscope focus set at the midplane of the microbubble. Spectral imaging was performed using a Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a 32-channel GaAsP detector array. C-Laurdan was excited at 405 nm, and the lambda detection range was set between 415 and 691 nm. This resulted in a single image stack with each image providing the intensity map at a single emission wavelength (n = 30). Approximately 20 images were taken per formulation, and each formulation was repeated three times using a freshly prepared microbubble suspension created from a new lipid film.
Generalized Polarization (GP) Calculation. GP is an optical observable that quantifies the shift in the emission spectra of the dye. It provides an order parameter for the phase transition discussed above and is related to the dielectric properties of its microenvironment, which in turn is directly affected by the presence of water in the lipid film. For example, an increase in hydration causes a red shift in the spectrum, which corresponds an increase in GP values. GP (ranging from −1 to +1) was utilized as a relative measure of lipid packing within the microbubble shell. To measure this, fluorescence microscopy images were processed using a custom script in MATLAB (The Mathworks Inc.). Briefly, images were first opened using the BioFormats for MATLAB script. Microbubbles were identified by a semiautomated implementation of the imfindcircles() function, and the background and internal volume of the bubble were removed by cropping, leaving only the pixels of the stained bubble coating. 21 This was done to reduce anisotropy effects and out-of-focal plane effects of the coating. GP values were calculated (per pixel) using intensity data from the 440 and 490 nm wavelength stacks by the following equation: 22 I I I I GP 440 490 440 490 Increased GP value corresponds to an increase in the lipid order of the coating layer, and vice versa. Whole microbubble GP values were calculated from the average of the GP per pixel values. Standard deviations, medians, and histogram plots were also generated from these data. Images with saturation (intensity of 255) in any wavelength were discarded. For spectral analysis, average intensity values of the coating per wavelength were calculated from pixel intensity values. 20 Lipid Domain Analysis by Langmuir Trough. Lipids from commercial stocks were used without further purification and were mixed in chloroform to obtain desired ratios at 25 mg/mL. Two microliters was then spread at the air−water interface in a Langmuir trough (microtrough G2, Kibron, Malminkaari, Helsinki, Finland) with an initial area of 212 cm 2 . The trough was compressed at 50 cm 2 /min, and the pressure was recorded using a Wilhelmy plate at room temperature. Fluorescence images of the DSPC:PEG-40stearate monolayer were obtained by titrating the lipids under a microscope in 100 mm diameter Petri dish. The amount of lipids to be titrated was estimated from the isotherm on the Langmuir trough.
Fluorescence Microscopy of Microbubbles. The stained microbubbles were pipetted onto a 1.5H 75 × 25 mm glass slide, and a coverslip was placed to prevent movement of the sample. A Zeiss LSM 780 confocal microscope equipped with a 63× Plan-Apochromat objective was used for imaging the microbubbles in the equatorial plane. The pinhole was adjusted to modify the thickness of the imaging plane and capture the phase separation on the microbubble surface. The imaging was performed on two separate Scheme 1. Synthesis of Fluorescein-PEG-stearate Langmuir Article channels: the first involved a 543 nm excitation and detectors set to a 553−673 nm range for detection of DiI; the second consisted of a 488 nm excitation with detectors set to acquire at 493−553 nm for detection of FITC. Intensity/pixel was determined using ImageJ public domain software (NIH, Bethesda, MD) by selecting the total area of the microbubble shell in midplane and measuring the raw intensity and the number of pixels.
Structure of Fluorescent PEG-40-stearate Analogue. Mass Spectroscopy Determination of Molecular Weight. Matrix-assisted laser desorption/ionization−time-of-flight (MALDI-TOF) spectra of fluorescein-PEG-stearate ( Figure 1) was performed in Voyager-DM-Bio spectrometer using 50 μL of 1 mg/3 mL sample mixed with 100 μL of DHB matrix, where the laser intensity was 1471. The numberaverage molecular weight (M n ) and average molecular weight (M w ) were calculated by the following equations: where M i is the molecular weight of the chain and N i is the number of chains of that molecular weight. 1 H NMR Spectra Determination of Molecular Structure. To determine the structure and confirm the synthesis had been successful, the 1 H NMR spectrum of fluorescein-PEG-stearate was recorded at room temperature using a Varian spectrometer operating at 500 MHz by using CDCl 3 as solvent. The relative frequency of a nucleus relative to a standard in a magnetic field or chemical shifts (δ) are given in parts per million (ppm) using tetramethylsilane (TMS) as an internal reference.
Microbubble Characterization. Microbubble Size and Stability. Microbubble suspension (10 μL) was added to a Neubauer hemocytometer (Sigma-Aldrich Ltd. Gillingham, Dorset, UK). This was then imaged using a Leica DM500 optical microscope (Larch House, Milton Keynes, MK14 6FG) and a 40× objective lens at room temperature. To obtain a representative size distribution for one batch of bubbles, at least 3 separate bubble samples must have 30 images taken via optical microscopy. 23 The bubble size distribution and concentration were then determined using a purpose written image analysis software in Matlab (2013b, The Mathworks, Natick, MA, United States). The software converts each micrograph to a binary image, and single spheroid shapes are then detected, measured and counted for each of the images analyzed. A size distribution and count are then produced for the microbubbles. Because the dimensions of the hemocytometer and the microscope field of view are known, the volume concentration of microbubbles can also be estimated.
Nanoparticle Size Measurement. To determine the presence and size of vesicles, measurements of lipid dispersions before sonication at the gas−water interface were performed via dynamic light scattering using a Zetasizer Nano-ZS, Malvern Instruments Ltd. (Worcestershire, UK) in a disposable capillary cell (DTS1070 Malvern Instruments Ltd., Worcestershire, UK). Each sample of the liposome or microbubble solution (60 μL) was added to 940 μL of 10% HEPES buffer.
Microparticle Size Measurement. Laser light obscuration and scattering of microbubbles was used to obtain accurate size and concentration using an Accusizer 780, NICOMP Particle Sizing System (Santa Barbara, CA). Samples (10 μL) of each microbubble suspension were diluted into a 50 mL flask under mild mixing during measurement.
Microbubble Stability in Serum. DSPC microbubbles at 9:1 molar ratio with PEG-40-stearate or DSPE-PEG-2000 and a set total concentration of 3 mg/mL were created, kept on ice, and added to serum. The microbubble size was then measured via the Accusizer. Ten microliters of the microbubble suspension was added to 500 μL of serum, giving a concentration of 10 7 microbubbles/mL. This is 10× the blood concentration of microbubbles in vivo, 24 but was used as lower concentrations could not be detected. The Accusizer was calibrated to remove the contribution of particles in the serum. The change in microbubble concentration was then measured over time 25 This was performed at both room temperature and 37°C. Measurement of Backscatter Acoustic Intensity of Microbubbles. Imaging of the microbubbles and measurement of their response to ultrasound was carried out using a tissue-mimicking agarose flow phantom model with an embedded 1.2 mm channel. 26 A peristaltic pump (Minipuls Evolution, Gilson Scientific UK, Dunstable, Bedfordshire) was used to flow the freshly prepared microbubble solution (500 μL) through the channel at a flow rate of 2 mL/min. An L12-5 probe of the iU-22 diagnostic ultrasound scanner (Philips Medical Systems, Bothell, WA) was used to interrogate and image the microbubbles at contrast mode and a low nondestructive mechanical index (MI = 0.04). The frame rate was set at 1 Hz to ensure replenishment of the channel with fresh microbubbles, and video loops of at least 10 images were captured. Quantification of the average backscatter intensity was done in Matlab (Mathworks, Natick MA, United States). The procedure was repeated for three separate solutions for each type of microbubble. Experiments were carried out in a degassed water tank at 37°C. Statistical analysis was performed using a one-tailed Student's t-test.
■ RESULTS AND DISCUSSION
Characterization of Fluorescein-PEG-stearate. Successful formation of the fluorescein-PEG-stearate was confirmed by 1 H NMR spectroscopy with Figure 1 showing the stacked spectra of the starting material fluorescein-PEG-NH 2 and the product fluorescein-PEG-stearate. Resonances in the downfield region (i.e., between 6.0 and 8.0 ppm) were attributed to the aromatic protons of the fluorescein ring and were largely unchanged in the spectrum of the product, given their distance from the site of conjugation. Examination of the upfield region, however, revealed the appearance of three new resonances in the spectrum of the product at 0.88, 1.40, and 2.10 ppm that were not present in the spectrum of the starting material and represent the methyl protons, the protons from the 15 methylene groups of the stearic acid aliphatic chain, and the methylene protons adjacent to newly formed amide carbonyl group, respectively. The broad peak at 3.9 ppm in both spectra was characteristic of methylene protons in the PEG polymer chain, and again, their chemical shift remained unchanged in the product when compared to the starting material. This was expected as their proximity to the newly created amide bond was separated by the two methylene groups, minimizing any change to their electronic environment following conjugation. The ratio of the integrals for the new methyl protons and the eight aromatic protons was also consistent with successful product confirmation.
The molecular weight of fluorescein-PEG-stearate was also analyzed by MALDI-TOF mass spectroscopy (Figure 2). Given the polymeric nature of the PEG component of fluorescein-PEG-stearate, it is difficult to identify an exact molecular weight for this compound. However, assuming the total repeat unit weight was exactly 2000 Da, the MW would be expected to be 2614 Da. Similarly, the fluorescein-PEG-NH 2 precursor would be expected to have a MW = 2347 Da. Analysis of the MALDI-TOF spectrum revealed the fluorescein-PEG-stearate had a M n and M w of 2619 and 2654 Da, respectively, with a polydispersity (M w /M n ) of 1.01. While there is a slight discrepancy between the expected 2614 Da and observed 2619 Da, the observed MW range strongly suggests the successful formation of fluorescein-PEG-stearate with the small difference attributed to variability in the PEG chain length. Combined, these analyses confirm the successful preparation of fluorescein-PEG-stearate.
Langmuir
Article 1% concentration of PEG-40-stearate. The lateral pressure vs area (π − a) behavior for several different lipid formulations containing DSPC lipids is shown in Figure 3A. The pure DSPC monolayer shows the well-known sublimation (gas to liquid condensed) transition at 0 mN/m where the pressure starts to rise abruptly. 27 The isothermal compressibility of the monolayer can be obtained from k T (eq 4). The data indicate that the DSPC monolayer is very stiff over the entire range (0−50 mN/m) of surface pressures. For DSPC:DSPE-PEG(2000), the compressibility is similar at high pressures, but the curve is shifted to the right. The DSPC, PEG-40-stearate (9:1) monolayer, on the other hand, is qualitatively different from both DSPC alone and DSPC:DSPE-PEG(2000). A distinct region of low compressibility appears around 35 mN/m. Such a plateau might be associated with a conformational transition in the components of lipid monolayers. 11 However, it would then be expected to show temperature, concentration, and lipid chain length dependence. 27 Previous work has shown that this is not the case for monolayers containing PEG-40-stearate 9 and instead that the plateau coincides with PEG-40-stearate driven expulsion of material from the interface. The evidence of vesicular structures appearing in Figure 3D supports this explanation.
Domain-like features were observed at pressures as low as 10 mN/m and could be clearly resolved at 25 mN/m. These domains continued to grow in size on further compression, showing extensive phase separation at 40 mN/m with evidence of vesicles forming at the interface. These results are consistent with those of Lozano and Longo wherein PEG-40-stearate was found to create microbubbles less stable than those of DSPE-PEG(2000). 11 These also concur with the results of Carugo et al., which indicated that PEG-40-stearate and lipids are able to leave the microbubble coating and pass into the surrounding liquid. 19 Impact on Coating Lipid Order. The packing density of the lipids in the microbubble coating was analyzed by calculating the GP from spectral microscope images of Claurdan ( Figure 4). GP values of microbubbles from spectral microscopy images were averaged and weighted to determine a mean GP for each formulation. Fluorescence microscopy images were also used to determine microbubble size and any relationship between mean GP and size. The GP values presented have not been weighted by pixel number, and values are pooled from all three repeats. Thus, GP values for microbubbles below 2 μm may be more affected by noise as fewer pixels are used to calculate mean GP values.
For the range of bubble sizes investigated, increasing the concentration of PEG-40-stearate had no statistically significant effect upon the packing density of lipids in the microbubble coating. This agrees with the previous study of Hosny et al. which indicated that there was greater variability within a microbubble population than between microbubble populations. 20 It is also consistent with the suggestion in Borden et al. 10 that PEG-40-stearate is excluded from the coating when the microbubble diameter is smaller than 2 μm, and thus, no difference in properties would be observed.
Fluorescence Microscopy Images of Bubbles. Fluorescence Microscopy with FlTC-PEG-40-stearate and DiI. Images were taken of DSPC:PEG-40-stearate microbubbles that had been stained with DiI and contained 1% PEG-40stearate-FITC ( Figure 5). Intensity analysis of the confocal image stacks indicated that larger microbubbles (>20 μm) had a higher intensity per pixel at 5570 ± 141 AU/pixel than smaller microbubbles (<20 μm) at 2644 ± 193 AU/pixel when the midplane was examined. The opposite effect was observed with DiI; smaller microbubbles (<20 μm) had an intensity per pixel of 5846 ± 1908 AU/pixel, whereas microbubbles larger than 20 μm had an intensity per pixel of 1546 ± 58 AU/pixel (Supporting Information Table 1). These observations are again consistent with the exclusion of PEG-40-stearate from smaller bubbles.
A further aim of these experiments was to determine whether the presence of the emulsifier gave rise to domain formation on the surface of microbubbles within the clinical size range. It was intended that the fluorescent PEG-40stearate analogue would provide a more direct means of determining emulsifier content and bubble coating structure compared with lipid analogue dyes. No domains were observed on microbubbles with diameters smaller than 5 μm. The DiI images indicated that some microbubbles with diameters between 5 and 20 μm did exhibit surface domains ( Figure 5K), similar to those reported previously. 11 The absence of a FITC signal in panel J, however, indicates that the formation of
Langmuir
Article domains was not necessarily associated with PEG-40-stearate (this assumes that there was no energy transfer between the two dyes potentially co-localised in the membrane, i.e. from FITC-PEG40-sterate to DiI). This was further suggested by the fact that there was limited overlap in the apparent domains visible in the DiI and FITC images (Figures 5G−I).
Examining PEG-40-stearate Content within Microbubbles. To investigate further whether PEG-40-stearate was excluded from microbubbles smaller than 5 μm in diameter, the microbubbles were centrifuged to remove any unincorporated PEG-40-stearate from the surrounding solution. Figures 6A−C indicate that the FITC-PEG-40-stearate was in fact still present in microbubbles after centrifugation. The greyscale intensity profile across the microbubble surface indicates the increase in fluorescence corresponding to the microbubble coating ( Figure 6C). This was observed for all microbubbles regardless of size (1−10 μm). Again, although there were variations in intensity across the microbubble coating, no domain-like features were visible on microbubbles smaller than 5 μm in diameter. This suggests that PEG-40-stearate was uniformly distributed throughout the microbubble coating and not entirely excluded by surface pressure.
Microbubbles were also created with fluorescent PEG-40 alone, without stearic acid, as a negative control. In this case, the fluorescent PEG-40 was found in solution and not in the microbubbles ( Figure 6D and E), as observed for the second greyscale values ( Figure 6F) after centrifugation. This suggests that PEG-40 does not passively attach to the microbubble surface and requires the stearic chain for incorporation into the microbubble coating.
Comparison of Microbubble Size and Concentration. Centrifugation of Microbubbles. DSPC-only microbubbles, DSPC:DSPE-PEG(2000) (9:1), and DSPC:PEG-40-stearate (9:1) were formulated in triplicate in PBS. These were analyzed for size and concentration, followed by centrifugation (1000 rpm, 10 min) whereby the supernatant and infranatant were also analyzed. Centrifugation has been used to clean microbubbles and size isolate certain populations, 28 and microbubble survival from centrifugation would provide information on stability. Results indicated that average size did not vary between microbubble samples within significance. However, after centrifugation, the DSPC-only microbubbles were more polydisperse relative to microbubbles formulated with DSPE-PEG(2000) and PEG-40 stearate ( Figures 7A and B). The highest concentrations ( Figure 7C) of microbubbles were achieved with PEG-40 stearate (1 × 10 11 microbubbles/ mL), followed by DSPE-PEG(2000) (1 × 10 10 microbubbles/ mL), and finally DSPC alone (1 × 10 9 microbubbles/mL). DSPE-PEG(2000) microbubbles were more stable to centrifugation, maintaining the same average concentration as before centrifugation within experimental error, whereas PEG-40-stearate microbubbles were lower in concentration (1 × 10 9 microbubbles/mL) after centrifugation, indicating microbubbles might be destroyed during the centrifugation process, as shown in Figure 7D. The data agree with results from Lozano et al. on the condensed phase of the lipids, resulting in increased stability when formulated with DSPE-PEG(2000). 11 Size of Vesicles. Vesicles present in the microbubble suspension were analyzed by dynamic light scattering by taking a fluid sample just before sonicating at the gas−water interface. The results shown in Figure 8 indicate that DSPC and PEG-40-stearate produces vesicles (mean diameter: 117 nm) in a smaller size range than DSPC alone (mean diameter: 1500 nm). DSPC and DSPE-PEG(2000) also produces smaller vesicles as well (mean diameter: 105 nm). This was as expected from previous literature. 31 The production of smaller vesicles could assist in the generation of the microbubble coating and the stability of the microbubble, according to the mechanism postulated by Li and Fogler 32 whereby vesicles break up through sonication at the gas−water interface and fuse to form the microbubble coating. Hence, the size of the vesicles plays an important role as smaller vesicles will have a higher surface energy, making microbubble formation more energetically favorable. These results are also consistent with observations of phospholipid coated microbubbles via electron microscopy by Owen et al., which also indicated that the coating comprised small vesicles. 29 The larger size of DSPConly vesicles might indicate that microbubbles generated from lipid fragments alone are less stable and in lower concentration, whereas microbubbles incorporating a PEG moiety contain Langmuir Article remained at a higher concentration than the PEG-40-stearate microbubbles ( Figure 9A). This agrees with the work of Lozano et al. 11 However, over the course of 10 min, the difference in residual microbubble concentration is not statistically significant, and microbubbles incorporating DSPE-PEG(2000) only showing a marked improvement at the 15 min time point. Microbubble size and concentration were also measured over a period of 7 days from samples stored at 4°C in a sealed vial (Supplementary Data) and showed similar trends. However, at 37°C, no difference between DSPE-PEG(2000) microbubbles and those formulated with PEG-40-stearate was observed at any time point. These differences in stability at different temperatures can be explained by comparing the isotherms of the two formulations. The isotherms for PEG-40-stearate containing monolayers show no significant change in form including the slope of the curves at all points and the pressure where the plateau appears, except that the size of the plateau shrinks. There exists a direct correspondence between the monolayer and bilayer surface pressure, and under equilibrium, the two are equal. 30−32 While the surface pressure for a bubble cannot be ascertained without a direct measurement, it can still be deduced from the isotherms that the compressibility of the film remains similar for all pressures when increasing the temperature from 18 to 37°C. In contrast, DSPE-PEG2000 containing films become more compressible with increasing temperature. 33 Statistical thermodynamics provide a direct relationship between lipid film permeability and its compressibility, 34,35 and thus, the gas permeability of PEG40S bubbles expected to remain the same while it is expected to increase for DSPE-PEG2000 bubbles as the temperature is increased from 18 to 37°C, leading to a reduction in microbubble stability.
It is important to note, however, that "stability" in the context of clinical applications is also determined by lung and liver capture, and further work is required to compare the in vivo circulatory stability of the two formulations.
Backscatter Acoustic Intensity Comparison of Microbubbles. The average backscatter intensity of 9:1 DSPC microbubbles with PEG-40-stearate and 9:1 DSPC microbubbles with DSPE-PEG(2000) was compared as shown in Figure 10. No significant difference was seen between the two microbubble formulations (p = 0.3719), indicating that both types of bubble would behave similarly as ultrasound contrast agents.
■ CONCLUSIONS
A fluorescent form of PEG-40-stearate was successfully synthesized and used to determine the location of the PEG-40-stearate molecule within a lipid microbubble coating. Langmuir trough measurements revealed that PEG-40-stearate changes the isotherm of DSPC alone, and images of the fluorescent PEG-40-stearate analogue indicated domain formation and vesicles entering the solution and leaving the lipid film at 30 mN/m in contrast to films of DSPE-PEG(2000) and DSPC alone, in which coating integrity was maintained.
Langmuir
Article Spectral imaging showed that PEG-40-stearate did not have a statistically significant impact on lipid order in the bubble coating at any concentration. This indicates that PEG-40stearate likely enhances microbubble formation without impacting shell properties and that most PEG-40-stearate is excluded from the coating and enters the solution. Fluorescence microscopy, however, revealed that some PEG-40-stearate does remain within the bubble coating.
Staining with DiI allowed observation of domains as previously reported by Borden et al. 9 In the present study, however, domains were also only observed on larger microbubbles (>5 μm). In addition, the proportion PEG-40stearate was higher in microbubbles with diameters >8 μm.
No differences in microbubble size or concentration were found relative to DSPE-PEG(2000) formulated microbubbles, and both PEG-40-stearate and DSPE-PEG(2000) were stable to centrifugation. Both formulations created ∼100 nm vesicles, indicating similar microbubble formation mechanisms.
At room temperature, DSPE-PEG(2000) microbubbles maintained a higher concentration for a longer period of time in human serum than PEG-40-stearate microbubbles. However, at 37°C, no statistically significant difference could be discerned. Furthermore, no statistical difference was found in acoustic intensity between the two microbubble populations in a flow phantom at 37°C. | v3-fos-license |
2020-02-13T09:12:14.827Z | 2019-01-15T00:00:00.000 | 214138486 | {
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} | pes2o/s2orc | Lateral Extraarticular Tenodesis in Combined ACL and ALL Reconstruction. Case presentation
The purpose of this paper was to evaluate the necesity of associating a lateral extraarticular tenodesis in patients that will undergo an anterior cruciate ligament reconstruction or revision and to briefly describe the surgical procedure. Multiple lateral extraarticular tenodesis techniques were described and also graft selection and fixation types are also important. In conclusion acute ACL tears with grade 3+ pivot shift can be succesfully treated by combined ACL reconstruction and LEAT association.
A Lachman test and pivot shift test are then performed. If there is any residual internal rotation we standardly perform an antero-lateral ligament reconstruction. A 5 cm incision is performed starting at the lateral epicondyle of the femur and running midway between Gerdys tubercle and the fibular head. A 14 cm graft of the ilio-tibial tract is harvested using a tendon stripper. The anatomical footprints of the ALL are then exposed. Two guide wires are inserted into the footprints and, using fiberwire, proper tension is checked (they should tension in extension and relax in flexion). Two tunnels are created in thefootprints and the graft is fixed using two PEEK anchors. The graft tension is then checked one more time [7,8].
Case presentation
We present the case of a 17 year-old male professional soccer player who presented in our clinic with instability in his left knee after an ACL reconstruction (hamstrings tendon graft), performed 1 year ago. Upon clinical examination we found a grade 3 Lachman test, positive anterior drawer sign and a grade 3 pivot shift test. We analyzed the Lysholm score [9] that is based on the following : limping, necesity of canes/crutches, locking sensation in knee, giving way sensation in knee, pain, swelling, climbing stairs and squating; and the Tegner activity score [10] that is based on the kind of lifestyle the patient has (from sick leave or disability pension to competitive sports). The Lysholm score was 61, and the Tegner activity score concurred that the patient was a level 9. We performed a series of radiographs on his left knee A-P, L and an MRI ( fig. 6 and fig. 7) showing a tear in the ACL graft. We decided to perform an ACL revision using BTB graft ( fig. 8) and an ALL reconstruction as shown in the technique previously presented. At the conclusion of the surgery the Lachman test, the anterior drawer sign and the pivot shift tests were negative. The drain was removed the next day after surgery and a hinged knee brace was applied for three weeks. Passive and active range of motion exercises were allowed the second day postoperative.
Results and discussions
In the presented case at 3 months post operative the Lysholm test was 82 and at 6 months it was 91 ( fig. 9). The patient returned to sport at 7 months post operation. The midterm results were very good also because of the integrity of the knee structures (no meniscus and cartilage lesions). The particularity of the case is represented by the fact that the patient is a professional athlete and he needed a fast recovery and a stronger stability of the knee. Due to the necessity of early return to high demanding activities the patient will undergo a continuing rehabilitation program and several other clinical evaluations during the first postoperative year.
The results obtained by associating ACL reconstruction with LEAT tenodesis depend on several factors, of which foremost are the pivot shift test results, with grade 3+ being the clearest indication of a good result. In our experience, another factor that influences the outcome is the overall status of the knee, the presence of osteoarthritis being a strong indication of a poor outcome in time, and is best managed with physical therapy and sometimes arthroscopy [11][12][13]. Another factor that we found to influence results is a limb axis deficiency, most often resulting from a varus or valgus deformation of the knee, and treated with an osteotomy procedure, or sometimes as a consequence of an ankle deformation, or a plantar arch deficiency, treated with physical therapy, laser therapy, medication, and sometimes open or endoscopic procedures [14][15][16].
A particular problem that we found in revision surgery, that directly influences the result of the combined procedure, is the altered bone stock in the targeted areas.
ADVANTAGES OF PEEK SCREWS
This may be grafted, but for a deep understanding of the defect and the manner in which it influences the overall procedure we recommend the use of a 3D reconstruction technique based on the patients MRI or CT scans, which leads to the creation of a 3D printed model of the bone defects [17][18][19][20][21][22]. The 3D printing technology is mostly used in bone defect reconstruction; although recent developments suggest that it may be used in other areas as well, such as prosthetics, in conjunction with other innovative solutions [23].
Different fixation devices have been described, the most commonly used ones being the PEEK screws and the Biocomposite interference screws. PEEK (Polyetheretherketone) is a very strong thermoplastic polymer. The mechanical properties of the PEEK has advantages for various orthopaedic applications. The tensile yield strength and shear strength are superior matches to cortical bone, especially when compared to titanium materials (table 1).
The Biocomposite interference screws used in ACL and PCL reconstruction are made of polylactide (PLA) and polyglycolide (PGA) which are easily degradable within the body. The PLA degrades into lactic acid and the PGA degrades into glycolic acid. Their osteoconductive properties allow for better growth and bone formation when proper growth factors are nearby. In a 2 year animal study (on sheep) biocomposite screws were found to produce new bone, no inflamatory response and little screw degradation [24].
A meta-analysis that included 10 studies published by Pubmed, Medline and the Cochrane database made by North America Arthroscopy Association evaluated the association of a lateral extraarticular tenodesis to the ACL reconstruction. The conclusion was that single extrarticular procedures on an ACL defficient knee did not restore the normal knee function, but when a lateral extraarticular tenodesis was associated with an ACL reconstruction the anterior tibial translation, internal rotation and graft tension were significantly reduced [25].
Another study made byOrtop J Sports Med in 2017 analised the role of LEAT in primary ACL reconstruction in patients with acute lesions (<12 months) and chronic lesions (>12 months). The results did not show a knee function improvement in patients with acute lesion, but showed a decreased lateral translation of the femur in chronic lesions [26].
In the end, we consider that an appropriate reconstruction of the ACL and ALL ligaments is crucial, as a permanent instability of the knee, not considering the patient's comfort, will eventually lead to an ever more advanced osteoarthritis of the knee, which may, in the end, need to be addressed with an uncemented or cemented prosthesis [27].
Conclusions
ACL tears are among the most frequent knee lesions. The gold standard treatment is the anatomical reconstruction of the ACL (single bundle or double bundle) using different tendinous grafts. Despite the anatomical reconstruction, anatomical function may not always be restored (increased internal rotation) and a lateral extraarticular technique is associated. We only use antero lateral reconstruction in patients with ACL reconstruction and pivot shift test positive. We prefer osteoinductive implants (PEEK screws) for their superior mechanical and non-mechanical properties: a superior match to cortical bone, shear strength and low friction coefficient.
In our opinion, the ALL reconstruction is mostly needed in high demand patients, with intraoperative pivot shift still positive after ACL reconstruction, which limits the indication to a select group of patients. | v3-fos-license |
2022-06-27T00:36:13.136Z | 2014-01-01T00:00:00.000 | 98699794 | {
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} | pes2o/s2orc | Solid Phase Extraction of Phospholipids from Brazil Nut ( Bertholletia excelsa ) and Their Characterization by Mass Spectrometry Analysis
: The Brazil nut ( Bertholletia excelsa - Lecythidaceae) is considered a product with high economic value, being a food widely appreciated for its nutritional qualities. Although previous studies have reported the biochemical composition of Brazil nut oil, the knowledge regarding the phospholipid composition exhibits a disagreement: the composition of fatty acids present in the structures of phospholipids is reported as being different from the composition of the free fatty acids present in the oil. In this work, solid phase extraction (SPE) was employed to provide a fast extraction of the phospholipids from Brazil nuts, in order to compare the phospholipid profile of the in nature nuts and their fatty acids precursor present in the oil. The major phospholipids were characterized by mass spectrometry approach. Their fragmentation pattern through direct infusion electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS 2 ) proved to be useful to unequivocal characterization of these substances. High resolution (HR) experiments through ESI using a quadruple time of flight mass spectrometry (QTOF) system were performed to reinforce the identifications.
Introduction
Bertholletia excelsa (Lecythidaceae), an endemic species of the Amazon region, is a large tree that reaches 40-50 m tall. Its fruits can weigh up to 2.5 kg and contain up to 25 seeds. 1,2 The Brazil nut (seeds) is considered a product of great economic value and an important source of nutrients, including protein, fiber, selenium, magnesium, phosphorus and thiamin. Due to its high nutrient content, the Brazil nuts are considered as a product with potential health benefits, including antioxidant, antiproliferative and cholesterol lowering effects. 3 Recent studies performed with Brazil nut oil revealed the presence of phospholipids in its composition. 4 Phospholipids are mostly glycerophospholipid compounds containing a phosphate ester in glycerol at the sn-3 position, being classified according by their polar head group constitution. 5 The main classes of phospholipids are: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), lysophosphatidylinosital (LPI) and phosphatidic acids (PA). 6 Like a dietary supplements, these substances have proven beneficial physiological effects, for example, PI plays a crucial role in cellular signal transduction 6 and PA has been associated with survival, proliferation, and reproduction of cell. 7 Beyond the cited biological activities, phospholipids are also known for their chemical properties, which are important in maintaining the integrity of the cell membranes. 8 As concerns the fractionation of phospholipids, has been employed various methods, such as thin layer chromatography (TLC), column chromatography and solid phase extraction (SPE). 9 Regarding the analysis of phospholipids, various techniques have been used, such as high performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS). 10 MS offers an attractive alternative to phospholipid analysis due to high sensitivity, specificity and simplicity of analysis from the complex biological matrix. 8 Recently, the use of MS to the analysis of phospholipids has become more frequent, 10 highlighting the use of electrospray ionization (ESI), which have facilitated the elucidation of the lipid structures through the increasing of the sensitivity for these substances. 11 *Reprint requests to Felipe M. A. da Silva E-mail: [email protected] All MS Letters content is Open Access, meaning it is accessible online to everyone, without fee and authors' permission. All MS Letters content is published and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org /licenses/by/3.0/). Under this license, authors reserve the copyright for their content; however, they permit anyone to unrestrictedly use, distribute, and reproduce the content in any medium as far as the original authors and source are cited. For any reuse, redistribution, or reproduction of a work, users must clarify the license terms under which the work was produced.
In the present work a selective solid phase extraction (SPE) procedure for phospholipid enrichment was employed for Brazil nut, being the identification of the main constituents performed by fragmentation pattern analysis through the direct infusion using electrospray ionization ion-trap tandem mass spectrometry (ESI-IT-MS 2 ) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS).
Chemicals and reagents
Methanol, acetone and hexane were purchase from Tedia (Faireld, OH, USA). The deionized water used in all of the analyses was obtained by a Milli-Q system, (Millipore, Bedford, MA, USA) (R = 18 MΩ cm).
Plant material and sample preparation
The Brazil nuts were purchased in Manaus Moderna market, Manaus, Amazonas state, Brazil. The proposed method 12 for extracting of compounds of intermediate polarity in Brazil nut was modified as described: The brown skin was removed from the kernel. The kernel was triturated (model TE-102, Tecnal) and then defatted with hexane (1:5 w/v, 5 min, three times) at ambient temperature (22 o C). The defatted samples were extracted with 70% acetone (10 g sample per 100 mL of solvent) over a vigorous stir in a shaker apparatus (model SL 223, Solab) for 30 min at 50 o C. The resulting was centrifuged at 4000 g (5810 R, Eppendorf) for 15 min, being the supernatants collected. The residue was re-extracted twice under the same conditions. The supernatants were combined and concentrated on a rotary evaporator. The residual water was subjected to solid phase extraction (SPE).
Solid-phase preparation and extraction of phospholipids compounds
The method for manual preparation of SPE described previously in the literature 13 was modified as described. A penicillin-type syringe (1 mL) was manually packed with 100 mg of octadecyl silica (C18). The cartridge was first eluted with methanol (3 mL) and then conditioned with deionized water (5% methanol) (3 mL). The aqueous sample was passed through the column. After the clean-up step through the elution with water (3 mL), the retained material was recovered with methanol (1 mL) and dried under a nitrogen gas stream.
ESI-IT-MS 2 analysis
The sample obtained was diluted until 10 ppm in methanol and analyzed by direct infusion into an ESI source of an LCQ Fleet ion trap spectrometer (Thermo Scientific), operating in negative mode at the range of m/z 100-1000 for the fullscan analysis. The ionization source working conditions were as follows: flow rate, 15 µL/min; spray voltage, 5 kV; sheath gas, 10 arb; auxiliary gas, 5 arb; sweep gas, 0 arb; capillary temp, 200 o C; capillary voltage, -40 V and tube lens, -115 V. Data acquisition was carried out with Xcalibur 2.07 software. For the MS 2 analysis of the phospholipids, helium was used as collision gas. The energy of collisional activation was obtained by setting the secular RF amplitude between 25-28%. The major ions were selected for fragmentation in an attempt to characterize them based on their fragmentation pattern.
Results and Discussion
A recent study performed with Brazil nut oil 4 described PI, PA, PC and PE as the major classes of phospholipids, which have occurred at 31%, 24%, 24%, and 21% proportions respectively. This previous work still presents the free fatty acids C18:1 (oleic acid; 39.3%), C18:2 (linoleic acid; 36.1%), C16:0 (palmitic acid; 13.0%) and C18:0 (stearic acid; 11.0%) as the major constituents in the oil, however the composition of the fatty acids moiety of phospholipids are not in accordance with the free fatty acids identified. As example, linoleic acid (C18:2), the second more abundant fatty acid in the oil, appears only in 6%, 8%, and 11% of PC, PE, and PI, respectively. Besides, it has not been detected in the PA composition. Considering such apparent disagreement, the composition of fatty acids moiety of phospholipids from Brazil nut oil was reassessed using a modified method for extraction of substances with low to intermediate polarity based on SPE procedures, targeting a better sample clean up, followed by spectrometric investigation in ESI-IT-MS 2 system.
The total ion spectrum ( Figure 1) obtained from the material recovered from the SPE cartridges displayed mainly high weight ions, with emphasis at m/z 671, 673, 697, 699, 833, 835, 861 and 863 ( Table 1). The less intensity signals observed for free fatty acid ions and the higher intensity for phospholipids ions highlighted the The MS 2 analysis of these ions revealed two well-defined patterns of fragmentation. While for the first four main ions in the spectrum it was observed initial neutral loss of 282 (oleic acid C18:1) or 280 Da (linoleic acid C18:2) followed by a neutral loss of 136 Da (phosphate) (Figure 2), the last four main ions have presented after the loss of 282 or 280, a neutral loss of 162 Da (inositol) followed by a neutral loss of 136 (Figure 3).
These losses of 162 and 136 have been elucidated 14 in mechanistic studies performed with PI compounds, being the first elimination associated with loss of inositol-H 2 O and the second associated with loss of phosphate group in a cyclic form. The absence of this 162 Da at the MS 2 experiments for the first four ions indicates the absence of inositol, suggesting a PA structure (Figure 4). The same sample when subjected to ESI-QTOF-MS analysis displayed the same ions, being the measured m/z compared to the exact theoretical masses for the identified PIs and Pas (Table 1).
After recognizing the classes of phospholipids present in fraction, well as the fatty acid group R2 (Figure 3), it was possible to predict the fatty acid group R1 (Table 1), based on the mass differences observed at the product ion spectra along with the knowledge of the free fatty acids present in Brazil nuts oil 4 , which was confirmed through the signals referents to R 1 CO 2 in spectrum. In this way were identified the presence of oleic acid (C18:1), linoleic acid (C18:2) and palmitic acid (C16:0), all previously described as major fatty acids in Brazil nut oil. 4
Conclusions
The combination of SPE to purify phospholipids and MS to analyze them revealed a useful methodology to improve the gain on sensitivity and has permitted unambiguous characterization of eight phospholipids, four PA and four PI in Brazil nut. For these phospholipids it was not observed disagreement in respect of the composition of fatty acids of the phospholipids and that commonly found in Brazil nut oil. The issue of the reported absence of the linoleic acid (C18:2) as moiety of PA was overcome in this work. This result may have been obtained due the well known accuracy of mass spectrometry for identification of substances, even in complex mixture. | v3-fos-license |
2019-04-10T13:13:20.009Z | 2019-01-08T00:00:00.000 | 104384402 | {
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} | pes2o/s2orc | Using Category Theory to Explore and Model Label Event Structures
The development of a concurrent system poses unique challenges, especially those related to correctness and consistency, as such a system usually involves several interactive processes executing simultaneously. To deal with some of these challenges, we resorted to Labeled Event Structures (LES) and category theory as the formal methods to model concurrent systems. Specifically, in this paper, we proposed an idea to define categories and corresponding con-structs, such as product and sum, to model events and relationships among events represented by LES. To explain the idea, several examples are devel-oped. Though a mathematical proof, the proposed idea helped to build a cor-rect-by-construction approach for formalizing LES models of concurrent systems.
Labeled Event Structure
LES is a mathematical model with true concurrency that describes a process in terms of relations between sets of events it generates. Loogen and Goltz used LES to analyze and model nondeterministic concurrent processes [8]. de León, Haar and Longuet proposed a theoretical framework based on LES for testing and verifying observable behaviors of concurrent systems from true concurrency models like Petri nets or networks of automata [9]. Castellan, Clairambault, Rideau and Winskel introduced a detailed, self-contained update to concurrent games on event structures which were preserved by composition with a copycat strategy, and the construction of a bicategory of these strategies [10]. Bruni, Melgratti and Montanari proposed a definition of a particular class of graph grammars that are expressive enough to model name passing calculi while simplifying the denotational domain construction, and applied this technique to derive event structure semantic [11].
Category Theory
For modeling concurrency, category theory is used to model, analyze, and compare Transition System, Trace Language, Event Structure, Petri nets, and other classical models of concurrency [12] [13] [14]. Sisiaridis, Kuchta and Markowitch proposed a framework for implementing Godement calculus and cartesian closed comma categories for information security management in the detection of threats and attacks in communication systems [15]. Paper [16] in-
Background
In this section, background and work related to our research are introduced.
Labeled Event Structure
An event structure expresses how these events are related to each other. In general event structures that are widely used, a concurrent system or a process can be represented as tuples ( ) ; ; E Con . Definition 1. An event structure is a tuple ( ) ; ; E Con consisting of • a set of events E, • the consistency predicate 2 E Con ⊆ , the set of conflict-free finite subsets of E, and • the enabling relation Con E ⊆ × which satisfies the following properties: • consistency of Con: , , that is, if X enables e so does any conflict-free superset Y of X.
Category Theory
Category theory focuses on the relationships (morphisms) between objects instead of their representations; the morphisms can determine the nature of interactions established between the objects. Definition 2. A category consists of the following data: • Objects: A, B, C, etc.
• For each arrow f, there are given objects: dom(f), cod(f) called domain as well as codomain of f, and f: A → B indicates that A = dom(f), B = cod(f).
• Given arrows f: A → B and g: B → C with cod(f) = dom(g), there is an given arrow: : called composite of f and g. • For each object A, there is an given arrow: 1 A : A → A called identity arrow of A.
These data need to satisfy the following laws: • Associativity: for all f: A → B. Example 2. Let (S; ≤) be a partially-ordered set (poset). Define the category C in which: each member x of S is an object of C; and each relation x ≤ y of (S; ≤) is a morphism x → y of C.
We can verify that C is a category as follows: • For every object x, there is an identity morphism x → x, corresponding to reflexivity, x ≤ x, in the poset.
• The morphisms (x → y) and (y → z) form a composition pair: ; corresponding to transitivity, x ≤ y, y ≤ z, and x ≤ z, in the poset.
• Composition is associative: • the property that, for any object C and morphisms Figure 2 commutes.
That is a a g f Definition 4. Let A and B be objects in a category Cat. Then a sum (or coproduct) of A and B consists of: • an object, A + B, • morphisms, often called inclusions or canonical projections, A B → + , and • the property that, for any object C and morphisms
An Overview of a Vending Machine Example
In this paper, we use a vending machine example to illustrate how to construct categories and the corresponding structures for models specified by LES which is defined and specified in section 3.
Example 3. There is a vending machine and a person. The vending machine can offer coke and pepsi. Each time, the vending machine accepts a coin first, then dispenses a bottle of coke or pepsi according to the person's choice.
In this example, the vending machine and the person are modeled as two processes. When the person inserts a coin and vending machine accepts the coin, this interaction can be represented by the shared event coin in person and vending machine. When person chooses coke or pepsi and vending machine accepts it, this interaction can be represented by the shared event coke or pepsi in person and vending machine. After that, the communications between person and vending machine terminate.
By using LES, the communications between person and vending machine in the example can be modeled as follows: { { } { }} , , , , , ,
Construct Categorical Structures for LES Models
In this section, we use category theory to construct structures for LES models. We first define categorical object and morphism for constructing categories for LES models, then we construct product and sum based on the categories, and we use the vending machine example in Section 3 to explain the categorical structures.
Object
An object is like an event structure, but simpler. Formally, an object (E, R) consists of a set of events, E, and an ordering relation R E E ⊆ × . If events e 1 and e 2 are related by R (that is, ( ) that indicates "e 1 precedes e 2 " or "e 1 happens before e 2 ".
The relation R is a partial order: reflexive, antisymmetric, and transitive. If R is reflexive, transitive. In Figure 3, we omit reflexive and transitive arrows. Note that some pairs of events, e.g., a and c, are unrelated: this means simply that there are no constraints on their order of occurrence.
Morphism
Let ( ) • Event trees are objects: the object shown above in Figure 3 has a tree with c as root.
• There is a morphism from any path in a tree to the tree itself.
Sum (Coproduct)
If S and T are objects, we define their sum(coproduct) S + T to be the smallest object that contains both S and T. The sum can be defined by set union as fol- X → , then X contains T. Thus X contains both S and T. Since S + T is the smallest object containing both S and T, it follows that X must contain S + T and therefore there is a morphism h S T X + → . The morphism h is unique. To illustrate the sum S + T, Figure 5 describes the sum and it commutes. We can use sum to build branching structures, which can be explained by using Example 4 as follows: Example 4. Given (E 1 , R 1 ) and (E 2 , R 2 ), where Then, there is a sum ( which can be represented by Figure 6.
In general, when S + T is formed, we assume that the event structures S and T belong to the same process. If e occurs in both structures, it refers to the same event. ,
Product
The purpose of the product is to combine concurrent processes, each defined by an object, i.e., event structure. To do this, two kinds of event must be distinguished: be objects corresponding to concurrent processes and let 1 e E ∈ . Then: • If 2 e E ∈ , it indicates that e is a communication event and e must occur simultaneously in both processes. • Otherwise, 2 e E ∉ , and e belongs to process P only. In this case, e is an independent event.
Product of P 1 and P 2 can be constructed as follows: be the set of events that communicate, namely the communication set. The communication set is as large as possible, which means that the only events that cannot be added to it are not communication events. T → . Then X must be contained in both S and T. Therefore it must also be contained in S × T which is the largest object contained in both S and T.
Hence the morphism h X S T → × exists and is unique.
Journal of Computer and Communications
To illustrate the sum S × T, Figure 7 describes the product and it commutes. We can use product to build largest communication structures, which can be explained by using Example 5 as follows:
Use Categorical Structures to Model the Vending Machine
In Section 4, this paper provided a vending machine in Example 3 which is modeled by LES. In this section, we use categorical structures defined in Section The above-mentioned sums are just some in the category, while there are other sums that are not listed specifically in above.
Conclusion
In view of the difficulties in the development of concurrent systems, the present Specifically, categorical object, morphism, product, and sum are constructed for events and relationships. To explain the work, a vending machine example is designed by LES, and the communications between vending machines and persons are modeled by category theory. By adopting the categorical approach, we can explore and model concurrent systems designed by LES with categorical structures, which can be specified, proved and composed formally with preserving their properties. In Future, we will explore the usage of more categorical structures, such as limit/colimit and natural transformation in LES which may be useful for the formalization and verification of communications. | v3-fos-license |
2018-12-05T21:42:35.308Z | 2002-11-01T00:00:00.000 | 54972517 | {
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} | pes2o/s2orc | THE ROAD HALF TRAVELED: AGRICULTURAL MARKET REFORM IN SUB-SAHARAN AFRICA
This article reviews the extensive evidence on agricultural market reforms in Sub-Saharan Africa and summarises the impact reforms have had on market performance, agricultural production, use of modern inputs, and poverty. It offers eight recommendations for completing the reform process and developing a new agenda for agricultural markets in Sub-Saharan Africa. The reform experience in Sub-Saharan Africa has varied widely across countries and crop subsectors. The available evidence shows clear progress in some areas and mixed results in others. Most reforms were only partially implemented and policy reversal was common. Once implemented, however, reforms have increased competition and reduced marketing margins, benefiting both producers and consumers. Reforms have also boosted export crop production. On the other hand, food crop production has stagnated and yields have not improved. Further expansion of private trade is constrained by lack of access to credit, uncertainty about the government’s commitment to reform, and high transaction costs.
performance has trailed that of other developing regions (Figure 1).At the beginning of the 21st century, Sub-Saharan Africa confronts a number of daunting problems: extensive hunger, malnutrition, poverty, resource degradation, and the spread of AIDS.Because the majority of the region's population remains dependent on agriculture for its livelihood, well-functioning and efficient agricultural markets continue to be key to improving Sub-Saharan Africa's economic health.This report reviews the extensive evidence on agricultural market reforms in Sub-Saharan Africa and summarizes the impact reforms have had on market performance, agricultural production, use of modern inputs, and poverty.The report offers eight recommendations for completing the reform process and developing a new agenda for agricultural markets in Sub-Saharan Africa.
The need for agricultural reform
Why were agricultural market reforms needed?Answering this question calls for a look at the agricultural policies of the 1960s and 1970s and the problems that resulted.
Agricultural policies before reforms
From independence through the 1970s, African governments played a relatively large role in national economies, and the agricultural sector was no exception.Policymarkers held the common view that private traders were exploitative and that markets could not be trusted with the critical task of feeding the nation.Furthermore, they equated economic development with industrialization, relegating agriculture to the role of supplier of labor, raw material, and cheap food to industry.Small-scale agriculture was seen as inherently inefficient because uneducated farmers were unable or unwilling to apply modern techniques such as mechanization.
Because of these views, state enterprises (often inherited from colonial powers) were given the responsibility of organizing food markers and fixing nationwide prices for farmers and consumers.Their success in doing so varied (Box 1).State enterprises also managed export crop production by farmers by providing inputs on credit, fixing crop prices, and monopolizing the processing and export of the crop.The prices farmers received were generally low because of taxation or high costs incurred by state enterprises, or both.In many countries, export crop prices averaged less than half the world market rate 2 .State enterprises also monopolized the import and distribution of fertilizer and other inputs, which were often supplied to farmers at subsidized prices and on credit.
Pressure for reform
Pressure for economic reforms came from several sources.In the 1970s, commodity prices boomed, allowing governments to expand their operations and greatly increase the size of the civil service.When commodity prices declined in the late 1970s, governments found it difficult to cut expenditures, resulting in large fiscal deficits.Significant losses incurred by state-owned enterprises exacerbated these deficits.Governments generally used monetary expansion to cover the deficits, thus causing inflation.Because exchange rates were fixed, inflation made export commodities less competitive on the international market, simultaneously increasing incentives to import goods that could be produced locally.Import tariffs and other barriers, already kept high to protect domestic industry, were increased further to stem the growing flow of imports.
These policies often had adverse effects on farmers and on the agricultural sector generally.Explicit taxation, the high marketing costs of state enterprises, and the over-valuation of the currency hurt export crop production in particular.In countries with repressive food marketing policies, farmers switched into unregulated crops such as roots and tubers.The emergence of parallel or black markets and cross-border smuggling provided additional evidence of the failure of interventionist policies.Although inputs were subsidized, budget constraints and bureaucratic problems often led to shortages and delays in delivery of these goods.
Inflation, stagnant economic growth, and shortages of consumer goods created doubts about the existing economic strategy.For many countries, however, significant reforms were postponed until trade deficits began depleting foreigh reserves and could no longer be covered by foreign borrowing.At this point, political leaders were forced to seek funding from the World Bank and the International Monetary Fund (IMF), accepting the policy conditions that were attached.Although the process was not uniform across the region, almost all countries adopted a series of economic reforms, including agricultural market liberalization, during the 1980s and early 1990s.
The nature of the reforms
The agricultural reforms introduced by the World Bank and IMF were designed to reduce or eliminate the bias against agriculture and open the sector to market forces 3 .The reforms were based on two beliefs: that reducing or eliminating state control over marketing would promote privatesector activity and that fostering competitive markets would lead to increased agricultural production.To these ends the reforms included four types of measures: * liberalizing input and output prices by eliminating subsidies on agricultural inputs such ad fertilizer and credit, by bringing domestic crop prices in line with world prices, and by ending the practice of imposing a single price for all regions and seasons; The pace and extent of reforms have varied widely across countries and crop subsectors (Tables 1 and 2).For the most part, reforms were not fully implemented.For example, many governments liberalized internal trade but maintained a state monopoly over external trade (Box 2).In other instances, although fixed prices were eliminated, price bands for food crops were imposed to limit market price fluctuations and protect consumers and producers from the allegedly "exploitative" behavior of private traders.State-owned enterprises remain active in several commodity subsectors, notably cotton in West Africa and maize in Kenya, Malawi and Zimbabwe.
Policy reversal was common
Many countries reversed reforms as a result of external shocks or changing economic conditions.Malawi, for instance, reinstated fertilizer subsidies that were to be phased out in the mid-1980s because currency devaluation and the severance of transport routes through Mozambique significantly raised fertilizer prices.Zambia reversed maize market liberalization under pressure from urban consumers who faced higher prices.In general, countries did not follow a linear path toward liberalization, and reforms often were not seriously implemented until the early to mid-1990s (Box 3).
Government commitment was weak
A slow and incomplete reform process resulted from several factors, including weak commitment on the part of African policy-makers to reforms imposed by donors, fear of disturbing existing patronclient relationships, and concern over losing important sources of public revenue.Reforms designed to eliminate the rents and privileges enjoyed by public enterprise employees met with strong resistance.And because governments negotiated and implemented the structural adjustment programs, they often continued the old ways of doing business 4 .For the most part governments did not encourage the participation of important constituents such as private businesses and nongovernmental organizations, choosing instead a top-down approach.At the same time, governments themselves rarely felt the sense of ownership necessary to sustain the reform effort 5 .The resulting climate of uncertainty and mistrust affected private investment, because private businesses were generally reductant to invest in countries where governments lacked credibility.
Reforms were most comprehensive in the food sector
In general market reforms were more comprehensive in food markets than in export crop or input markets.The main reason for this difference was that the purchase and sale of export commodities brought considerable revenue to many governments, while food-crop marketing typically brought losses 8 .In much of Africa, government intervention is still pronounced in the input sector, which traditionally has been more heavily controlled than cereal trading and distribution.Although multinationals and private traders have penetrated the fertilized and seed markets in many countries, state-owned enterprises still dominate those markets in Benin, Ethiopia, Mali, Malawi, Senegal and elsewhere.In Mali, for example, two state enterprises account for 95 percent of fertilizer distribution 9 .In Benin, the cotton parastatal distributes 85 percent of the fertilizer.Although it contracts private companies to import and deliver fertilizer, it fixes prices based on the average bid 10 .
Impact of the reforms
How successful has agricultural market reform been in Sub-Saharan Africa?How has it affected market performance, agricultural production, input use, farm productivity, and poverty?The available evidence shows clear progress in some areas and mixed results in others.
Market performance
Assessments of market performance since the reforms have focused on the expansion of private trading, reductions in marketing margins, and increases in market efficiency (measured by the degree of market integration).In general all three areas have seen improvements since the 1980s.However, further expansion of private trade faces many constraints and marketing boards are still active in some countries.
Private trade has expanded
Market liberalization has encouraged private trade, even in cases where parastatals are still active.Small private traders have emerged in response to increased market opportunities 11 .In certain export markets, the presence of multinationals has fostered a well-coordinated domestic privatetrading sector.In Tanzania, multinationals contract with private, domestic traders to buy tobacco and cashew nuts from small farmers, and these traders have little difficulty accessing credit or finding buyers.In Malawi, small farmers sell their tobacco on auction floors to international buyers, something only large estate farms could do before the reforms.
Further expansion is constrained
Private dealers are constrained by lack of credit and uncertainty about the government's commitment to reform.The lack of access to credit and working capital is a significant barrier to further expansion of private trade, preventing traders from moving into areas such as wholesale commerce, transport and storage 12 .Uncertainty about the role of the state has led to a vicious cycle of mistrust and speculative behavior that hampers the reform process in East and Southern Africa.Other constraints to private-sector expansion include lack of adequate market information, poor roads and communications infrastructure, limited access to storage and transport facilities, and unclear property rights (Box 4).
Marketing boards are still active
Marketing boards also continue to constrain private trade.Grain marketing boards in most countries of Sub-Saharan Africa were restructured and given responsibility for a few functions developing a market information system, stabilizing prices, and ensuring food security, in part by maintaining stocks 13 .But many marketing boards lack the money to support prices and in some countries are still active in the grain trade, competing with private dealers.The combination of the private sector's uncertainty about future policy and state involvement in grain trading makes for volatile grain markets.In Benin, Côte d'Ivoire, Mali and Senegal, parastatals continue to handle the majority of marketing and distribution for export crops.
Marketing margins have narrowed
Marketing margins defined as the spread between producer and consumer prices have fallen from prereform levels, reflecting the lower marketing and processing costs of a more competitive private sector.In turn, this has improved transmission of world prices to farmers and forced real consumer prices downward 15 .
Two main factors have helped reduce marketing margins in maize markets.First, in East and Southern Africa, private-sector participation in grain marketing and milling and the liberalization of interregional trade have narrowed the margins between producer and consumer prices for maize meal and increased consumer access to maize markets.When the government stopped subsidizing large maize mills, many households began buying from smaller mills where maize meal was cheaper, raising market share for these small enterprises 16 .Second, price spreads between surplus and deficit regions have declined (Figure 2).Cereal market liberalization in Mali, for instance, has reduced transaction costs for private traders (who no longer need to operate on the black market) and increased the flow of cereals from surplus to deficit areas.
Marketing margins have decreased similarly in the export crop sector, in countries where export market liberalization has occurred.The producer's share of the f.o.b. (free on board) price is the best measure of how well the reforms have succeeded in passing on the benefits of liberalization to producers.The producer's share of the f.o.b. price has ranged from 64 to 98 percent in Cameroon, Malawi, Tanzania and Uganda, where markets have been widely liberalized.But in Benin, Côte d'Ivoire, Ghana and Senegal, where reform has been slow or nonexistent, the producer's share has remained low between 37 and 62 percent.
Markets have become increasingly integrated
In general, market integration is measured by how well price signals are transmitted among markets.Integrated markets allow for the efficient flow of commodities from surplus to deficit regions.While the level of market integration in Africa remains lower than in other developing countries, it has improved since the reforms were instituted 17 .In part this improvement has depended on the prereform situation.For example, countries such as Benin and Ghana, where the private sector controlled food marketing before liberalization, have developed better-integrated grain markets than countries such as Malawi and Madagascar, where parastatals dominated food marketing 18 .Most of the improvement is the result of increased private-sector participation in trading activities participation that has fostered the efficient transmission of information and prices among markets.
Agricultural production
The basic premise of agricultural market reform is that improving the incentive structure for small farmers (in the form of higher prices and well-functioning markets) will generate a positive supply response, increasing both agricultural output and income levels.But the average growth of agricultural production per capita has been negative in Sub-Saharan Africa since the 1970s (Figure 1).For small farmers in some countries, reform has meant the elimination of government input and credit subsidies a loss that has kept yields stagnant or reduced them.Where production growth has occurred, it is the result of increases in the amount of land under cultivation rather than of yield increases.And where producers have benefited, the bulk of the gains have gone to export and cashcrop farmers with access to credit and modern inputs (such as fertilizer).
Food production has stagnated
Although positive changes have occurred in some sectors and countries, overall food production per capita in Sub-Saharan Africa has stagnated.West African countries have performed better in terms of food production than countries in East and Southern Africa, for several reasons (Figure 3).First, the food markets in West Africa have traditionally been more open than those in other parts of the continent.Second, the 1994 devaluation in West Africa increased the relative price of imported foodstuffs, boosting the demand for local food crops.Finally there is some evidence that because of higher prices received by cash-crop producers, fertilizer use on cash crops has increased in some West African countries (Benin, Burkina Faso, Mali).Increased access to fertilizer and cash income from export crop production has also allowed farmers in parts of West Africa to use more fertilizer on food crops.In contrast, food crop production in several East and Southern African countries has declined because the elimination of food subsidies has resulted in lower farm-gate prices for maize.
The food supply response in Sub-Saharan Africa has been limited by structural and institutional constraints that have persisted despite market reforms.Nonprice factors can have a more profound impact than prices on aggregate agricultural output 19 .These factors include the condition of infrastructure (such as roads, irrigation schemes, and communication networks); the availability of marketing services, modern inputs, and credit (especially in rural areas); and government support in the form of research and extension services, human capital development, and commitment to reform.Physical factors such as the weather and soil quality also affect output.
Export crop production has increased
Export crops have responded more strongly to liberalization than foodgrains.Most price changes have favored tradables, making export crops more attractive than domestic staples.Export crops were also taxed more heavily before the reforms, making their postreform response particularly vigorous.In addition, price control was far less effective for food than for export crops, so that farmers growing food crops were less affected by official prices (and more responsive to unofficial prices) than farmers growing export crops.Cash-crop sectors such as cotton in Benin and Mali, cashew nuts in Mozambique, and Tanzania, and coffee in Uganda have been among the most responsive to reforms, largely because of higher producer prices, exchange rate liberalization, privatization, infrastructure investment, and improved input supply.
Input use
The use of modern inputs declined in some countries more so for food than for cash crops, and in part because of the elimination of input subsidies.The lack of well-functioning agricultural credit markets to finance purchases of inputs has exacerbated the problem.In general, the use of fertilizer, arguably the most important purchased input in African agriculture, is still very low, especially compared with other developing countries (Table 3).
Fertilizer prices have risen
A number of reforms have affected fertilizer prices, including the elimination of fertilizer subsidies, the depreciation of the real exchange rate, and liberalization of fertilizer imports and distribution.The fertilizer-crop price ratio has more than doubled in four out of ten countries examined (Benin, Ghana, Nigeria, and Tanzania) and increased at least 50 percent in three more (Malawi, Senegal, and Zambia).On the other hand, the fertilizer-crop price ratio fell in Ethiopia, Kenya, and Zimbabwe.
Fertilizer use declined before rebounding slightly
Fertilizer use rose steadily from 1970 until 1992 (Figure 4).The positive trend stopped in 1992 as fertilizer use dropped more than 20 percent in 1994 and 1995 before partially rebounding in 1996.
The drop in Nigerian fertilizer use, which accounts for more than one-third of fertilizer use in Sub-Saharan Africa, explains the decline in fertilizer consumption in 1994 and 1995.Fertilizer application rates, expressed as kilograms of nutrients per hectare of arable land, generally followed the trend in use.
Declining demand for fertilizer since liberalization has led to speculation that fertilizer market reform has had adverse effects on agricultural productivity and rural incomes.Indeed, some of the effects of fertilizer reform have been less positive than expected.Cost savings from privatization have not been enough to prevent prices from rising as subsidies were removed.Despite the increased availability of fertilizer through new, more efficient delivery networks, demand remains weak.Increased fertilizercrop price ratios mean that many farmers have been reluctant to increase their use of fertilizer, and in some countries farmers use less fertilizer than they did before the reforms.
On the other hand, fertilizer market reform has fulfilled expectations in some respects.Comparing the early 1980s and the mid-1009s, fertilizer use has fallen in 7 of the main fertilizer-using countries, but increased in 14.Countries devoting a higher share of fertilizer to cash crops have seen fertilizer use grow relatively rapidly through the reform period.This includes most of the cotton-producing countries of West Africa.In contrast, countries using fertilizer mainly on maize, such as Ghana, Malawi, Tanzania, and Zambia have seen slower growth and some declines in absolute fertilizer use.
Effects on production and income are negligible
It is difficult to find evidence of any adverse impact of fertilizer market liberalization on agricultural production in Sub-Saharan Africa.Agricultural production appears to be more sensitive to weather, agricultural policy, and shifts in exchange rates than to fertilizer policy.The removal of subsidies and subsequent increases in the fertilizer-crop price ratios have had negligible impacts on welfare.The changes affected approximately 15-35 percent of rural households (that is, those that used fertilizer) and had a greater impact on relatively larger farms located in high-potential areas because these farms benefited the most from fertilizer subsidies.Several estimates suggest that fertilizer subsidy removal reduced rural income by 1-2 percent at most.On the other hand, the positive effects of better availability, timing, and product choice have not been evaluated.
Access to credit for input use has decreased
Access to credit for input use has declined in cases where state-sponsored credit systems have collapsed.The decline in input use in Sub-Saharan Africa is most serious in countries where input and credit subsidies were eliminated at the same time, and where fertilizer is mostly applied to maize (such as in Malawi).In Tanzania, where inputs on credit are not available and export markets are no longer dominated by state-owned enterprises, input use on cotton has also declined.In contrast, in countries such as Benin and Burkina Faso, where cotton inputs are available on credit through the government, input use has increased and has had positive spillover effects on food production.
The integrated cash-crop marketing and input distribution system provided by state-owned enterprises allowed inputs to be purchased on credit.But the private sector has not been able to provide inputs on credit to farmers because of its inability to enforce loan repayment.Under parastatal marketing arrangements, farmers often received seed, pesticides, and fertilizers from the parastatal.The cost of those inputs was later deducted from what the parastatal paid for the crop.
With multiple outlets now available, the farmer is not obliged to sell the crop to the entity that advanced funds for inputs.This situation has prevented the private sector from offering inputs on credit.
Total factor productivity has increased
Changes in total factor productivity, defined as the amount of total output generated by all factors used in production, provide the most accurate measure of national and regional productivity trends.Total factor productivity stagnated throughout the mid-1960s and 1970s but appears to have increased in the 1980s and 1990s.The lagged effect of research expanditures and policy reform account for as much as two-thirds of the growth in total factor productivity during the 1980s 20 .This raises questions about whether the recovery in the 1980s is sustainable in the long run given the reduction in research expenditures throughout the reform period.Countries such as Kenya and Zimbabwe, for example, have not experienced significant increases in food productivity since liberalization.In these countries the elimination of public support for small farmers cost there producers access to affordable modern inputs.Land productivity stagnated or declined as a result 21 .
Poverty
Agricultural production constitutes the most important source of income and employment for the majority of households in Sub-Saharan Africa.By stimulating agricultural production, market reforms were expected to improve rural incomes and alleviate poverty.In many Sub-Saharan African countries, rural poverty rates have declined since the 1980s.Although not all of the decline can be attributed to agricultural reforms, this trend challenges the view that the rural poor have been adversely affected by agricultural market liberalization.
Changes in producer food prices have had mixed effects
The impact of market reforms on income through changes in food prices has varied across countries and crops, in large part owing to differences in prior conditions and levels of intervention.Many smallholder farmers in Sub-Saharan Africa are net foodgrain buyers.Therefore, in the short run, higher farm prices could be detrimental to these households unless market reform can reduce the margin between producer and consumer prices.For example, one-third of rice farmers that fell below the poverty line were hurt by higher and more variable rice prices following agricultural market liberalization in Madagascar 22 .However, in countries such as Côte d'Ivoire, where both agricultural production and household food consumption are diversified, increases in producer prices had no significant impact on the rural poor 23 .
A larger marketable surplus is usually associated with larger and wealthier farms, and therefore higher farm prices tend to favor the better-off farmers more than the poor ones 24 .In East and Southern Africa, however, reforms have resulted in lower farm prices, thereby hurting commercial farmers.Some households may produce mainly for subsistence especially in remote areas and therefore will have little or no contact with agricultural markets and will be insulated from changes in product prices 25 .
Market reforms have reduced consumer food prices
Although market reforms could lead to higher farm prices they could simultaneously reduce consumer prices by decreasing marketing costs.Increased competition and greater efficiency brought about by liberalization have reduced marketing margins in many African countries, thereby benefiting both food producers and consumers 26 .In Ghana food prices have decreased since the reforms, in part because investments in roads and better trucks have reduced marketing costs 27 .In East and Southern Africa maize meal production has shifted to cheaper mills, offsetting the price increases that came with the elimination of consumer price subsidies 28 .Overall, consumer prices for major food crops have fallen in a number of countries, including Ethiopia, Ghana, Kenya, Mali, Tanzania, and Zambia.
Reforms reduced market inefficiencies, offsetting some of the negative effects of price rises
Even if food crop prices did rise, their negative impact on net food buyers and poor consumers was mitigated by a reduction in market inefficiency following liberalization.Before reforms, many governments were ineffective in providing cheap food to the most needy and created many marketing inefficiencies 29 .As a result, many poor consumers had to rely on parallel markets to meet their food requirements because government-subsidized grain was often rationed or poorly targeted.
Higher export crop prices have benefited export crop farmers
Devaluation and export market liberalization increased the income of small export growers by about 20 percent on average between 1990 and 1997, although this varied greatly across countries and crops.The income of poor and nonpoor rural households has increased in several countries, including Benin, Cameroon, Gambia, Madagascar, Malawi, Niger, and Uganda.Small-scale cotton growers in Benin and producers of cashew nuts and tobacco in Tanzania have benefited from higher producer prices thanks to declining marketing margins and the depreciation of the real exchange rate.Similarly, following the liberalization of burley tobacco production and marketing in Malawi, increased tobacco production increased the cash income of smallholder farmers in that country (Box 5) 30 .
Rural poverty has declined in many African countries
Contrary to conventional wisdom, the evidence indicates that rural poverty has fallen in many African countries.In Tanzania, for example, a comparison of pre-and postreform household surveys suggests that rural incomes and poverty declined between the late 1970s and the early 1990s 33 .Similarly, a comparison of two household surveys in Uganda estimates that the incidence of poverty fell from 56 percent in 1992 to 46 percent in 1996 34 .Using an index that combines information on the ownership of household assets and housing characteristics, another study findsthat rural poverty declined in Ghana, Kenya, Madagascar, Mali, Tanzania, Uganda, and Zambia 35 .In half of the countries, poverty fell more than 9 percentage points.The evidence was mixed in Senegal, and poverty increased in Zimbabwe.
The future of agricultural market reform in Sub-Saharan Africa
The reform efforts of the 1980s and late 1990s have generated a positive response in the agricultural sector of Sub-Saharan Africa.Despite the progress that has been made, however, the results of market reform have generally not met expectations, and much remains to be done.
The reforms focused on eliminating government control and increasing the producer price of tradable agricultural commodities but placed little emphasis on developing the institutions needed to support private sector activity.Improving price incentives and liberalizing markets were expected to be enough to generate a supply response and create well-functioning markets.The private sector was expected to take over the institutional functions the state had been providing.The reality has been quite different.While private trade has increased in virtually all agricultural markets, the private sector has been unable or unwilling to supply credit and marketing services in remote areas.And although the elimination of policies enforcing a uniform, nationwide price has been a boon for many producers and consumers close to markets, it has often left farmers in remote areas worse off than they were before liberalization.
Constraints to further reform
Sub-Saharan Africa faces a number of constraints in its efforts to reduce poverty through agricultural market development.These constraints include: What must be done to overcome the remaining constraints and make agricultural market reform more effective?
A new agenda
Further progress in developing well-functioning markets will require not only further liberalization but also a more concerted effort to go beyond the withdrawal of the public sector from agricultural marketing.The state must assume a new, supportive role as market facilitator.One aspect of this role is to strengthen investment in public goods such as infrastructure, research and extension, and public market information.The second is to foster institutions required for the development of competitive and efficient markets.The new agenda for market development in Sub-Saharan Africa includes the following eight priorities:
Fully implement all reforms
Experience shows that market performance improves and marketing costs fall once the government no longer monopolizes trade.
Find institutional solutions to provide input credit fo farmers
Credit for input use can be provided through a number of institutional innovations, including contract farming, credit associations, group lending, and farmers' organizations.
Develop a legal infrastructure for market transactions
This long-term step will reduce the risk of investment and decrease transaction costs for both farmers and traders by clarifying property rights, enforcing contracts, ensuring quality control, and establishing rules of market conduct, among other legal concerns.
Increase investment in infrastructure and institutions
Higher productivity and effective markets require investment in research and extension, access to market information, and efficient transportation and communication networks.
Promote effective governance and state capacity to monitor market development
Proper governance will prevent investment from being channeled to rentseeking groups and will ensure that funds are distributed to their intended uses.Improved state capacity to monitor market development would allow governments to anticipate undesirable market developments and devise appropriate responses to eventual short-term difficulties in a timely and effective manner.
Encourage smallholder production of export crops
In many areas, food and export crop production are highly complementary and export crop production has positive spillover effects on input use and food crop productivity.Therefore, promoting smallholder production of export crops should have beneficial impacts on agricultural production in general and on the food security and income of smallholder farmers in particular.
Address the problems of vulnerable groups in remote areas
Farmers in remote rural areas have suffered from the loss of parastatal activity and official pricing that effectively subsidized high transportation costs.Short-term targeted interventions may be needed to alleviate these problems.
Institute credible, sustainable macroeconomic policies
Indirect taxation through overvalued exchange rates and protective industrial policies can have a more negative effect on agricultural incentives than direct taxation.In addition, stable and predictable macroeconomic policies encour-age savings and investment and focus private sector effort on efficiency rather than on anticipating and reacting to macroeconomic shocks.
Notes : 1 Agriculture is the mainstay of African economies.In Sub-Saharan Africa it represents between 27 and 42 percent of gross domestic product, employs between 65 and 80 percent of the labor force and in more than half the countries accounts for as much as 60 percent of export revenue.Notes: With the exception of Ethiopia, margins are the relative margin, defined as the price spread divided by the producer price.For Benin, the price spread for maize is between Parakou and Cotonou for the periods 1985-1989 and 1990-1995.For Malawi, price spread for maize is between Nikhotakota and Lilongwe for the period 1984-1987 and 1988-1991.For Ethiopia, the price spread for teff is the absolute margin between Addis Ababa and Bako for the period 1986-1996.
See A. Abdulai and C.L. Delgado, Re-establishing agriculture as a priority for development policy in Sub-Saharan Africa (Washington, DC: International food policy research institute, 1995). 2 See, for example, World Bank, Accelerated development in Sub-Saharan Africa: an agenda for action (Washington, DC, 1981); U. Lele, Agricultural growth and assistance to Africa: lessons of a quarter century, International center for economic growth sector studies 2 (San Francisco: ICS Press, 1990); W.K. Jaeger, The Effects of economic policies on African agriculture, Discussion Paper 147, Africa technical department series (Washington, DC, World Bank, 1992). 3World Bank, Accelerated Development in Sub-Saharan Africa.
Figure 2 .
Figure 2. Difference in marketing margins between central markets in selected countries.Sources: O. Badiane, F. Goletti, M. Kherallah, P. Berry, K. Govindan, P. Gruhn and M. Mendoza."Agricultural input and output marketing reforms in African countries", final donor report (IFPRI, Washington, DC, 1997); A. Negassa and T. S. Jayne, The Response of Ethiopian grain markets to liberalization, Working Paper 6 (Addis Ababa: Grain market research project, Ministry of Economic Development and Cooperation, 1997).
Figure 3 .
Figure 3. Net per capita food production, 19961-1997.Source: FAOSTAT 1998 (statistical database of the Food and Agriculture Organisation of the United Nations).Notes: Each graph line represents the index for region divided by the index for that region divided by the index for all regions.
Figure 4 .
Figure 4. Global fertilizer application rates, by region.Source: FAOSTAT 1998 (statistical database of the Food and Agriculture Organisation of the United Nations). | v3-fos-license |
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} | pes2o/s2orc | Evaluation of the Interaction Among Microalgae Spirulina sp , Plastics Polyethylene Terephthalate and Polypropylene in Freshwater Environment
The annual plastic production in Indonesia has exceeded 4.6 million tons and accumulated in the aquatic system. Polyethylene terephthalate (PET) and Polypropylene (PP) are the most widely used plastics in manufacture of packaging, fibres, and drinking bottles, etc. The degradation of these plastics to micro sizes leads to environmental threats, especially when the micro plastics interact with fresh water microorganism such as microalgae. Therefore, the study on the interaction between micro plastics and microorganisms is really important. The aim of this study was to evaluate the impact of microplastics on the growth of microalgae Spirulina sp and also to evaluate the contribution of microalgae Spirulina sp to the plastic degradation. The interaction between microalgae and microplastics was evaluated in a 1 L glass bioreactor contained microalgae Spirulina sp and PP and PET microplastics with the size of 1 mm at various concentrations (150 mg/500 mL, 250/500 mL and 275 mg/500 mL) for 112 days. The results showed that the tensile strength of micro plastic PET decreased by 0.9939 MPa/day while PP decreased by 0.1977 MPa/day. The EDX analysis of microplastics showed that the decreasing carbon in PET (48.61%) was higher as compared to PP (36.7%). FTIR analysis of Spirulina sp cells showed that the CO2 evolution of cells imposed by PET microplastic was higher than imposed by PP. The growth rate of Spirulina sp applied by micro plastic was lower than the control and the increase of microplastic concentration significantly reduced the growth rate of algae by 75%. This research concluded that biodegradation has important role in the degradation process of plastic.
INTRODUCTION
Indonesia is facing plastic waste threats which currently reach 2.4 million tonnes and spread to aquatic environments.These plastics contaminate the natural environment and also endanger the ecosystem, especially when they degrade into small pieces (Moore, 2008).The plastic debris in the environment includes ropes, plastic bag or packaging, whichj are available in the form of macroplastics and microplastics.Large plastic items (macroplastics) have been indicated in the marine system for long time period (Derraik, 2002).Microplastics (< 5 mm) have recently attracted attention because of their ingestbility by organisms as well as transport for the pollutants into food chain (Teuten et al., 2009).Microplastics in the aquatic environment that can originate from various sources such as UV degradation and fragmentation of plastics or caused by damage during the transportation process, mechanical damage and also aquatic environment direct release (Andrady, 2003;Cole et al., 2011;Erikson et al., 2013;Rezania et al., 2018).
The most common and abundant polymers are high-density polyethylene (HDPE), lowdensity polyethylene (LDPE), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) and polyethylene terephthalate (PET), and among these plastic, PET and PP are the major plastic wastes which have different chemical-physical properties and functions (Andrady and Neal, 2009).PET and PP are derived from thermoplastic polymers which are mostly used for packaging materials.For example, single use plastic bottles for mineral water and soft drinks are made from polyethylene terephthalate (PET) which is highly recyclable with a density of 1.41 g / cm 3 (Mortula, 2013).Furthermore, PET contains two hydroxyl (OH) groups and dicarboxylic aromatic acid, which comprises a large, six-sided carbon (or aromatic) ring and two carboxyl (CO 2 H) groups.Under heat and catalysts, the hydroxyl and carboxyl groups will react to form ester (CO-O) (Venkatachalam et al., 2012;Farzi et al., 2019).The presence of a large aromatic ring in PET makes it stiff, strong, tough, hydrolytic, solvent resistant (Venkatachalam et al., 2012) and hygroscopic (Ma and Buhshan, 2003; CWC Best Practices in PET Recycling, 1997).In turn, polypropylene is polyolefin with linear hydrocarbon polymers (Arutchelvi, 2008) has the lowest density among commodity plastics that is about 0.94 g / cm 3 (Howe, 1999;Cole, 2002 andSchimanski, 2018).PP has three stereo configurations that can be distinguished, namely the isotactic, the syndiotactic and the atactic form.The isotactic PP (i-PP) contributes the most to the consumption of polypropylene because of its properties: ease to handle, stablility in aqueous solutions and organic solvents and also thermal-stability (Schimanski, 2018;Bertin et al., 2010).PP also has an excellent property regarding on chemical and temperature resistance which makes PP particularly suitable for application in many food packaging purposes especially that have to be sterilized frequently (Maddah, 2016).
The degradation process is the most important fate of plastics in the environment.Degradation of plastics may occur through some mechanisms that involve thermal, chemical, photo and biological degradation (Alshehrei, 2017;Gewert et al., 2015).Biodegradation of plastics occurs due to the action of extra cellular enzymes secreted by the microorganisms when the organisms attach to the surface of plastics leading to physical and chemical change of the latter (Lucas et al., 2008;Alshehrei, 2017;Arutchelvi et al., 2008).The growth of microorganisms utilising the plastics polymer as a carbon source and with the availability of oxygen, plastics will degrade completely by using CO 2 and the biomass as an ultimate product (Shah et al., 2008;Muthukumar and Veerappapillai, 2015;Arutchelvi et al., 2008;Arkatkar et al., 2009).
The hydrophilicity of plastic has important role in attaching microorganism cell to the plastic surface and therefore will affect the biodegradation process of plastic.PP which has a CH 2 group will be prone to attaching to hydrophobic polymeric surfaces (Arutchelvi et al., 2008).Due to the presence of ester and terephthalate group in PET, this plastic it polar molecule and therefore it is more hydrophilic (Venkatachalam et al., 2012).On other hand, Lucas et al. (2008) assumed that the esters group in PET makes it more resistant to biodegradation compared to other polymers.The extracellular polymeric substances produced by microorganisms can play a role as surfactants contain both hydrophobic and hydrophilic groups that support the exchanges between hydrophilic and hydrophobic phases (Lucas et al., 2008).
Microalgae are photosynthetic microorganisms which utilize CO 2 as carbon source to form biomass. Microalgae are mostly used as food sources in which they contain hydrocarbon, lipids and protein and other high added value compounds (Habib, 2008 andHariyati, 2008).Since they are used for food products, microalgae must be free from pollutants, including microplastics.Plastic can be used as carbon sources for microalgae and the release of plastic additives promote the growth of microorganisms by supplying the nutrient source (Rummel et al., 2017).Since micro-plastics have sizes of 1 to 5 mm (Lee et al. 2013), they are a suitable medium for bacteria and microalgae to form a bio-fouling.Bio-fouling that occurs on micro plastic surfaces causes shading effects which decrease light intensity, thus affecting the photosynthesis of microalgae (Sjollema et al., 2015;Yurtsever et al., 2017).Furthermore, the impact of micro plastics on microalgae growth is also affected by the availability of oxygen and CO 2 evolution as the consequence of plastic degradation (Shah et al., 2008;Arutchelvi et al., 2008).
The study on the biodegradation of PET and PP carried out simultaneously has not been found by the authors but some reports about PET and PP biodegradation in separate experiment have been observed.Farzi et al., (2019) studied the kinetic modelling in the process polyethylene terephthalate biodegradation waste using Streptomyces sp.The results showed that particle sizes and time of reaction were the most important parameters affecting biodegradation and the Michaelis-menten model can predict precisely the experimental results.Auta et al. (2018) evaluated the growth kinetics and biodeterioration of polyethylene microplastics by Bacillus sp and Rhodococcus sp and concluded that these microorganisms could modify and utilize PP microplastics as carbon source.
Lagarde et al. (2016) investigated the effect of polymer type during the interaction between micro plastic and freshwater microalgae.They reported that microalgae were over-expressed of sugar biosynthesis in HDPE rather than in PP.Furthermore, Yoshida et al., (2016) isolated a bacterium to breakdown the PET within 6 weeks.Long et al. (2015) evaluated the interaction between microplastics and phytoplankton aggregates.The result showed that marine aggregates can be an efficient sink for the microplastics.
Sharon and Sharon (2012) studied the plastic biodegradation of polyethylene terephthalate plastic in microbial culture and the degradation was slow and weak.It also demonstrated that microbes could act on the polyethylene terephthalate to form biofouling. Nowak et al. (2011) studied the biodegradation of modified PET by using polyester in Penicillium funiculosum culture.The result showed that modified PET was not significantly degraded in the presence of the culture.Since the interaction of micro plastic and microorganism is relatively strong, further research is required.The purposes of this research were to evaluate the contribution of Spirulina sp in the process of plastic degradation and to observe the impact of microplastics on the Spirulina sp growth.
Materials
Polyethylene Terephthalate (PET) used in this research was obtained from Danone Indonesia that already standardized by SNI 19-4370-2004 (Nasional Indonesian Standart for Plastic bottle Single-use).Polypropylene (PP) 15 μm thick was provided by PT.Indofood Sukses Makmur Tbk.
Spirulina sp cultivation
Spirulina sp used in this experiment was supplied by C-Biore Laboratory, Diponegoro University.The experiment was started by testing the physiological form of Spirulina sp which included maximum cell density by using a spectrophotometer, measurement of pH, temperature, oxygen content and CO 2 content.Furthermore, Spirulina sp is placed in 2 pieces of 35 cm x 25 cm glass ponds and 7 pieces of 500 mL Erlenmeyer, each equipped with an aerator as a source of oxygen and LED lights (3000 lux) to provide light intensity, then the temperature is maintained at 24-26 o C and pH between 7-8.Nutrition needed to maintain the Spirulina sp growth is given every 2 days with a mix of TSP and Urea 12.5 mg / 250 mL of Spirulina sp and 100 mg NaHCO 3 / 250 mL Spirulina sp (Hadiyanto and Azim, 2012).
Sample Preparation
PET and PP plastics were washed with etanol and dried at room temperature for 24 hours and then the plastic was cut to the size of 5 x 5 cm to be applied in a glass ponds containing Spirulina sp.Microplastics were obtained by cutting a PET and PP plastic in the same size between 1-2 μm.The microplastic was weighted carefully at 150 mg, 200 mg and 275 mg and mixed into 500 mL of Spirulina sp culture.Stirring was done with the aerators so that microplastics were distributed into the Spirulina sp culture properly.Nutrient, pH, temperature, light intensity and oxygen supply were maintained and the growth was measured by using a spectrophotometer (Spectroquant pharo 300 M and Spectrophotometer SP -300 Optima) at 560 nm wavelength and ultrapure water was used as a blank solution (Hadiyanto et al., 2012).
Tensile strength measurement
In SNI 7818: 2014 and SNI 7188.7.2011, it was stated that one of the degradability tests for plastic is by using tensile strength test and it was also supported by ISO 527-3 (Lucas et
Morphologies
The morphology of PET and PP was observed using scanning electron microscope (SEM) and combination with Energy Dispersive X-ray spectroscopy (EDX) to determine the inorganic elements contained in the material (Lucas et al., 2008).The analysis was conducted at room temperature and metalized using Au.A Jeol (model JSM-6510 LA ) at 3000x magnification.
FTIR analysis
FTIR is a common technique used for the study of macromolecules such as PET and PP polymer that was recommended for investigation of plastic degradation as mentioned in ISO 4582 and ISO 4892 for UV exposure, and for microorganism's surface colonization in ISO 846 and ISO 11266 (Lucas et al., 2008, Melissa et al., 2018, Schmitt et al., 1998).PET and PP plastics that were applied in Spirulina sp were taken every 7 days for about 112 days.Prior to the FTIR test, plastics were rinsed with aquadest and left to dry for 24 hours, then the plastic was cut at a size of 1.5 x 2 cm.A Perkin Elmer Type Frontier was used to collect spectra from 4000-200 cm -1 (SNI 19-4370-2004 method) and ASTM D6288-89.FTIR test was also conducted in Spirulina sp which had interacted with microplastic treatment for 7 days.Filter Spirulina sp containing micro plastic with diameter of 200 mm / 8 "stainless steel 40 mesh sifter sieve fine wire strainer to obtain Spirulina sp without microplastic.
Contribution of Spirulina sp in the plastic degradation processes
The standard test of the elasticity (elongation) properties for degraded plastics in Indonesia is regulated by SNI 7188.7:2011 (BSN, 2011) which requires tensile elongation, while on an international scale, it is regulated in ASTM D3826 concerning the procedures for determining the end point of degradation in Polyethylene and Polypropylene plastic using tensile test.The quantitative relationship between tensile strength and degradation is the first step in the process of investigating yjr plastic degradation (Guo et al., 2016).When plastic changes both due to biotic and abiotic factors, the strength of stress and the versatility of plastic will alter in line with changes in the molecular structure of the polymer.Therefore, the initial identification of the plastic degradation process is by measuring the tensile strength.There are several factors that influence the tensile strength of plastic, namely the molecular structure that also affects the density of the plastic, the temperature at which the plastic is applied and the chemical composition of the plastic itself.Measurements of tensile strength were carried out on two types of plastic and the measurement results can be shown in the curve of tensile strength degradation over time.
The change in mechanical polymer property by tensile strength measurement
Figure 1 depicts the tensile strength degradation over time of two plastics (PP and PET).The decrease in tensile strength in PET is far greater than the rate of decrease in tensile strength of PP.The density of PET is higher (1.37 kg / m 3 ) than PP density (0.94 kg / m 3 ), which leads to higher tensile strength value.
The tensile strength of both plastic during interaction with Spirulina sp for 112 days showed a greater rate of tensile strength decrease of PET compared to PP.The hydrophilic and hydrophobic nature of polymers plays a role in biofilm and hetero-aggregation formations in the biodegradation process of polymers in the aquatic system (Merina, 2014; Cerca et al., 2005;Lobelle et al., 2011).The formation of biofilms by microorganisms can improve the hydrophilic properties of the plastic surface (Lobelle et al., 2011).PET is more hydrophilic than PP, as it contains polar group (C = O bond) (Lai et al., 2006).Therefore, PET has greater potency to experience exopolysaccharide (EPS) heteroaggregation produced by Spirulina sp.Spirulina sp has both hydrophilic and hydrophobic properties because of its high protein contents (Bashir et al., 2015).The exopolysaccharide heteroaggregation that occurs in PET surfaces happened when microalgae reach the stationary growth phase (Long et al., 2017) where (EPS).Heteroaggregation is very important in determining the fate, transportation, transformation and toxicity of nanoparticles in aqua phase (Wang et al., 2015).Heteroaggregation in PET results in more brittle PET which causes the tensile strength of PET decrease faster than in PP.
The difference in the decrease rate of tensile strength in PET and PP can also be caused by chemical composition, especially the amount of additives added in the process of plastics production (Lagarde et al., 2016).
The change of organic groups investigation by FTIR Analysis
FTIR analysis is a suitable technique for investigating the degradation of plastics in the environment through the determination of changes in various functional groups that contribute to the plastic polymer compound (Loakeimidis et al., 2016).Some results of research on the FTIR's ability in analyzing organic functional groups in plastic polymers were reported in a review of Jung et al., (2018) who concluded that FTIR spectroscopy is able to provide a simple, effective and non-destructive method for identifying and distinguishing functional groups organics widely in most plastic polymers with high accuracy results.
Figure 2 shows the FTIR analysis of PET before and after interaction with Spirulina sp.The characteristic of PET were identified by its functional groups of for C = O stretch (ketone) at wavelength of 1718 cm -1 , C=C aromatics at 1505 cm -1 , 1523 cm -1 , 1578 cm -1 and 1613 cm -1 , (CO) aliphatic ether at 1125 cm -1 , aromatics (CH) at 874.5 cm -1 and aromatic bonds (CH) at 733 cm -1 .
The spectrum of PET after interaction with Spirulina sp is characterized by the decreasing the peak intensities of the band located at 1613-1505 cm -1 (aromatic C=C) (Fig. 2).It also shows there is no progressive reduction in the relative intensity of the peak carbonyl and the appearance of new absorption bands was observed.
Figure 3 shows the FTIR-ATR spectrogram of the PP surface layer before and after interaction with Spirulina sp.Before the interaction with Spirulina sp, the results of the PP spectrogram control gave an important peak in the wavelength region of 3100-3700 cm -1 (water OH stretch, 1456 cm -1 (CH 2 bend), 1377 cm -1 (CH 3 bend), 1166 cm -1 (CH bend, CH 3 rock, CC stretch), 997 cm -1 (CH 3 rock, CH 3 bend, CH bend), 840 cm -1 (CH 2 rock, C-CH 3 stretch), 809 cm -1 (CH 2 rock, CC stretch).After interaction with Spirulina sp, the appearance of absorption bands located at 1599 and 1534 cm -1 corresponds to Amide (C=O) and very strong peak at 1731, 48 cm -1 that corresponds to an ester and keton (C=O).Domagala (2012) found a new absorption band within the wave number range of (1730-1680)cm -1 that corresponds to a carbonyl group as a results of the nucleophilic substitution of PP.
Moreover, the absorption band of carbonyl group in the PP spectrum is broad which indicates the presence of carbonyl group in various products of oxidation such as aldehydes and ketons (Carlsson and Wiles 1969).Figure 3 also shows the new peak at 3343 cm -1 , which reveals the presence of Spirulina sp as it was also reported by Theivandran et al., (2015).The results of FTIR-ATR Spectrogram of PP after interaction with Spirulina indicate a particular activity of oxidative degradation process of PP in Spirulina sp medium.
Morphological evaluation of microplastics using SEM/EDX Analysis
The SEM analysis was conducted to investigate the changes in the surface morphology of the plastics.Nauendorf et al., (2016) proved in his study that biofilm formation in surface of plastic depends on several factors such as plastic surface roughness, plastic hydrophilic surface properties, the properties of substratum and also the bulk liquid properties as well as on cell surface properties.Figure 4 shows the SEM micrographs of the PET and PP surfaces before and after 112 days of interaction with Spirulina sp.Before interaction with Spirulina sp (Fig. 4a) the PET samples had a smooth area among the blisters and (Fig. 4c) the PP samples had a coarser and more textured surface with defects.However, after incubation with the Spirulina sp, surface erosion and the formation of pits and cavities on the surface of the samples were observed (Figs.4b and 4d).The presence of pits and cavities may be because of the absence of biofilm that become the areas colonized by microorganism, also suggesting that the fungus penetrated into the PET and PP matrix and a bacterial biofilm formed on the surface of plastics.Nakkabi et al.(2015) found that Bacillus subtilis strain has an effect on the change of PET surface morphological heterogenecity and signs of erosion which show the ability to degrade the PET.This experiment revealed that PET and PP also contribute a carbon source for Spirulina sp to form carbon dioxide as one of the major metabolic end products under aerobic conditions (Shah et al., 2008).In ISO 14852, it was depicted that the identified ultimate aerobic biodegradability of plastic materials in an aqueous medium can be performed by analysing the evolved CO 2 and this can be used as a reference in considering the amount of carbon from EDX investigation on PET and PP before and after interacting with microalgae.
Table 1 shows a decrease in carbon concentration by 48.61% in PET while in PP there is a carbon decline of 36.7%.The plastics that contain chemical compounds in the manufacturing process also have the ability to release and distribute contaminants to the environment as well as contaminate the environment by harmful chemical pollutants, and are able to absorb contaminants from the environment (Teuten et al., 2009).Rummel et al., (2017) reported the occurrence of chemical pollutants transport through the biofilms formed on the surface of the plastic.This is evident from the results of EDX that after plastic treatment with Spirulina sp, several inorganic elements were newly identified in plastics.These inorganic elements can be derived from the nutrients added to Spirulina sp media and from the release of additive compounds from the plastic which are added during the process of making plastic itself.
Optical density measurement for Spirulina sp growth
In this part of the experiment, we characterized the impact of PET and PP microplastics to Spirulina sp growth by measuring the optical density of Spirulina sp in various concentrations of microplastics (Fig. 5).
The decreasing microalgae growth is statistically significant (Fig. 6 after 72 hours in the presence of 250 g/L of polystyrene.However, exposing different microplastics concentrations level gives different microalgae growth rates whereas the higher microplastics concentration in the microalgae led to lowering the growth rate of the microalgae.Microalgae with the addition of PET and PP (Figure 8), generally have lower growth rate constant as compared to the microalgae without microplastic addition (0.399 day -1 ).This is because of the presence of microplastics in Spirulina sp culture may cause shading effects and lead to the inhibition of light intensity which is important in the process of microalgae photosynthesis (Hadiyanto et al., 2012).The microplastics dosage applied with a concentration of 150 mg/mL results in higher microalgae growth rates in PET (0.3584 day -1 ) compared to PP (0.2647 day -1 ) whereas at higher concentrations of 200 mg/mL and 275 mg/mL the opposite results were found in which the microalgae with the addition of PP microplastics (0.1229 day -1 and 0.907 day -1 ) have a higher growth rate than the microalgae with the addition of PET microplastics (0.1144 day -1 and 0.0832 day -1 ).
FTIR analysis
The FTIR analysis of Spirulina sp in fresh water without any treatment (Fig. 7 and Fig. 8), represent the following associated functional groups: at the wavelength of 3572 cm -1 representing the O-H stretching vibration and thus presence of alcohols and phenols.The peak at 3436 cm -1 represents the strong N-H (amine) and then at 1651 indicates -C=C-stretching vibration presence of alkenes, and peak of 1271 cm -1 presence of C-H wag (-CH 2 X) stretching of alkyl halides.Furthermore, the frequency ranges of 1200-1020 cm -1 is the presence of C-OH stretching and peak of 838 cm -1 present N-H symmetric stretching vibration primary of secondary amines.The last peak at 618 cm -1 representing C-Br stretching vibration indicates the presence of alkyl halides compounds.Some new peaks emerge and change of their intensity after additions of microplastics in Spirulina sp culture.The appearance of the absorption spectra in the region of 2300 -2400 cm -1 only occurs in PET under microplastics treatment.The highest concentration of PET was shown by the higher intensity of the spectra in that region (from 150 mg/500mL to 275 mg/500 mL), and this is slightly different to the PP application.The appearance of spectra in the 2300-2400 cm -1 region only occurs in Spirulina sp by applying 200 mg/500 mL and 275 mg/500 mL of microplastics.Gerakines et al. (1995) reported the appearance spectra at peak 2343 cm -1 , which was identified as the existence of CO 2 .The fact that higher CO 2 intensity in Spirulina sp added by PET and PP is related to the availability of carbon supplied by microplastic which lead high amount to be converted to CO 2 .This conversion process is part of the mineralization in the biodegradation process.Gupta et al. (2007) explained that the mineralization process in plastic biodegradation will occur in fragmented plastics where the microplastic residues produced are carbon as food sources and the converted energy produces CO 2 .Furthermore, Shah et al., (2008) reported that under aerobic conditions oxygen was used by microbes to oxidize carbon to produce CO 2 as a major metabolic end product and this is also sup- The decrease of intensity in some peaks and the emergence of new peaks after application of PET and PP in Spirulina sp is an indication of the interaction between microplastics and Spirulina sp.The appearance of FTIR absorption peak in the region of 3700-3800 cm -1 in Spirulina sp under addition of both microplastics is in line with the increasing microplastics dosage applied.The absorption is due to O−H stretching modes in the range of 3800-3000 cm -1 .The IR spectra can be represented as a sum of contributions from interfacial and bulk like water (Profio et al., 1998)
CONCLUSION
In this study, the measurements of tensile strength, analysis with FTIR-ATR and SEM-EDX were carried out on PET and PP after interacting with Spirulina sp for 112 days to understand the biodegradation process in PET and PP.From the measurement results of tensile strength, PET appears to provide a greater decrease in tensile strength compared to PP, but the opposite condition occurs in FTIR-ATR analysis, which shows a more significant change in functional groups in PP compared to PET.Furthermore, both PET and PP surface imaging with SEM after interaction with Spirulina sp in 112 days showed signs of surface alteration.Although the signs of biodegradation in PET and PP are indicated by the results of the analysis, but it still cannot be concluded that the process of biodegradation with microalgae provides the most effective results.Further research is required to obtain more infromation on the effectivity of microalgae Spirulina sp on its involved in biodegradation processes.Another study of PET plastic and PP impact to Spirulina sp growth shows a strong influence of PET and PP on the growth of Spirulina sp.The interaction between plastic and microalgae provides phenomena that need to be further studied to devise a solution in handling the abundance of plastic waste in the aquatic system and maintaining the survival of organisms in waters.
Fig. 1 .
Fig. 1.The changes of tensile strength of polyethylene terephthalate and polypropylene upon degradation time under Spirulina sp influences
Fig. 2 .
Fig. 2. The FTIR comparative spectra of the PET : (a) before treatment and (b) after treatment with Spirulina sp.
Fig. 3 .
Fig. 3. FTIR comparative spectra of the PP: (a) before treatment and (b) after treatment with Spirulina sp.
) among PET, PP and control during 7 days cultivation.Lagarde et al. (2016) found the decreasing microalgae growth after 78 hrs contact and Besseling et al., (2016) resulted in the decrease of microalgae growth
Fig. 4 .
Fig. 4. Morphological analysis using SEM in magnification x 1000 visualizations of the surface topography and roughness of the (a)PET without treatment, (b) PET after treatment with Spirulina sp, (c) PP without treatment, (d)PP after treatment with Spirulina sp for about 112 days.
Fig. 6 .Fig. 7 .
Fig. 6.Comparison of effect of microplastics in various concentrations to Spirulina sp growth rate (μ) ported by the research of Hoffmann et al. (1997) and Lucas et al. (2008).Arutchelvi et al. (2008) and Rummel et al. (2017) explained that biomass accumulation characterized by the growth of microorganisms was capable to utilize polymers as a carbon sources and this cause the main chain cleaves that leading the formation of low molecular weight compounds as impact of the extra cellular enzymes secreted by the microorganism, called as bio-fragmentation.This step is followed by diffusing oligomers into the microorganism to obtain assimilation.When the biodegradation is accomplished, it will produce CO 2 , H 2 O and biomass under aerobic conditions.
Fig. 8 .
Fig. 8. FTIR comparative of spectra of Spirulina sp under micro plastic Polypropylene influence in various concentrations.
al., 2008, Alvarez-zeferino et al., 2015, Guo Meng et al., 2016, Hoffmann et al., 1994, Hongliang et al., 2017, Strapasson et al., 2005).Tensile strength tests were conducted by tensile meter (Brookfield CT3 -4500) which were carried out on plastic before and after treatment by Spirulina sp.The plastics exposed by Spirulina sp treatment were measured every 7 days for 112 days to measure their tensile strength.
Table 1 .
The EDX result for PET and PP before and after treatment with Spirulina sp | v3-fos-license |
2019-12-05T09:05:22.602Z | 2019-12-04T00:00:00.000 | 208642752 | {
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} | pes2o/s2orc | The E3 ubiquitin ligase TRIM7 suppressed hepatocellular carcinoma progression by directly targeting Src protein
Aberrant Src kinase activity is known to be involved in a variety of human malignancies, whereas the regulatory mechanism of Src has not been completely clarified. Here, we demonstrated that tripartite motif containing 7 (TRIM7) directly interacted with Src, induced Lys48-linked polyubiquitination of Src and reduced the abundance of Src protein in hepatocellular carcinoma (HCC) cells. We further identified TRIM7 as a tumor suppressor in HCC cells through its negative modulation of the Src-mTORC1-S6K1 axis in vivo and in vitro in several HCC models. Moreover, we verified the dysregulated expression of TRIM7 in clinical liver cancer tissues and its negative correlation with Src protein in clinical HCC specimens. Overall, we demonstrated that TRIM7 suppressed HCC progression through its direct negative regulation of Src and modulation of the Src-mTORC1-S6K1 axis; thus, we provided a novel insight into the development of HCC and defined a promising therapeutic strategy for cancers with overactive Src by modulating TRIM7.
Introduction
The proto-oncogene tyrosine kinase Src is the prototype of the membrane-bound, nonreceptor tyrosine kinase family that includes Fyn, Yes, Lyn, Hck, Yrk, Lck, and Blk [1][2][3]. The Src protein is composed of several functional domains including a membrane-targeting myristoylated or palmitoylated SH4 domain, an SH3 domain, an SH2 domain, a protein tyrosine kinase domain (SH1) and a regulatory tail in the C-terminus [4]. Intermolecular autophosphorylation of Tyr419, which is located in the activation loop of the kinase domain, significantly promotes Src kinase activity [5]. Src kinase functionally interacts and cooperates with several receptor and nonreceptor protein tyrosine kinases to provide a diversity of signals for the regulation of cellular functions [6].
In the past decade, remarkable progress has been achieved in understanding the diverse functions of Src. As a nonreceptor protein tyrosine kinase, Src plays a broad role in cellular biological processes including proliferation, differentiation, survival, and metabolism. Activation of Src kinase promotes multiple downstream transduction cascades including phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), mitogen-activated protein kinase, and signal transducer and activator of transcription 3, which are vital regulators of survival, proliferation, and gene expression [4].
There is a large body of evidence showing that aberrant expression and activation of Src is involved in a variety of human malignancies and that Src dramatically facilitates Edited by V. D'Angiolella cancer cell division, proliferation, and invasion [7,8]. It has been reported that cytoplasmic Src family kinase activity is associated with more aggressive disease and shorter patient survival in several types of cancers [9][10][11]. The oncogenic role of Src in cancers has been well recognized whereas the regulatory mode of Src in human malignancies is far from being clarified.
In this study, we identified tripartite motif containing (TRIM) 7 as a novel negative regulator of Src protein.
TRIM7 is a new member of TRIM family proteins, and it was first identified as glycogenin-interacting protein in consideration of its role in binding and activating glycogen proteins [12][13][14]. TRIM7 possesses an N-terminal RING (really interesting new gene) domain, a B-box domain (zinc-finger motif), a coiled-coil domain, and a C-terminal B30.2/SPRY domain, which indicates its role as a RINGtype E3 ubiquitin ligase to directly bind with target proteins for ubiquitination [15].
Here we showed for the first time that TRIM7 directly interacted with Src and acted as a negative regulator of Src protein in hepatocellular carcinoma (HCC) cells. We demonstrated that TRIM7 suppressed oncogenesis and progression of HCC by modulating the Src-mTORC1-S6K1 axis. Moreover, experiments including in vivo xenograft tumor models and clinical liver cancer specimens further validated the tumor suppressor role of TRIM7 via its negative regulation of Src. Altogether, this study demonstrated a novel regulatory effect of TRIM7 on Src and provided a therapeutic strategy to manipulate Src-overactive cancers by regulating TRIM7.
TRIM7 directly bound to the Src protein
To investigate whether TRIM7 has a potential regulatory effect on Src, we first performed endogenous and exogenous co-immunoprecipitation (co-IP) assays to determine whether TRIM7 could interact with Src. Our data showed that TRIM7 interacted with both exogenous and endogenous Src protein (Fig. 1a, b). Moreover, endogenous TRIM7 could bind to endogenous Src (Fig. 1c, d), which indicated their interaction in natural conditions. Then, we obtained TRIM7 and Src proteins by an in vitro transcription and translation system and further demonstrated that TRIM7 could directly interact with Src protein in vitro (Fig. 1e).
To investigate the molecular basis of the interaction between TRIM7 and Src, we generated a series of domaindeleted mutants of both TRIM7 and Src, and tried to define a Co-IP analysis of the binding between exogenous TRIM7 and exogenous Src in HEK293T and Huh7 cells co-transfected with Flag-TRIM7 plasmid and HA-Src plasmid. b Co-IP analysis of the binding between exogenous TRIM7 and endogenous Src in HEK293T and Huh7 cells transfected with Flag-TRIM7 plasmid or mock control plasmid. c Co-IP analysis of the binding between endogenous TRIM7 and endogenous Src in Huh7 cells. d Co-IP analysis of the binding between endogenous TRIM7 and endogenous Src in Huh7 cells. e Co-IP analysis of the direct binding between Flag-TRIM7 and HA-Src proteins obtained from an in vitro transcription and translation system. f Schematic illustration of Src, showing wild-type and truncation mutants of Src. g Co-IP analysis of the interaction between TRIM7 and Src or Src truncation mutants in HEK293T cells co-transfected with HA-Src plasmid or HA-Src truncation mutant plasmids together with Flag-TRIM7 plasmid. h Schematic illustration of TRIM7, showing the wild-type and truncation mutants of TRIM7. i Co-IP analysis of the interaction between Src and TRIM7 truncation mutants in HEK293T cells co-transfected with Flag-TRIM7 truncation mutant plasmids together with HA-Src plasmid. The presented figures are the representative of data from at least three independent experiments their interacting domains (Fig. 1f, h). Our data showed that the SH2 domain of Src (Fig. 1g) and the B30.2/SPRY domain of TRIM7 (Fig. 1i) were required for the interaction between TRIM7 and Src. Thus, these data demonstrated that the binding between the SH2 domain of Src and the B30.2/SPRY domain of TRIM7 was the molecular basis for the direct interaction between TRIM7 and Src.
TRIM7 reduced the abundance of Src protein
Since we identified a direct interaction between TRIM7 and Src, we were further interested in defining whether TRIM7 regulated the abundance and stability of Src protein. First, we constructed gain-of-function models by transient and stable transfection of a TRIM7 expression plasmid into HCC cells (Fig. 2a, b, Supplementary Fig. S1a, b). Our data showed that after successful overexpression of TRIM7 by both transient and stable transfection of the TRIM7 plasmid ( Fig. 2a, b, Supplementary Fig. S1a, b), the protein levels of both Src and p-Src were significantly reduced (Fig. 2a, b). Second, we constructed three different loss-of-function models including Si-TRIM7 transiently transfected cells (Fig. 2c, Supplementary Fig. S1c), lentivirus-mediated Sh-TRIM7 stably transfected cells (Fig. 2d), and TRIM7knockout cells based on the CRISPR-Cas9 system (Fig. 2e). Our data showed that in all of these cell models of TRIM7 inhibition, the protein levels of both Src and p-Src were significantly increased (Fig. 2c, d, e). All these data Fig. 2 TRIM7 reduced the abundance of Src protein and directly induced K48-linked polyubiquitination. a Western blot analysis of Src and p-Src protein levels in HepG2 and Huh7 cells transiently transfected with Flag-TRIM7 plasmid or mock control plasmid. b Western blot analysis of Src and p-Src protein levels in Huh7 cells stably transfected with Flag-TRIM7 plasmid or mock control plasmid. c Western blot analysis of Src and p-Src protein levels in HepG2 and Huh7 cells transiently transfected with Si-NC, Si-TRIM7-1, or Si-TRIM7-2. d Western blot analysis of Src and p-Src protein levels in Huh7 cells stably transfected with lentivirus-mediated Sh-NC, Sh-TRIM7-1, or Sh-TRIM7-2. e Western blot analysis of Src and p-Src protein levels in TRIM7-knockout Huh7 cells. f HepG2 and Huh7 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid and were cultured for 24 h before being further incubated with CHX for 0, 3, 6, and 9 h. The Src protein level of the transfected cells was detected by western blot. g HepG2 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid and were cultured for 24 h before being further incubated with MG132 (10 μM) for 4 h or chloroquine (25 μM) for 6 h. The Src and p-Src protein levels of the transfected cells were detected by western blot. h Co-IP analysis of the ubiquitination of Src in HEK293T cells co-transfected with Flag-TRIM7 plasmid, HA-UB plasmid and HA-Src plasmid. i Co-IP analysis of ubiquitination of endogenous Src in HEK293T cells cotransfected with Flag-TRIM7 plasmid and HA-UB plasmid. j In vitro ubiquitination analysis of ubiquitination of Src in the presence of in vitro-translated Flag-TRIM7 and HA-Src, E1, UbcH5a, and ubiquitin. k Co-IP analysis of ubiquitination of Src in HEK293T cells cotransfected with Flag-TRIM7 plasmid, HA-Src plasmid, and HA-UB-K11, HA-UB-K48, or HA-UB-K63 plasmid. l Co-IP analysis of ubiquitination of Src in HEK293T cells co-transfected with Flag-TRIM7 plasmid or Flag-TRIM7 truncation mutant (TRIM7ΔRING) plasmid as well as HA-Src plasmid and HA-UB plasmid. The band intensities of the key proteins were further quantitatively analyzed in the indicated groups and were presented in the right panel. ***P < 0.001 for statistical analysis of the indicated groups. The presented figures are representative of data from at least three independent experiments indicated that TRIM7 inhibited the proto-form as well as the activated form of Src.
Our data further showed that the TRIM7-mediated negative regulation of Src occurred at the translational level but not the transcription level of Src ( Supplementary Fig. S1a, b, c). After blocking de novo protein synthesis in the TRIM7-overexpressing cells by cycloheximide, we found that TRIM7 significantly facilitated the decrease of Src protein level (Fig. 2f). Further investigation suggested that the TRIM7-mediated negative regulation of Src could be significantly rescued by the proteasome inhibitor MG132 (Fig. 2g), which indicated that Src was degraded by TRIM7 through proteasome-mediated degradation. Altogether, these data indicated that TRIM7 significantly reduced the abundance of Src protein via proteasome-mediated degradation.
TRIM7 induced Lys48-linked polyubiquitination of Src
Considering that TRIM7 belongs to the TRIM family, most of which are RING-type E3 ligases, we tried to define whether TRIM7 negatively regulated Src protein by its E3 ligase activity. Our data showed that TRIM7 was able to attach a polyubiquitin chain to Src protein (Fig. 2h, i), and further investigation confirmed the polyubiquitin chain could be attached to Src protein by TRIM7 in an in vitro ubiquitination system (Fig. 2j), which suggested that TRIM7 directly degraded Src protein through polyubiquitination.
Further investigation revealed that TRIM7 could attach a Lys48 (K48)-linked but not Lys11 (K11)-linked or Lys63 (K63)-linked polyubiquitin chain to Src protein, which implied that TRIM7 could induce Lys48-linked polyubiquitination of Src (Fig. 2k). When we transfected HCC cells with the RING domain-deleted TRIM7 mutant plasmid, the polyubiquitination of Src was significantly reduced in comparison with that of the wild-type TRIM7 plasmidtransfected group (Fig. 2l). Thus it indicated that TRIM7 induced ubiquitination of Src via its RING domain. Taken together, these data demonstrated that TRIM7 directly interacted with Src protein and induced Lys48-linked polyubiquitination of Src via its RING domain.
TRIM7 suppressed the Src-mTORC1-S6K1 axis
We determined that TRIM7 directly bound to Src and mediated Lys48-linked polyubiquitination of Src protein, so we were interested in defining the downstream molecular pathway of the TRIM7-Src axis. Our data showed that both transient and stable overexpression of TRIM7 efficiently reduced the protein levels of p-mTOR, p-S6K1, p-S6, and p-4E-BP1, which indicated its inhibitory effect on the mTORC1 pathway (Fig. 3a, b). In contrast, in TRIM7knockdown and -knockout cells, the protein levels of p-mTOR, p-S6K1, p-S6, and p-4E-BP1 were significantly upregulated ( Fig. 3c, d, e). Our data further showed that overexpression of Src could rescue the TRIM7-mediated suppression of the mTORC1-S6K1 signaling pathway (Fig. 3f), while the mTORC1 pathway inhibitor rapamycin did not change the protein levels of either Src or p-Src (Fig. 3g), which indicated that mTORC1-S6K1 signaling was downstream of the TRIM7-mediated negative regulation of Src. In summary, our data demonstrated that TRIM7 inhibited Src protein as well as its downstream mTORC1-S6K1 signaling in HCC cells.
TRIM7 acted as a tumor suppressor through its suppression of the Src-mTORC1-S6K1 axis in HCC cells
Since we defined the TRIM7-mediated negative regulation of Src, we were further interested in investigating whether TRIM7 had any effect on HCC cells. In HCC cells transiently or stably overexpressing TRIM7 (Fig. 4a, e), the proliferation (Fig. 4b, f), invasion (Fig. 4c, g), and colony formation (Fig. 4d, h) were significantly inhibited. In contrast, in the TRIM7-knockdown and -knockout HCC cells ( Fig. 4i, m, q), the proliferation (Fig. 4j, n, r), invasion ( Fig. 4k, o, s) and colony formation (Fig. 4l, p, t) were all significantly promoted. Thus, these data suggested that TRIM7 acted as a tumor suppressor and inhibited the malignant behaviors of HCC cells.
To define whether TRIM7-mediated degradation of Src protein was responsible for its antitumor effect, we cotransfected TRIM7 and Src plasmids into HCC cells and further detected the malignant behaviors of HCC cells. After successful overexpression of both TRIM7 and Src proteins ( Fig. 5a), the results showed that exogenous overexpression of Src significantly rescued the TRIM7-mediated suppression of the proliferation (Fig. 5b), invasion (Fig. 5c), and colony formation (Fig. 5d) of HCC cells. These data verified that TRIM7 exerted its antitumor effect through its negative regulation of Src.
Next, we attempted to define whether TRIM7 acted as a tumor suppressor through its suppression of the mTORC1-S6K1 signaling pathway. Our data showed that treatment with the mTORC1 inhibitor rapamycin significantly abolished the increased protein levels of p-S6K1 and p-S6 but not Src and p-Src in Si-TRIM7-transfected HCC cells (Fig. 5e), which indicated that TRIM7 induced suppression of the mTORC1 pathway downstream of its negative regulation of the Src protein. Moreover, the proliferation ( Fig. 5f), invasion (Fig. 5g), and colony formation (Fig. 5h) of Si-TRIM7-transfected cells were strikingly suppressed by rapamycin. Altogether, these data confirmed that TRIM7 exerted its antitumor effect through its suppression of the Src-mTORC1-S6K1 axis.
TRIM7 played a tumor suppressor role in HCC through its functional domains
We identified the B30.2/SPRY domain as the binding domain of TRIM7 with Src, while the RING domain was necessary for the TRIM7 ubiquitination function. Thus, we further examined whether TRIM7 exerted its antitumor effect on HCC cells through these two domains. Our data showed that deletion of either the B30.2/SPRY domain or the RING domain in TRIM7 efficiently rescued the TRIM7mediated inhibition of the Src-mTORC1-S6K1 axis (Fig. 6a), which indicated that TRIM7 suppressed the Src-mTORC1-S6K1 axis through these two domains. We further transfected these domain-deleted mutant plasmids into HCC cells and verified the successful overexpression of these two proteins in HCC cells by western blot (Fig. 6b). Our data showed that the overexpression of either of these two mutants could significantly abolish the TRIM7-mediated suppression of the proliferation (Fig. 6c), invasion (Fig. 6d), and colony formation (Fig. 6e) of HCC cells. Altogether, these data verified that both the B30.2/SPRY and RING domains were necessary for the tumor suppressor role of TRIM7 in HCC cells.
Exogenous overexpression of TRIM7 suppressed tumorigenesis in xenograft tumor models
To investigate the effect of the TRIM7-Src-mTORC1 axis in an animal model, we constructed xenograft tumors in nude mice as described before [16,17]. After visible tumors appeared, we injected Flag-TRIM7 plasmid into tumors in the left flanks and mock control plasmid into tumors in the right flanks of the mice every other day before the mice were sacrificed for analysis. Growth kinetics of the formed tumors showed that exogenous overexpression of TRIM7 significantly suppressed the growth of xenograft tumors overexpressing TRIM7 compared with the mock control tumors (Fig. 7a). The excised tumors from each group were compared, and the results indicated that tumors or Sh-TRIM7-2. e Western blot analysis of Src, p-mTOR, p-S6K1, p-S6, and p-4E-BP1 protein levels in TRIM7-knockout Huh7 cells. f Western blot analysis of Src-mTORC1-S6K1 signaling in Huh7 cells transiently co-transfected with Flag-TRIM7 plasmid and HA-Src plasmid. g Western blot analysis of Src-mTORC1-S6K1 signaling in Huh7 cells transiently transfected with Si-NC, Si-TRIM7-1, or Si-TRIM7-2 before treatment with rapamycin (100 nM) for 24 h. ***P < 0.001 for statistical analysis of the indicated groups. The presented figures are representative of data from at least three independent experiments with TRIM7 overexpression were much smaller than those of the mock group (Fig. 7b, c). In addition, detailed analysis of the excised tumors showed that the exogenous overexpression of TRIM7 significantly reduced the volume and weight of xenograft tumors (Fig. 7d, e). The successful overexpression of TRIM7 in the TRIM7 plasmidtransfected group was verified by analyzing the extracted tumors from each group by western blot (Fig. 7f). Moreover, the Src level in the TRIM7-overexpressing tumors was significantly decreased, and the activation of the mTORC1-S6K1 pathway was also significantly inhibited (Fig. 7f), which further verified the TRIM7-mediated negative regulation of the Src-mTORC1 axis in vivo.
To further verify the tumor suppressor role of TRIM7 and its inhibitory effect on the Src-mTORC1 axis in vivo, we constructed another xenograft tumor model by subcutaneously injecting TRIM7 stably overexpressing Huh7 cells into one flank and mock control cells into the other flank of nude mice. The growth status of the formed tumors was monitored every other day before the mice were sacrificed. Our data showed that the stable overexpression of TRIM7 significantly suppressed tumor growth and that TRIM7 stably overexpressing tumors were significantly smaller than those of the mock group (Fig. 7g, h). Statistical analysis showed that both the volume and weight of the TRIM7 stably overexpressing tumors were significantly analyzed. e-h Huh7 cells were transfected with Flag-TRIM7 plasmid or mock control plasmid, and the stably transfected cells were selected and further cultured. Effective stable overexpression of TRIM7 in these transfected cells was verified by western blot (e). The proliferation (f), invasion (g), and colony formation (h) of HCC cells stably overexpressing TRIM7 were further detected and analyzed. i-l HepG2 and Huh7 cells were transiently transfected with Si-TRIM7-1, Si-TRIM7-2, or Si-NC, and the successful knockdown of TRIM7 in the transfected cells was verified by western blot (i). The proliferation (j), invasion (k), and colony formation (l) of the TRIM7-knockdown cells were further detected and analyzed. m-p Huh7 cells were transfected with lentivirus-mediated Sh-TRIM7-1, Sh-TRIM7-2, or Sh-NC, and the stably transfected cells were selected and further analyzed. The successful knockdown of TRIM7 in the transfected cells was verified by western blot (m). The proliferation (n), invasion (o), and colony formation (p) of the TRIM7-knockdown Huh7 cells were further detected and analyzed. q-t TRIM7-knockout Huh7 cells were generated by the CRISPR/Cas9 system, and stable TRIM7-knockout clones were selected and further analyzed. The successful deletion of TRIM7 in these cells was verified by western blot (q). The proliferation (r), invasion (s), and colony formation (t) of the TRIM7knockout cells were further detected and analyzed. **P < 0.01 and ***P < 0.001 for statistical analysis of the indicated groups. Scale bar in c, g, k, o, s: 100 μm. The presented figures are representative of data from at least three independent experiments decreased compared with those of the mock group (Fig. 7i, j). Western blot assay verified the successful overexpression of TRIM7 and its significant suppressive effect on Src protein as well as its downstream mTORC1 pathway in TRIM7 stably overexpressing tumors (Fig. 7k).
To further confirm the effect of TRIM7 in vivo, we performed another set of xenograft tumor model by injecting TRIM7-knockout Huh7 cells generated by the CRISPR/ Cas9 system into one flank of nude mice and the mock control cells into the other flank of nude mice. The growth status of the formed tumors was monitored before the tumors were excised. Our data showed that growth of the TRIM7-knockout tumors was significantly promoted (Fig. 7l, m). Statistical analysis demonstrated that the volume and weight of the TRIM7-knockout tumors were significantly increased compared with those of the mock The Xenograft tumor model with TRIM7 plasmid injection was constructed as described before. When visible tumors appeared, 30 μg of TRIM7 plasmid or equal amount of mock control plasmid were injected into tumors in either flank once every other day. Tumors were measured every other day before the mice were sacrificed (a). Images were presented to show the representative of the mice with subcutaneous xenograft tumors in the TRIM7-transfected group and mock control group (b). Images were presented to show formed tumors isolated from sacrificed mice in the TRIM7-overexpressing group and mock control group (c). Statistical analysis of the volume (d) and weight (e) of formed tumors in the TRIM7-overexpressing group or mock control group. The protein levels of TRIM7 and the Src-mTORC1-S6K1 signaling in the TRIM7overexpressing tumors and mock control tumors were detected by western blot (f). Band intensities of the key proteins were further quantitatively analyzed in the indicated groups and were presented in the right panel. g-k The TRIM7 stably overexpressing xenograft tumor model was constructed by subcutaneously injecting 1 × 10 7 TRIM7 stably overexpressing Huh7 cells into one flank of athymic nude mice, and injecting equal amount of mock control cells into the other flank of the athymic nude mice. Growth curves of the TRIM7 stably overexpressing tumors and mock control tumors were monitored every other day before the mice were sacrificed (g). Images were presented to show the formed tumors isolated from the TRIM7 stably overexpressing group and mock group (h). The volume (i) and weight (j) of the formed tumors in the TRIM7 stably overexpressing group and mock control group were statistically analyzed. The protein levels of TRIM7 and the Src-mTORC1-S6K1 signaling in the TRIM7 stably overexpressing tumors and mock control tumors were analyzed by western blot, and intensities of the key proteins were further quantitatively analyzed and were presented in the right panel (k). l-p The TRIM7-knockout xenograft tumor model was constructed by injecting 1 × 10 7 TRIM7-knockout cells into one flank of the nude mice and equal amount of control cells into the other side of the mice. The formed tumors were measured every other day before the mice were sacrificed (l). Images were presented to show the formed tumors isolated from the sacrificed mice in the TRIM7-knockout tumors and mock control tumors (m). The volume (n) and weight (o) of formed tumors in the TRIM7-knockout group and mock control group were statistically analyzed. The protein levels of TRIM7 and the Src-mTORC1-S6K1 signaling in the TRIM7-knockout tumors and mock control tumors were analyzed by western blot (p). Band intensities of the key proteins were further quantitatively analyzed in the indicated groups and were presented in the right panel. q IHC staining of TRIM7 in HCC tissues and corresponding noncancerous liver tissues from clinical HCC patients. Presented images are representative of figures from the investigated HCC patients. r Statistical analysis of TRIM7 expression by IHC assay in HCC tissues and corresponding noncancerous liver tissues from the investigated HCC patients. s Real-time PCR analysis of TRIM7 mRNA level in HCC tissues and corresponding noncancerous liver tissues from clinical HCC patients. t Western blot analysis of TRIM7 protein level in HCC tissues and corresponding noncancerous liver tissues from clinical HCC patients. Presented images are the representative of blots from the investigated HCC patients. u Statistical analysis of TRIM7 expression by western blot assay in HCC tissues and corresponding noncancerous liver tissues from the investigated HCC patients. v IHC staining of Src expression in HCC tissues from clinical HCC patients. Presented images are representative of figures from the investigated HCC patients. w Correlations analysis of TRIM7 and Src expression by IHC assay in HCC tissues from the investigated HCC patients. x Schematic illustration of the interaction between TRIM7 and Src proteins, as well as the related signaling pathway. *P < 0.05, **P < 0.01, and ***P < 0.001 for statistical analysis of the indicated groups. Scale bar in q, v: 50 μm group (Fig. 7n, o). Western blot analysis of these excised tumors demonstrated successful knockout of TRIM7 and subsequent activation of the Src-mTORC1 axis in response to TRIM7 knockout (Fig. 7p).
Altogether, these in vivo data verified that TRIM7 acted as a Src inhibitor and inhibited HCC tumor growth in xenograft tumor models through its suppression of the Src-mTORC1-S6K1 axis.
Loss of TRIM7 expression contributed to hepatocarcinogenesis in clinical HCC tissues
We determined that TRIM7 exerted antitumor effect through its negative regulation of Src in vitro and in vivo, and we were further interested in investigating whether abnormal TRIM7 expression contributed to tumorigenesis in clinical HCC patients. Thus, we assessed the expression level of TRIM7 in HCC tissues and corresponding distal noncancerous liver tissues by immunohistochemistry (IHC), real-time PCR, and western blot. First, the location and the expression of TRIM7 were detected by IHC in paired tissues from 80 HCC patients. The IHC data showed that TRIM7 was mainly expressed in the cytoplasm of HCC cells and hepatocytes, and the expression level of TRIM7 in HCC tissues was significantly downregulated compared with that in the corresponding distal noncancerous liver tissues (Fig. 7q, r, Supplementary Table S1). Next, we measured TRIM7 mRNA and protein levels in paired tissues from one cohort of 64 HCC patients and another cohort of 52 HCC patients, respectively. Similarly, both real-time PCR and western blot assays confirmed the IHC data, showing that both TRIM7 mRNA and protein levels in HCC tissues were significantly decreased compared with those in the corresponding distal noncancerous liver tissues (Fig. 7s, t, u). To verify the TRIM7-mediated negative regulation of Src in clinical HCC specimens, we further detected Src expression in HCC tissues. Our data showed that Src protein accumulated significantly in HCC tissues with the lower expression of TRIM7 (Fig. 7v), and statistical analysis showed that TRIM7 expression was significantly inversely correlated with the abundance of Src (Fig. 7w), which verified the TRIM7mediated negative regulation of Src in clinical HCC patients. Altogether, the clinical investigations demonstrated that the loss of TRIM7 expression in HCC tissues might be involved in HCC progression and further verified the TRIM7-mediated negative regulation of Src in clinical specimens.
Altogether, we demonstrated that TRIM7 acted as an important negative regulator of Src as well as its downstream mTORC1-S6K1 signaling, thus suppressing HCC progression (Fig. 7x).
Discussion
Src interacts with several types of tyrosine kinase receptors, and it has been defined as a key molecule in tumor progression that can provide oncogenic signals for mitogenesis, cell survival, invasion, and metastasis [18]. Emerging evidence has shown that Src is hyperactive in the development of several types of cancers, including HCC [4,[19][20][21][22]. It has been reported that increased Src expression and activation are detected in more than 60% of HCC patients and is involved in disease progression [23,24]. Due to the positive correlation between increased Src activity and cancer progression, targeting Src is emerging as a promising attractive strategy for improving the clinical therapeutic effects for cancer patients. Bosutinib, dasatinib, ponatinib, and vandetanib have been approved by the Food and Drug Administration as Src inhibitors for drug therapy of malignant diseases [4], which indicates the great potential of targeting Src in clinical treatment. Furthermore, Src mutants are very uncommon in tumors [4], thus discovering novel regulators of Src provides great therapeutic potential for cancers.
In this study, we demonstrated that TRIM7, a newly identified TRIM family member, is a novel negative regulator of Src. To date,~100 human TRIM genes have been identified, and studies by us and others have indicated that alterations of TRIM proteins are involved in diverse pathological conditions, including carcinogenesis [16,17,[25][26][27]. Many TRIM proteins act as E3 ubiquitin ligases and induce ubiquitination by directly interacting with their substrates and further regulating multiple biological processes, including protein stability, transcriptional control, signaling transduction, and cell cycle progression [14,28,29]. TRIM7 belongs to class IV of the TRIM family proteins, and the exact role of TRIM7 in physiological and pathological conditions remains largely unknown. In this study, we verified that TRIM7 directly interacted with Src protein both in vivo and in vitro, and the B30.2/SPRY domain of TRIM7 and the SH2 domain of Src protein were the molecular basis for the direct binding between TRIM7 and Src. We further showed that TRIM7 induced Lys48linked polyubiquitination of Src via its RING domain, which ultimately led to the significant inhibition of Src activity. Thus, we have identified TRIM7 as a novel inhibitor of Src protein and defined the direct binding domains as well as the functional domains of TRIM7 and Src, which provided the molecular basis for further development of targeted therapy for the treatment of cancers.
Based on the critical involvement of Src activity in multiple cancers and the role of TRIM7 as a negative regulator of Src, we expected an antitumor effect of TRIM7 on cancers. Therefore, we tested the effect of TRIM7 on malignant behaviors of HCC cells, and our data demonstrated that TRIM7 suppressed the proliferation, invasion, and colony formation of HCC cells via its direct inhibition of Src protein in several cellular and animal models. We further demonstrated abnormal loss of TRIM7 expression in clinical HCC tissues and verified the negative correlation between TRIM7 and Src protein in clinical specimens, which indicated the involvement of the TRIM7-Src axis in HCC progression.
The contribution of Src activity in cancers is mainly attributable to its powerful activation of multiple downstream oncogenic signaling pathways; thus it has been recognized that targeting Src together with its downstream signaling is an attractive therapeutic strategy for cancer patients. In this study, we demonstrated that TRIM7 directly targeted Src for degradation and thus further led to efficient suppression of its downstream mTORC1-S6K1 signaling. Over the past decade, the critical role of mTORC1 signaling in HCC progression has been widely accepted. It has been reported that the mTORC1 pathway is frequently activated in up to half of HCCs and is highly associated with poor prognosis in HCC [30]. Thus, targeting the mTORC1 pathway provides an attractive strategy for improving clinical therapeutic effects for HCC patients. The critical role of Src in activating mTORC1 signaling has been demonstrated in several recent studies [31,32], whereas the TRIM7mediated regulation of the Src-mTORC1 signaling axis in HCC cells is reported here for the first time. Thus, this study provided evidence to define TRIM7 as a novel inhibitor of Src and opened a new avenue for treating mTORC1-overactive cancers by modulating TRIM7.
In conclusion, we identified TRIM7 as a novel negative regulator of Src. TRIM7 induced Lys48-linked, RING domain-dependent polyubiquitination of Src protein, which further led to the suppression of its downstream mTORC1-S6K1 signaling pathway, and acted as a tumor suppressor in HCC. Thus, this study identified a novel TRIM7-mediated regulatory mechanism of Src, and it also has clinical significance by providing a novel therapeutic strategy for Srcoveractive cancers by modulating TRIM7.
Cell culture and transfection
Human HepG2 and Huh7 HCC cells and human embryonic kidney 293T cells were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China). All mycoplasma-free cells were cultured in DMEM-High Glucose medium (HyClone, Massachusetts, USA) supplemented with 10% fetal bovine serum and were authenticated by STR profiling by HKGENE (HKGENE Genetechnology, Beijing, China). The small interfering RNA targeting TRIM7 was synthesized by RIBOBIO (RIBOBIO, Guangzhou, China). TRIM7 and Src plasmids were synthesized by OriGene (OriGene Technologies, Maryland, USA) and Vigene (Vigene Biosciences, Rockville, USA), respectively. Truncation mutants of TRIM7 were generated using a KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacturer's protocol. All of the transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
Co-IP, western blot assay, immunofluorescence, and IHC Co-IP, western blot assay, immunofluorescence, and IHC were performed as described previously [17,33,34]. Immunofluorescence and IHC assays were conducted in a manner blinded to the sample identity. The specific primary antibodies used in these assays included antibodies against
Cell proliferation, invasion, and colony formation assays
HepG2 and Huh7 cells were plated in 96-well plates at a density of 10 4 cells/well, and the proliferation of HCC cells was detected by a CCK-8 Kit (Dojindo, Kumamoto, Japan) at 0, 24, 48, and 72 h according to the manufacturer's instructions. Invasion assays and colony formation assays were performed as previously described [35,36].
Relative mRNA levels of the genes were normalized to βactin. Primers for the β-actin gene were forward: 5′-GG CACCACACCTTCTACAATG-3′, reverse: 5′-TAGCACA GCCTGGATAGCAAC-3′. The relative mRNA levels of target genes were obtained by using the 2 −ΔΔCt method, and all assays were performed in triplicate.
In vitro binding and ubiquitination assay
The direct interaction between TRIM7 and Src proteins was assessed by a TNT Quick Coupled Transcription/Translation System (Promega, Madison, WI, USA) according to the manufacturer's protocol. The Flag-TRIM7 and HA-Src proteins were expressed in vitro, mixed together, and analyzed by co-IP assay with anti-Flag antibody, followed by western blot assay with anti-HA antibody to determine the direct binding between TRIM7 and Src proteins. In vitro ubiquitination was analyzed with a ubiquitination kit (Boston Biochem, Cambridge, MA, USA) following the protocol recommended by the manufacturer. Briefly, Flag-TRIM7, and HA-Src proteins were expressed in vitro and further incubated with ubiquitin conjugation mixture at 30°C for 60 min, followed by subsequent ubiquitination analysis of the Src protein by co-IP assay.
Generation of genetically modified stable HCC cell lines
To construct stable TRIM7-overexpressing HCC cells, a TRIM7 plasmid was transfected into Huh7 cells using Lipofectamine 2000. The successfully transfected cells were selected by complete medium containing 2 μg/mL of puromycin at 48 h after the transfection. The surviving colonies of transfected cells were further amplified, followed by validation by western blot. To generate a stable TRIM7knockdown cell line, lentivirus carrying TRIM7-RNA interference sequence (Sh-TRIM7-1 and Sh-TRIM7-2) or its mock control (Sh-NC) were transfected into Huh7 cells using lentivirus (GeneChem Co., Ltd., Shanghai, China) according to the manufacturer's protocol. The transfected cells were further selected with puromycin (2 μg/ml) and stable TRIM7-knockdown clones were validated by western blot. TRIM7-knockout cells were constructed with the CRISPR/Cas9 system according to the procedure described in the reference [37]. Briefly, a single-guide RNA (sgRNA) targeting TRIM7 (forward: 5′-CACCGAGAGAGGATG AGGCGCGGGT-3′, reverse: 5′-AAACACCCGCGCCTC ATCCTCTCTC-3′) was transfected into Huh7 cells by using the pLentiCRISPRv2 system (Addgene). The sgRNA-transfected cells and the pLenti-V2-transfected mock control cells were further selected with puromycin (2 μg/ml) and the selected positive clones were cultured for further experiments.
In vivo tumor growth assay
All of the xenograft tumor models were constructed with 5week-old immunodeficient BALB/c athymic male nude mice (Huafukang Biotechnology Ltd, Beijing, China). The xenograft tumor model with TRIM7 plasmid injection was constructed and analyzed in seven nude mice as described before [16,17]. To construct the xenograft tumor model with TRIM7 stably overexpressing cells, 1 × 10 7 TRIM7 stably overexpressing Huh7 cells were injected into one flank, and equal amount of cells stably overexpressing mock plasmid were injected into the other flank of five athymic male nude mice. To construct the xenograft tumor model with the TRIM7-knockout cells, we injected 1 × 10 7 CRISPR/Cas9-based TRIM7-knockout Huh7 cells into one flank of five athymic male nude mice, and injected equal amount of mock control cells into the other flank. The formed tumors were measured and analyzed as previously described [33]. All mice for xenograft tumor models were randomly assigned. All of the protocols dealing with the animals were in accordance with guidelines of the Institutional Animal Care and Use Committee and were approved by the Medical Ethics Committee of Shandong University.
Clinical liver cancer specimens
Paired samples of HCC tissues and corresponding noncancerous liver tissues were collected from the Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University. Among these samples, 80 pairs of HCC tissues and corresponding noncancerous tissues were used for the IHC assay, 52 pairs of matched specimens were used for the western blot assay to detect the protein level of TRIM7, and 64 pairs of matched specimens were used for realtime PCR to determine the mRNA level of TRIM7. Details of the clinicopathologic characteristics of the recruited HCC patients were shown in Table 1.
Written informed consents were obtained from all patients before the study was initiated. All of the protocols were in accordance with the Helsinki Declaration and approved by the Medical Ethics Committee of Shandong University.
Statistical analysis
Data were statistically analyzed using SPSS 22.0 software (SPSS, IL, USA) and GraphPad Prism 5 software (Graph-Pad, CA, USA). Quantitative variables were evaluated with two-tailed Student's t test or one-way analysis of variance, while categorical variables were analyzed by χ 2tests. Correlations for categorical and continuous variables were evaluated by Spearman's rank correlation test and Pearson's correlation test, respectively. Data were presented as mean ± SEM. A P value < 0.05 was considered statistically significant, and the P value used in all analyses was two-tailed.
Data availability
All relevant data that support the findings of this study are available from the corresponding author upon request. Supplementary information is available at Cell Death & Differentiation's website. | v3-fos-license |
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} | pes2o/s2orc | Mechanical properties of one and two-step fluoridated orthodontic resins submitted to different pH cycling regimes
The aim of this study is to assess the in vitro shear bond strength and adhesive remnant index (ARI) of one and two-step fluoridated orthodontic resins under conditions that simulate high cariogenic challenge. Edgewise brackets for maxillary central incisors were randomly bonded to 80 bovine incisors, using either TransbondTM Plus Color Change orthodontic resin and a self-etching primer adhesive (G1; n = 40) or Orthodontic Fill Magic with a conventional acid-etch technique (G2; n = 40). Each group of resin (n = 10) was divided into: immediate shear (Apre-cycling control), immersion in artificial remineralizing saliva (neutral saliva) for 14 days (Bpost-cycling control) and pH cycling with high cariogenic challenge (Cacid saliva with pH 5.5 and Dacid saliva with pH 4.5). After 14 days of pH cycling, the shear bond strength and ARI were evaluated. Considering the shear bond strength, TransbondTM Plus Color Change resin was stronger than Orthodontic Fill Magic when it was submitted to high cariogenic challenge (p < 0.05). Also TransbondTM Plus Color Change resin showed better adhesion to enamel than Orthodontic Fill Magic, in all situations evaluated (p < 0.05). It could be concluded that TransbondTM Plus Color Change resin presented better shear bond strength and adhesive remnant index when submitted to high cariogenic challenge, in comparison with Orthodontic Fill Magic. Descriptors: Orthodontic brackets; Composite resins; Shear strength; Tooth demineralization. Introduction In orthodontics practice, white spot lesions are observed around orthodontic appliances with relative frequency.1-3 Caries lesions adjacent to brackets can be reduced or even completely inhibited when a fluoride dentifrice is used.4 However, its use depends on the patient’s compliance, which is usually inadequate.5 Therefore, preventive measures that do not depend on an individual’s compliance were developed to solve this problem, such as bonding dental materials with fluoride-releasing properties,6,7 which exhibit an additional source of fluoride locally, near the brackets.2,8 Glass ionomer cement was presented as the first material with potential cariostatic properties, however, its use in orthodontics has the disadvantage of low bond strength to dental substrate.2,9,10,11 Orthodontic composite resins generally present high bond strength when compared Mechanical properties of one and two-step fluoridated orthodontic resins submitted to different pH cycling regimes Braz Oral Res. 2010 Apr-Jun;24(2):197-203 198 with glass ionomer orthodontic cements used for this purpose.12 The development of materials with high bond strength associated with fluoride release, such as fluoride-releasing composite resins, began to solve these problems.13 The literature has demonstrated that resins for bonding procedures present satisfactory mechanical properties.14,15 Fluoride release of bonding materials16,17 and their release mechanisms18,19 have been extensively studied. Few in vitro studies have evaluated the real influence of cariogenic challenge that simulates oral environment in shear bond strength tests. The traditional system of bonding orthodontic brackets is a three-step mechanism that involves three separate agents: an enamel conditioner, a priming agent, and an adhesive resin.20 To reduce chair time and improve effectiveness, a two-step mechanism, which combines a primer and adhesive agent, and recently, self-etching primers (SEPs) have been developed. These systems combine the conditioning and priming agents into a single acidic primer solution for simultaneous use on both enamel and dentin.21 However, there are no studies in the literature that assess bonding material properties under conditions that simulate the oral environment, such as using a pH cycling with a high cariogenic challenge model. Thus, the purpose of the present study is to evaluate the shear bond strength and adhesive remnant index of one and two-step fluoridated orthodontic resins submitted to two pH cycling regimes with different demineralization potentials, simulating a high cariogenic challenge. Materials and Methods Sample preparation Eighty bovine incisors were randomly divided into 8 groups, n = 10 (Table 1), and sectioned along the cemento-enamel junction (Figure 1A). Next, the crowns were submersed in epoxy resin with the buccal surface facing the glass plate (Figure 1B,C). Silicon carbide abrasive papers with successive grits (180, 400 and 600 3M, Rio de Janeiro, RJ, Brazil) were used to expose the bonding area (Figure 1D,E). After this, the coronal portion was submitted to prophylaxis with prophylactic rubber cups (KG Sorensen, Rio de Janeiro, RJ, Brazil) at low speed for 5 seconds. Samples were washed in deionized water and dried using an oil-free air jet and water vapor for 15 seconds. Maxillary central incisor brackets (Edgewise system – Morelli, Rio de Janeiro, RJ, Brazil) were bonded in the most central area of the middle third of the bovine incisor buccal surface (Figure 1F) with the two different orthodontic light-polymerized fluoridated resins (Table 1): TransbondTM Plus Color Change (G1) using a one-step self-etching primer adhesive (TSEP; 3M Unitek, Monrovia, California, USA) and Orthodontic Fill Magic (G2) with a conventional acid-etch technique consisting of two steps (Vigodent, Rio de Janeiro, RJ, Brazil). For G1, TransbondTM Plus Self Etching Primer (3M Unitek, Monrovia, California, USA) was used. For G2, the enamel was previously etched with 37% phosphoric acid (SSWhite, Rio de Janeiro, RJ, Brazil) for 30 secGroups (n = 10) Orthodontic fluoride resins Treatments G1A (Pre-cycling control) TransbondTM Plus / Self-Etch Primer Immediate shear bond* G2A (Pre-cycling control) Orthodontic Fill Magic Immediate shear bond * G1B (Post-cycling control) TransbondTM Plus / Self-Etch Primer Artificial saliva pH 7.0* G2B (Post-cycling control) Orthodontic Fill Magic Artificial saliva pH 7.0* G1C (Experimental group) TransbondTM Plus / Self-Etch Primer pH Cycling 5.5 G2C (Experimental group) Orthodontic Fill Magic pH Cycling 5.5 G1D (Experimental group) TransbondTM Plus / Self-Etch Primer pH Cycling 4.5 G2D (Experimental group) Orthodontic Fill Magic pH Cycling 4.5 *Groups not submitted to pH cycling. Table 1 Sample division in the TransbondTM Plus Color Change (G1; n = 40) and Orthodontic Fill Magic (G2; n = 40) groups. Passalini P, Fidalgo TKS, Caldeira EM, Gleiser R, Nojima MCG, Maia LC Braz Oral Res. 2010 Apr-Jun;24(2):197-203 199 onds, followed by the application of a one-component adhesive resin, according to the manufacturer’s instructions. The samples were stored in deionized water at room temperature for one day. pH cycling Exactly 24 hours after bonding, negative pre-cycling control groups (G1A and G2A) were submitted to immediate shear bond strength testing, without undergoing modified cariogenic pH cycling according to Queiroz et al.22 (2008). Negative post-cycling control groups (G1B and G2B) remained in artificial remineralizing/neutral saliva (1.54 mmol/L calcium, 1.54 mmol/L phosphate, 20 mmol/L acetic acid and 0.308 g ammonium acetate, adjusted to pH 7.0 with potassium hydroxide; VETEC, Rio de Janeiro, RJ, Brazil)23 for 14 days. Experimental groups (G1C/ G2C and G1D/G2D) were submitted to pH cycling, simulating two different cariogenic challenges, using artificial remineralizing and demineralizing saliva (3 mmol/L calcium, 3 mmol/L phosphate, 50 ml/L acetic acid and 0.308 g ammonium acetate; VETEC, Rio de Janeiro, RJ, Brazil)24 with medium and high demineralizing potential (pH adjusted to 5.5 and 4.5 with sodium hydroxide; respectively). The experimental groups submitted to pH cycling remained in demineralizing saliva daily for 22 hours consecutively, and after being washed with deionized water, they were kept in contact with remineralizing saliva for 2 hours, completing a cycle of 24 hours. During the period of pH cycling, the specimens were kept in an incubator (Fanem Ltd., São Paulo, SP, Brazil), at a constant temperature of 37°C in order to simulate the oral environment. These dynamics were reproduced for the period of 14 days, during which the artificial saliva (neutral and acid) was changed every 2 days. Shear bond strength The shear tests were performed in a Universal Test machine (EMIC, São José dos Pinhais, SP, Brazil), at a constant speed of 0.5 mm/min. The force required to dislodge the bracket was recorded in Newtons (N) and converted into megapascals (MPa) as a ratio of Newtons to the bracket surface area (MPa = N/mm2). Figure 1 Sample preparation. (A) Positioning of buccal surface of bovine incisors facing the glass plate; (B) Insertion of apparatus support; (C) Submersion in epoxy resin; (D) Bond area exposure; (E) Washing of the samples; (F) Bonding of incisor brackets. A B C
Introduction
2][3] Caries lesions adjacent to brackets can be reduced or even completely inhibited when a fluoride dentifrice is used. 4However, its use depends on the patient's compliance, which is usually inadequate. 5Therefore, preventive measures that do not depend on an individual's compliance were developed to solve this problem, such as bonding dental materials with fluoride-releasing properties, 6,7 which exhibit an additional source of fluoride locally, near the brackets. 2,8lass ionomer cement was presented as the first material with potential cariostatic properties, however, its use in orthodontics has the disadvantage of low bond strength to dental substrate. 2,9,10,11Orthodontic composite resins generally present high bond strength when compared with glass ionomer orthodontic cements used for this purpose. 12he development of materials with high bond strength associated with fluoride release, such as fluoride-releasing composite resins, began to solve these problems. 13The literature has demonstrated that resins for bonding procedures present satisfactory mechanical properties. 14,15Fluoride release of bonding materials 16,17 and their release mechanisms 18,19 have been extensively studied.Few in vitro studies have evaluated the real influence of cariogenic challenge that simulates oral environment in shear bond strength tests.
The traditional system of bonding orthodontic brackets is a three-step mechanism that involves three separate agents: an enamel conditioner, a priming agent, and an adhesive resin. 20To reduce chair time and improve effectiveness, a two-step mechanism, which combines a primer and adhesive agent, and recently, self-etching primers (SEPs) have been developed.These systems combine the conditioning and priming agents into a single acidic primer solution for simultaneous use on both enamel and dentin. 21However, there are no studies in the literature that assess bonding material properties under conditions that simulate the oral environment, such as using a pH cycling with a high cariogenic challenge model.
Thus, the purpose of the present study is to evaluate the shear bond strength and adhesive remnant index of one and two-step fluoridated orthodontic resins submitted to two pH cycling regimes with different demineralization potentials, simulating a high cariogenic challenge.
Sample preparation
Eighty bovine incisors were randomly divided into 8 groups, n = 10 (Table 1), and sectioned along the cemento-enamel junction (Figure 1A).Next, the crowns were submersed in epoxy resin with the buccal surface facing the glass plate (Figure 1B,C).Silicon carbide abrasive papers with successive grits (180, 400 and 600 -3M, Rio de Janeiro, RJ, Brazil) were used to expose the bonding area (Figure 1D,E).After this, the coronal portion was submitted to prophylaxis with prophylactic rubber cups (KG Sorensen, Rio de Janeiro, RJ, Brazil) at low speed for 5 seconds.Samples were washed in deionized water and dried using an oil-free air jet and water vapor for 15 seconds.
Maxillary central incisor brackets (Edgewise system -Morelli, Rio de Janeiro, RJ, Brazil) were bonded in the most central area of the middle third of the bovine incisor buccal surface (Figure 1F) with the two different orthodontic light-polymerized fluoridated resins (Table 1): Transbond TM Plus Color Change (G1) using a one-step self-etching primer adhesive (TSEP; 3M Unitek, Monrovia, California, USA) and Orthodontic Fill Magic (G2) with a conventional acid-etch technique consisting of two steps (Vigodent , Rio de Janeiro, RJ, Brazil).For G1, Transbond TM Plus Self Etching Primer (3M Unitek, Monrovia, California, USA) was used.For G2, the enamel was previously etched with 37% phosphoric acid (SSWhite, Rio de Janeiro, RJ, Brazil) for 30 sec- onds, followed by the application of a one-component adhesive resin, according to the manufacturer's instructions.The samples were stored in deionized water at room temperature for one day.
The experimental groups submitted to pH cycling remained in demineralizing saliva daily for 22 hours consecutively, and after being washed with deionized water, they were kept in contact with remineralizing saliva for 2 hours, completing a cycle of 24 hours.During the period of pH cycling, the specimens were kept in an incubator (Fanem Ltd., São Paulo, SP, Brazil), at a constant temperature of 37°C in order to simulate the oral environment.These dynamics were reproduced for the period of 14 days, during which the artificial saliva (neutral and acid) was changed every 2 days.
Shear bond strength
The shear tests were performed in a Universal Test machine (EMIC, São José dos Pinhais, SP, Brazil), at a constant speed of 0.5 mm/min.The force required to dislodge the bracket was recorded in Newtons (N) and converted into megapascals (MPa) as a ratio of Newtons to the bracket surface area (MPa = N/mm²).Adhesive Remnant Index (ARI) The brackets and enamel surfaces were analyzed by two trained and calibrated examiners (Kappa = 1.00).An optical microscope (Eclipse E600, Nikon, Melville, NY, USA) was used at 4x magnification, with ARI scores according to Artün and Bergland, 25 (1984) (Figure 2) as follows: in the range from 0 to 3, where 0 = No adhesive on enamel surface; 1 = Less than 50% of the adhesive on enamel surface; 2 = More than 50% of the adhesive on enamel surface; 3 = 100% of the adhesive on enamel surface.
Statistical analysis
The shear bond strength test results were inserted in the database of the statistical program SPSS 16.0 (SPSS Inc, Chicago, IL, USA) and submitted to analysis of variance (ANOVA) and the Tukey test.For ARI evaluation, Mann-Whitney and Kruskal-Wallis tests (p < 0.05) were applied.
Results
The high cariogenic challenge was able to induce white spot formation around orthodontic brackets (Figure 3).
Shear bond strength
The results showed that G1 had higher shear bond strength than the mean of G2 (Table 2).
There was statistically significant difference among the materials when immediate shear bond strength before pH cycling was assessed (G1A > G2A, p = 0.0001).When comparing the pre-cycling and post-cycling control groups, an increase in the shear bond strength could be noted when artificial remineralizing saliva was used, however, without significant difference (p > 0.05) for the same material (G1A, G1B and G2A, G2B) (Table 2).
No statistical difference was noted in the behavior of the experimental groups in both G1 (G1C and G1D) and G2 group (G2C and G2D).Although there was no difference within the same material, G1 presented a higher shear bond strength, showing statistical difference when was compared with G2 group (G1D and G2D, p = 0.0001) under cariogenic challenge with the demineralizing solution at a pH of 4.5.The same was not found for medium cariogenic challenge (G1C and G2C) with demineralizing solution at a pH of 5.5 (Table 2).
Discussion
Bonding materials used in orthodontics must have ideal physical-chemical and mechanical characteristics, as well as sufficient bond strength to resist chewing forces. 1,26Representative values of a satisfactory bonding to the teeth to resist orthodontic forces vary from 2.86 to 7.59 MPa. 27lthough shear bond strength is usually evaluated under neutral conditions, the high prevalence of white spot lesions around orthodontic appliances 2 has aroused interest in the study of this mechanical property in the face of the adverse conditions in the oral environment.In the present study, both bonding materials presented shear bond strength values capable of resisting orthodontic forces, 27 nevertheless, Transbond TM Plus Color Change presented higher shear bond strength.It is essential to highlight that high shear bond strength values are important to keep orthodontic bracket adhered to the enamel surface during orthodontic treatment, particularly in patients that present high susceptibility to white spot lesions.Future studies should be conducted with regard to this condition.
In our study, shear bond strength values, using a high cariogenic challenge model, varied from 7.15 to 16.43 MPa under all situations analyzed.There was no difference in the adhesive behavior when different conditions were assessed in the same material.On the other hand, the shear bond strength of onestep was statistically higher than that of two-step agent when immediately submitted to shear testing and after high cariogenic challenge (pH 4.5).This result showed the better adhesive stability of onestep when compared with two-step agent, under conditions that simulate great fluctuations of pH in the oral environment.According to the literature, 20,21 the bond strength of self-etching primers is similar to that of the twostep agents.However, the present study exhibited higher shear bond strength for TSEP/adhesive, Transbond TM Plus Color Change, when compared with Orthodontic Fill Magic, showing better properties for the one-step system than for the two-step agents, probably due to being less sensitive to technique.
Furthermore, bonding orthodontic attachments with composite resins requires conditioning of the enamel surface with phosphoric acid, leading to a substantial loss of enamel by etching. 28The procedure of debonding orthodontic accessories may result in enamel alterations. 25Several variables that may influence bond strength have been investigated, 29 however, high cariogenic challenge has not been assessed.In the current study, one-step also showed better performance when compared with two-step agents, reinforcing the adhesive properties of the former in comparison with the latter resin.
When the ARI was assessed, one-step had 90% of scores 2, under most of the studied conditions.It is worth emphasizing that high cariogenic challenge at pH 4.5 reached score 2 in most specimens, showing that the adverse oral conditions did not interfere negatively in the adhesive quality of the material, as it remained stable in an acidic environment, with ARI equal to that of the controls.Furthermore, the behavior of two-step mechanism tended to improve when the material was exposed to simulated oral conditions, especially high cariogenic challenge.However, most of its scores were 1, showing its higher tendency to adhere to brackets and damage the dental structure.
With regard to ARI, a previous study 21 demonstrated that TSEP presented all the adhesive remained on the tooth and another study reported that more than 90% of the adhesive remained on the tooth, 30 corroborating the present study, which showed most of the Transbond TM Plus Color Change specimens with over 50% of the adhesive on enamel.On the other hand, the two-step agents presented less than 50% of the adhesive on enamel.This could be explained by the simultaneous action of phosphoric acid attack and primer that dissolve the enamel surface and allow penetration of the primer monomers into the demineralized enamel. 21This could reduce the possibility of contamination during the bonding procedure, and for this reason, improve adhesion on enamel when compared with the two-step agents.
Conclusion
The present study analyzed the mechanical properties of two adhesive systems of fluoridated orthodontic bonding resins, using pH cycling simulating high cariogenic challenge to reproduce in vivo conditions.Based on the methods applied, it was shown that Transbond TM Plus Color Change resin presented better shear bond strength and adhesive remnant index when submitted to high cariogenic challenge, in comparison with Orthodontic Fill Magic.
Figure 1 -
Figure 1 -Sample preparation.(A) Positioning of buccal surface of bovine incisors facing the glass plate; (B) Insertion of apparatus support; (C) Submersion in epoxy resin; (D) Bond area exposure; (E) Washing of the samples; (F) Bonding of incisor brackets.
Figure 3 -
Figure 3 -Representative image of white spot formation after cariogenic challenge.
Table 2 -
Shear Bond Strength values (MPa) of fluoridated orthodontic resins submitted to high cariogenic challenge.Equal letters = absence of statistically significant difference (Variance and Tukey test) p < 0.05. *
Table 3 -
Adhesive remnant index (ARI) of different fluoridated orthodontic resins submitted to high cariogenic challenge. | v3-fos-license |
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} | pes2o/s2orc | Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100.
Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.
Isolation and oxidation of LDL
Isolation of LDL. LDL was isolated by ultracentrifugation from healthy blood donors at the A. Vesale Hospital (Charleroi, Belgium) ( 27 ). Protein content was measured using the Lowry technique ( 28 ). ( 29,30 )], equivalent to a molar ratio of oxidant/lipoprotein of 25:1 and 50:1, respectively. Controls were performed in the absence of MPO. A more intensive enzymatic oxidation was performed as described previously ( 11 ). Briefl y, MPO-LDL was generated by mixing 8 µl of HCl 1 M (fi nal concentration: 4 mM), 45 µl of MPO (fi nal concentration: 250 nM), a volume containing 1.6 mg LDL (fi nal concentration: 0.8 mg/ml in PBS, pH 7.4), and 40 µl of H 2 O 2 50 mM (fi nal concentration: 1 mM). The volume was adjusted to 2 ml with PBS (pH 7.4) containing 1 g/l of EDTA. In this oxidation condition, the oxidant/lipoprotein molar ratio is 625:1.
Chemical oxidation of LDL (HOCl-LDL
Copper oxidation. Briefl y, LDL (1 mg/ml) in PBS was incubated with 10 µM CuSO 4 for 24 h at 37°C ( 18 ). The oxidation was stopped by the addition of 25 µM BHT and incubation on ice for 1 h.
Isolation of apoB-100 from LDL
ApoB-100 was isolated from LDL as described previously ( 31 ). Briefl y, LDL was precipitated with 500 µl trichloroacetic acid peroxide (H 2 O 2 ) and chloride ions (Cl Ϫ ), and this potent oxidant contributes to the antimicrobial activity of phagocytes ( 1,2 ). However, evidence has emerged that either chronic or prolonged production of HOCl by the MPO-H 2 O 2 -Cl Ϫ system contributes to tissue damage and the initiation and propagation of vascular diseases ( 3 ). HOClmodifi ed epitopes were present in acute and chronic vascular infl ammatory diseases where staining was found to be associated with monocytes/macrophages, smooth muscle cells, and endothelial cells (4)(5)(6). As human atherosclerotic lesions contain elevated levels of MPO, the enzyme may act as a catalyst for LDL oxidation ( 7 ). Furthermore, the oxidation of LDL/apoB-100, leading to species called "oxidized LDLs (OxLDLs)," plays a crucial role in the pathogenesis of atherosclerosis (8)(9)(10). Fingerprints for in vivo modifi cations of apoB-100 by the MPO-H 2 O 2 -Cl Ϫ system were observed by immunohistological analyses ( 4,11 ) and GC-MS ( 12 ). Observations that an increasing oxidant/ LDL molar ratio of HOCl-modifi ed apoB-100 is paralleled by a decreased ligand interaction by the classical LDL receptor ( 13 ) suggested that scavenger receptors on macro phages mediate the uptake of HOCl-modifi ed LDL (HOCl-LDL) ( 14,15 ). In addition to its capacity to promote lipid accumulation in monocytes/macrophages ( 16 ), HOCl-LDL adversely affects biological properties of smooth muscle cells and endothelial cells ( 13 ), thus favoring progression of atherosclerosis. Furthermore, LDL oxidized by the MPO-H 2 O 2 -Cl Ϫ system (MPO-LDL) accumulates in macrophages and exerts proinfl ammatory effects on monocytes and endothelial cells ( 17,18 ). Modifi cation by reagent HOCl alters the lipid moiety of LDL but primarily leads to amino acid oxidation favoring posttranslational modifi cation (PTM) of the protein moiety. Lysine (Lys), histidine (His), and the N-terminal ␣ -amino group may form reactive chloramine species, which may lead to secondary oxidation processes ( 13 ). Methionine (Met) can be converted into sulfoxide form while tyrosine (Tyr) may be converted into 3-chlorotyrosine (Cl-Tyr), a specifi c marker for the MPO-H 2 O 2 -Cl Ϫ system-mediated oxidation in vivo ( 12,19,20 ) and in vitro ( 19 ). Furthermore, it has been reported that MPO, probably due to its charge, can bind LDL ( 21,22 ). That binding, which seems to be mediated via the protein moiety of LDL ( 23 ), may result in localized formation of oxidants and hence sidespecifi c damages ( 22,24 ). ApoB-100, the unique protein of LDL, is a highly hydrophobic protein with 4,536 amino acid residues (molecular mass 550 kDa). Furthermore, apoB-100 contains a high number of amino acid residues prone to be modifi ed by HOCl.
In the present work, we have studied the impact of adsorption of MPO on native and HOCl-modifi ed LDL and on its structural and enzymatic features. Using highresolution MS, we then performed a comprehensive survey of PTMs on apoB-100 treated with HOCl added as reagent or generated enzymatically. Numerous modifications were identifi ed including methionine sulfoxide (O-Met), (di)-oxidized tryptophan [(di-)OxTrp], and Cl-Tyr. Finally, we compared these in vitro fi ndings with oxidation patterns of LDL that has been isolated from placed into the vial, 200 µl of acid mixture [6 M HCl supplemented with 10% (v/v) thioglycolic acid, 0.1% (m/v) phenol, and 0.1% (m/v) benzoic acid] and internal standards were added, and hydrolysis was carried out for 40 min at 160°C. 13 C 9 -Tyr and 13 C 15 N-Lys were used as internal standards. Samples were thereafter purifi ed using Si-SCX solid phase extraction cartridges. Briefl y, columns were fl ushed twice with 1 ml methanol and then equilibrated with 2 ml FA (0.2 M). After hydrolysis, the samples were loaded and washed with 2 ml FA (0.2 M). Amino acids were eluted with 2 ml methanol containing 5% (w/v) NH 4 OH. Samples were evaporated to dryness using a vacuum centrifuge and fi nally dissolved in 50 µl water before injection of 10 µl into the LC-MS system.
The LC system was a rapid resolution LC (RRLC) 1200 series using a Zorbax Eclipse XDB Phenyl RR column [4.6 × 150 mm inner diameter (ID), 3.5 µM particle size] and coupled to an electrospray ion source (ESI) in positive mode quadrupole TOF (QTOF) 6520 series mass spectrometer from Agilent Technologies. Amino acid residues were separated by an acetonitrile gradient, and amino acids of interest were analyzed by MS/MS using Mass Hunter Acquisition ® and Qualitative Analysis ® (Agilent Technologies).
Identifi cation of PTMs of ApoB-100
Digestion. Protein pellets from native or oxidized LDL preparations were treated using an optimized method that ensures an optimal protein recovery ( 31 ). Briefl y, apoB-100 was unfolded using 250 µl of RapiGest SF (Waters) 0.2% (w/w) in 50 mM ammonium bicarbonate buffer (pH 7.8), reduced using dithiothreitol (20 mM) at 37°C during 30 min, and fi nally, alkylated with iodoacetamide (60 mM) for 30 min in the dark. The solution was heated at 100°C (5 min), and apoB-100 was then digested by trypsin (enzyme-protein = 1:10, w/w) at 37°C for 24 h. The reaction was then stopped by heating the sample at 100°C for 30 min. Deglycosylation of tryptic peptides was performed with PNGase F (10 U/mg of protein) during 24 h at 37°C. The sample was then adjusted to 0.5% FA, incubated (30 min, 37°C), and centrifuged (10 min, 13,000 g ). The supernatant was evaporated to dryness in a centrifugal vacuum evaporator. Finally, peptides were dissolved in 50 µl FA [0.1% (v/v) in water] before analysis. Additionally, unfolding of protein was performed by 2,2,2-trifluoroethanol. Briefl y, to 1 mg protein, 50 µl of 2,2,2-trifl uoroethanol, 50 µl of ammonium bicarbonate buffer (100 mM, pH 7.8), and 2 µl of 200 mM dithiothreitol were added. The sample was heated at 60°C (1 h) and then cooled to 20°C. Eight microliters of 200 mM iodoacetamide was added, and the sample was kept in the dark at 20°C (1 h). Finally, 2 µl of 200 mM dithiothreitol was added, and the sample was kept at 20°C (1 h) in the dark before dilution with 600 µl of water and 200 µl of bicarbonate buffer (100 mM). ApoB-100 was then digested at 37°C (24 h) by adding 50 µl of trypsin (enzyme-protein = 1:10, w/w). Trypsin was then inactivated by heating the sample (100°C, 30 min). After cooling to 20°C, deglycosylation was performed at 37°C (24 h) by adding 10 units of PNGase F. Finally, the sample was acidifi ed by addition of 2 µl of FA (100%) and evaporated to dryness in a centrifugal vacuum evaporator. Peptides were dissolved in 50 µl of FA [0.1% (v/v) in water] before analysis.
LC-MS/MS process, data acquisition, and analysis. Ten microliters of the resulting samples was injected into the LC system and analyzed as described previously ( 31 ). Briefl y, analyses were performed with the same RRLC 1200 series mentioned previously. Peptides were separated on a Fused Core Ascentis ® Express C18 column (100 × 2.1 mm ID, 2.7 µm particle size) from Supelco (Bellefonte, PA) using a 105 min gradient of FA and acetonitrile. The ESI-QTOF mass spectrometer mentioned previously was used for the MS/MS analyses. Auto-MS/MS spectra were (10%, v/v) and centrifuged for 10 min at 4,500 g . The supernatant was discarded, and the precipitate was treated one more time with trichloroacetic acid. Pellets were then mixed with 1.1 ml of watermethanol-diethyl ether (1:3:7, v/v/v) to remove lipids. The mixture was sonicated for 30 min and then centrifuged for 10 min at 4500 g . The supernatant was discarded, and the procedure was repeated.
Isolation of apoB-100 from patients
Alternatively, LDL was isolated from patients on chronic maintenance hemodialysis therapy (n = 9) or healthy volunteers (n = 9). Briefl y, blood (5 ml) was drawn before dialysis in patients or in volunteers. Four hundred microliters of LDL solution from each patient/volunteer was treated as described previously. This study conforms to the Declaration of Helsinki, and its protocol was approved by the Ethics Committee of the ISPPC ("Intercommunale de Santé Publique du Pays de Charleroi") Hospital. Finally, all subjects gave their written informed consent.
Amperometric measurements of MPO activity
The chlorination activity of MPO (100 nM) in the absence or presence of indicated lipoprotein concentrations was measured by continuously monitoring H 2 O 2 consumption by amperometry using a combined platinum/reference electrode, which was covered with a hydrophilic and a dialysis membrane (cutoff 3,600 Da), fi tted to the Amperometric Biosensor Detector 3001 (Universal Sensors Inc.). The applied electrode potential at pH 7.4 was 650 mV, and the electrode fi lling solution was freshly prepared half-daily ( 32 The peroxidase activity of MPO was measured in 10 mM PBS (pH 7.4) using 100 µM of Tyr as the one-electron donor. One unit of peroxidase activity is defi ned as the consumption of 1 µmol H 2 O 2 per minute at 25°C in the presence of 100 µM Tyr.
ApoB-100 hydrolysis for amino acid analysis
ApoB-100 protein pellets (obtained from isolated native or oxidized LDL samples) were hydrolyzed using a StartS microwave oven and a protein hydrolysis reactor according to the manufacturer's protocol (Milestone, Italy). Briefl y, apoB-100 (1 mg) was
Activity and structure of MPO at the surface of LDL
Measurement of MPO activity. MPO interacts preferentially with the protein moiety of native LDL ( 23,34 ). It is likely that the enzyme may interact in a similar manner with modifi ed LDL; we therefore studied whether this interaction is paralleled by changes in the activity of MPO. Figure 1A shows that the chlorinating activity of MPO increases as a function of increasing LDL concentration; at the highest LDL concentration, the chlorinating activity of MPO increased by 90%. In order to investigate whether an increased consumption of H 2 O 2 could result from scavenging of HOCl by LDL (which would protect MPO from inhibition by its own product), Met was added. However, the presence of this HOCl scavenger had no impact on the observed kinetics when compared with samples containing MPO only.
Second, the effect of HOCl-LDL on H 2 O 2 consumption by MPO was investigated. Compared with native LDL (0.3 mg/ml), the chlorinating activity of MPO incubated with HOCl-LDL at identical concentrations was lower ( Fig. 1B ). Although at low oxidant/lipoprotein molar ratios (83:1 and 166:1) the chlorinating activity of MPO was significantly higher ( ف 20%) compared with MPO alone, a high acquired in positive and high-resolution acquisition mode (4 GHz) ( 31 ). Data were acquired by the Mass Hunter Acquisition ® software and analyzed by the Mass Hunter Qualitative Analysis with Bioconfi rm ® and by Spectrum Mill ® software (Agilent Technologies). Peptide identifi cation and validation were based on mass error (ppm), peptide scores, and % score peak intensity (SPI), which are essential to validate peptide mapping as previously described ( 31 ).
To improve the sensitivity for the analysis of the samples from patients, a nanoLC system coupled to the QTOF-MS was used. Tryptic peptides were separated on an Agilent Technologies nanoLC Chip Cube II system using a Polaris HR nanochip column. This consists of a 360 nl enrichment column and a 75 µm × 150 mm separation column, both packed with Polaris C18 phasis 3 µm particle size, 180 Å pore size. Mobile phases and gradient were identical to those used for the RRLC separation mentioned previously. Samples were loaded on the enrichment column of the chip using the capillary pump in 97% of the aqueous mobile phase at a fl ow rate of 1.5 µl/min. The nanofl ow pump was used to generate the analytical gradient with a fl ow of 0.40 µl/min. MS/MS parameters were identical to those used for RRLC analyses.
Statistical analysis
Data were analyzed using the SigmaPlott ® 12.0 software (Systat ® , San Jose, CA). Differences were considered statistically signifi cant with a two-tailed P < 0.05. Comparisons were made using one-way ANOVA and a Dunnett's post hoc test. Next, we focused on oxidation of Met and tryptophan (Trp). Independent of whether apoB-100 was native or modifi ed (either chemically or enzymatically), Met was widely oxidized. Formation of OxTrp and di-OxTrp was found in all LDL/apoB-100 samples with no statistical difference even if there was a tendency to increased levels of di-OxTrp under both oxidative conditions (twice the level observed in native LDL).
To confi rm that chlorination is specifi c for HOCl treatment, apoB-100 was oxidized by copper ( 18 ) and then subjected to hydrolysis. No Cl-Tyr formation was observed, while low levels of (di-)OxTrp may also occur, data that parallel previous fi ndings ( 37 ).
Mapping of in vitro modifi cations on apoB-100. Next, we analyzed the location of PTMs on apoB-100. Instead of RapiGest SF ( 31 ), 2,2,2-trifl uoroethanol was also used as the unfolding agent to recover apoB-100 with the same results on protein sequence recovery (79%) or even higher.
Analyses revealed that few residues (Met 4 , Met ) were already oxidized in native LDL. The latter residues seem thus to be highly sensitive to oxidation.
Third, we tested whether the chlorinating activity of MPO is dependent on the concentration of HOCl-LDL at a given oxidant/lipoprotein molar ratio (333:1). Preincubation of MPO with HOCl-LDL did not affect the chlorinating activity at low concentrations of HOCl-LDL, whereas consumption of H 2 O 2 by MPO was decreased by ف 35% at the highest lipoprotein concentration ( Fig. 1C ).
Next, we investigated whether the observed behavior was independent of an electron donor and thus independent of the nature of the oxidation product released by MPO. Peroxidase activity in the presence of Tyr showed similar effects on H 2 O 2 consumption by MPO, namely increased activity in the presence of native LDL but decreased peroxidase activity in the presence of highly modifi ed LDL ( Fig. 1D ).
These data illustrate that native LDL enhances its ability to be oxidized by MPO apparently due to a specifi c interaction between MPO and nonmodifi ed apoB-100. In contrast, HOCl-LDL may decrease the activity of MPO, suggesting that oxidation of apoB-100 by HOCl may modulate the MPO-apoB-100 interaction.
Structural features of MPO adsorbed on LDL. Next, we investigated whether altered activity of MPO after preincubation with native LDL or HOCl-LDL ( Fig. 1 ) could also be refl ected by changes on a spectroscopic basis. Electronic CD spectroscopy was chosen because it allows analysis of both the overall secondary structure and the (asymmetric) environment of the prosthetic heme group. In the far-UV region, the spectra of MPO in the presence of LDL or HOCl-LDL exactly matched the sum of spectra of individual lipoproteins at the same protein concentrations (data not shown). This suggests that the structural changes must be local and that the overall content of the secondary structure of MPO (which is mainly ␣ -helical) is not affected.
However, this observation seems to be in contrast to the Soret region. Native MPO has a Soret minimum at 410 nm, whereas the ellipticity of native LDL and HOCl-LDL is negligible at this wavelength region ( Fig. 2 ). Upon incubation of MPO with native LDL, ellipticity is lost in the Soret region, whereas in the near-UV range (260-350 nm) the difference was very small. This observation is indicative for structural changes in the immediate surroundings of the prosthetic group. The effect on the CD of the heme group was smaller when MPO was incubated with HOCl-LDL. The Soret minimum shifted to 411 nm with native MPO-like ellipticity, whereas between 300 and 350 nm ellipticity was lost. This indicates that HOCl-LDL has some local impact on the tertiary structure of MPO but not on the architecture of the active site. In any case, the observed changes in the enzymatic activity in the presence of native-or HOCl-LDL are refl ected by structural changes at the active site of the peroxidase with native LDL having a stronger impact than HOCl-LDL.
MS analysis of apoB-100 modifi ed chemically or enzymatically
Total hydrolysis of apoB-100. In order to investigate whether modifi cations of apoB-100 differed when LDL was oxidized under several conditions, we investigated the (oxidant/lipoprotein molar ratio = 625:1) previously used to raise monoclonal antibodies to detect MPO-LDL in vivo and in vitro ( 11 ). In addition to oxidative modifi cations listed in Table 1 , 41 tryptic peptides carrying PTMs (n = 46) were identifi ed ( Table 3 ). These peptides further include 3 residues of 3-hydroxytyrosine (HO-Tyr), 5 O-Met residues, 10 OxTrp residues, 18 di-OxTrp residues, and 10 Cl-Tyr residues, respectively. Again, in most peptides a single amino acid modifi cation (primarily di-OxTrp in this case) was found, while few peptides included either two (O-Met and HO-Tyr or O-Met and Cl-Tyr) or three modifi cations (two O-Met and HO-Tyr). These fi ndings indicate that a more pronounced enzymatic oxidation of apoB-100 further targets Trp and Tyr residues.
Mapping of MPO-mediated PTMs of apoB-100 isolated from patients undergoing hemodialysis or healthy volunteers.
To confi rm whether our in vitro fi ndings may occur also in vivo, LDL was isolated from patients (n = 9) who have high levels of MPO ( 38,39 ) and circulating MPO-LDL ( 25 ) and compared with healthy volunteers (n = 9). As levels of oxidized LDL in patients with hemodialysis are still low when compared with levels of native LDL, apoB-100 was analyzed using a nano-LC-MS/MS with high-resolution analytical column (Polaris CHIP), which is more sensitive than an LC system.
In sum, 90 PTMs were identifi ed over patients and volunteers. Again ( Table 3 ). Cl-Tyr 125 might also be interesting as it is specifi c to the MPO-H 2 O 2 -Cl Ϫ system and was never detected before. Results represent mean ± SEM of three experiments. * P < 0.001, ** P < 0.005, and *** P < 0.05 versus natLDL; # P < 0.001 and ## P < 0.05 versus HOCl-LDL 50 µM; and P < 0.001 versus HOCl-LDL 100 µM. generated enzymatically. Furthermore, oxidative PTMs occurring in vitro were compared with those occurring under in vivo conditions. Finally, we were interested in whether the extent of HOCl modifi cation of apoB-100 can in turn modulate the activity of MPO to generate oxidants. ApoB-100 is probably the most diffi cult protein for structural analysis because of its huge size and its insolubility in aqueous buffer after delipidation ( 43 ). However, studying modifi cations of apoB-100 in relation to cardiovascular diseases is of major importance ( 40 ). Yang and colleagues ( 44 ) were the fi rst to identify 88% of the sequence of native apoB-100 by HPLC analysis of tryptic peptides coupled to an automatic sequencer (different than the MS technique). Oxidation of LDL with HOCl produced a diverse array of 2,4-dinitrophenylhydrazinereactive peptides, with little indication of selectivity ( 24 ). HOCl treatment of apoB-100 resulted in modifi cation at cysteine (Cys), Trp, Met, and Lys. Thirteen out of 14 modifi ed biomarkers are becoming more apparent ( 40 ). Because OxLDL impairs the physiological functioning of various cells ( 13,17 ) and plays a causal role in atheroma plaque formation, both the nature of oxidants as well as the modifi ed entity of the lipoprotein particle are of importance. Oxidation of LDL can be carried out by, among others, transition metals, hemoglobin, lipoxygenases, and reactive oxygen species generated by vascular endothelium or phagocytes, and HOCl can modify LDL at the lipid and the protein moieties in vitro and/or in vivo ( 13 ). Modifi ed lipids were preferentially identifi ed by MALDI-TOF-MS and 31 P NMR spectroscopy (40)(41)(42), while MALDI-TOF-MS, MS/MS, and LC-MS/MS are suitable techniques to identify the respective protein modifi cations basically after tryptic digestion ( 31,40 ). Here, we have used an LC-MS/ MS method with a high-resolution MS (a QTOF) to identify PTMs on tryptic peptides from delipidated apoB-100 samples ( 31 ) treated with HOCl added as reagent or ]. Tyr 76 , which was chlorinated by the MPO-H 2 O 2 -Cl Ϫ system, was also reported to act as a specifi c target for LDL nitration ( 45 ). Met, apparently the fi rst target for oxidation, is highly reactive toward HOCl-mediated attack ( 13 ), and Met residues may protect proteins from critical oxidative damage ( 46 were reported to be specifi c MPO-mediated modifi cations of apoB-100 ( 22,24 ). Our results suggest that O-Met 4 and O-Met 4192 are already present on non-in vitro oxidized LDL isolated from healthy volunteers. These data also refl ect the high sensitivity of those residues to oxidative damages. Modifi cations of residues Met 3719 and Cys 61/3734/4190 were not found under our experimental conditions. Cys residues have been reported to rapidly react with HOCl ( 47,48 ). Despite careful analysis of possibly oxidized Cys residues, none were detected under our experimental conditions.
DISCUSSION
We further show that oxidation of apoB-100 by HOCl (added as a reagent) leads to a higher content of aminoadipic acid compared with MPO-mediated oxidation. On the other hand, no statistical difference in chemical and enzymatic oxidation was seen for Trp and Met residues, a fact underlining their high sensitivity toward oxidation. So again, oxidation patterns are dependent on the oxidant, suggesting that reagent HOCl does not exactly mimic the respective MPO enzymatic oxidation.
Both the extent of modifi cation as well as the difference in the charge of amino acid side chains may alter the binding properties of MPO to the respective LDL particle in vivo or in vitro. Adsorption of MPO on native LDL increased both chlorinating and peroxidase activities ( Fig. 1 ), and this was refl ected by structural changes in the heme cavity using CD spectroscopy ( Fig. 2 ). These fi ndings are in accordance with Sokolov et al. ( 49 ) ( 22 ). To analyze PTMs on apoB-100, we used an RRLC system coupled to high-resolution MS/MS detection. This methodology is extremely effi cient because LC enables high resolution of tryptic peptides by C18 column, while highresolution MS/MS provides high accuracy of m/z values and a fragmentation pattern of the compounds (i.e., peptides). The latter technique enables both mapping of peptides and detection of untargeted PTMs. Here, we are the fi rst to present the most comprehensive pattern of tryptic peptides of apoB-100 treated by reagent HOCl. In sum, 97 tryptic peptides carrying at least one PTM could be identifi ed. Although others ( 10 ) have tried to differentiate between trypsin-releasable, nontrypsin-releasable, and mixed fractions of apoB-100, we directly performed delipidation of total LDL prior to trypsin digestion. Using two different concentrations of reagent HOCl (50 or 100 µM), 46 modifi ed tryptic peptides were found.
Although some modifi cations (n = 15) were also present when LDL was modifi ed by the MPO-H 2 O 2 -Cl Ϫ system ( Table 1 were omitted, indicating PTMs to be MPO specifi c. Each modifi ed peptide is characterized by its peptide score, % SPI, mass error, fragmentation, and position(s) of modifi ed residue(s).
protocol, a total of 41 peptides carrying at least one PTM (e.g., primarily modifi ed Trp and Tyr residues but also O-Met residues) were detected ( Cl-Tyr, a protein modifi cation specifi c for MPO-catalyzed oxidation, has previously been identifi ed on LDL isolated from human plaque material or LDL plasma samples from patients with cardiovascular disease ( 12 ). Although several Tyr residues are prone to be modifi ed either enzymatically or nonenzymatically by HOCl, we are the fi rst to identify Cl-Tyr residues on apoB-100 at posi tions Tyr 76, 102, 749, 1575, 1687, 1747, 1901, 2341, 2405, 2732, 3653, and 4242 . An increased level of Cl-Tyr, apparently a consequence of high plasma MPO levels ( 50,51 ), was also reported in plasma samples from dialysis patients ( 38,39 ). This observation prompted us to follow PTMs on apoB-100 isolated from plasma of hemodialysis patients who have high plasma MPO-LDL levels ( 25 ) interaction between LDL and MPO that differs from MPO interaction with high-density lipoprotein ( 34 ). An increase in the activity of MPO at the surface of LDL might indicate that native LDL enhances its own oxidation by MPO and that MPO activity could be underestimated in vitro and/or in vivo. On the other hand, MPO activity decreased with increasing HOCl/LDL molar ratios and HOCl-LDL concentration. The more wild-type-like Soret CD spectrum of MPO in the presence of HOCl-LDL is further support. Apparently, oxidative LDL modifi cations (e.g., O-Met, Cl-Tyr, OxTrp, or N-chloramine Lys) alter the interaction between apoB-100 and MPO and, at least at higher concentrations, diminish MPO activity to some extent.
Recently, monoclonal antibodies were raised against LDL that had been modifi ed by MPO under conditions using the drastic molar ratio 625:1 ( 11 ) as performed in the present study. The respective epitope identifi ed by one of the antibodies corresponded to a 66 kDa fragment of apoB-100 starting with Gly 1612 ( 11 ). Using this oxidation and compared with apoB-100 of healthy volunteers. Among the observed PTMs, O-Met and OxTrp residues were detected, as well as only one Cl-Tyr. This suggests that Cl-Tyr formation on apoB-100 is rare, but Tyr 125 is an interesting PTM for future studies in MPO. However, only one patient among nine carried this MPO-specifi c modifi cation. Furthermore, O-Met 2499 was only observed in patients and so is an interesting PTM that should be investigated in future studies.
Although we could not fi nd PTM in the close vicinity of the LDL receptor binding domain [between residues 3,346 and 3,369 of apoB-100 ( 52,53 )], the remaining PTMs, markers of oxidative stress and/or lipoprotein abnormalities ( 54 ), could still contribute to impaired binding of MPO-LDL to the LDL receptor favoring scavenger receptor-mediated uptake as reported for nitrated LDL ( 45 ). Furthermore, Trp 3606 is close to the binding domain and might interact with the latter when oxidized, and OxTrp 3606 was observed in more patients (n = 4) than volunteers (n = 1).
Summarizing, using LC-MS/MS we further identifi ed in vitro a series of modifi ed tryptic peptides (n = 97) that are indicative of modifi cation by HOCl, added as reagent or generated enzymatically. Among these modifi cations, several have been identifi ed in vivo from patients suffering from kidney failure and undergoing hemodialysis therapy where high MPO levels are causally linked with LDL modifi cations and abnormalities in clearance of the modifi ed LDL particles. Our data also highlight that, when studying LDL oxidation, HOCl added as a reagent does not completely mimic enzymatic modifi cation via the MPO-H 2 O 2 -Cl Ϫ system and that MPO activity could be misestimated under in vitro or in vivo experiments due to the fact that MPO/apoB-100 interaction is specifi c and changes the enzymatic activity. Future experiments mapping PTMs of LDL in different cardiovascular diseases are also of importance to link individual oxidative modifi cations with disease severity. | v3-fos-license |
2021-09-09T13:11:21.589Z | 2021-09-06T00:00:00.000 | 239679886 | {
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} | pes2o/s2orc | Immunomodulatory Effects of Pentoxifylline: Profiling Data Based on RAW 264.7 Cellular Signaling
Pentoxifylline (PTX) is a methylxanthine derivative that has been developed as an immunomodulatory agent and an improvement of microcirculation. Osteoradionecrosis (ORN) is a serious complication of radiation therapy due to hypovascularity. Coronavirus disease 2019 (COVID-19) has spread globally. Symptoms for this disease include self-limiting respiratory tract illness to severe pneumonia and acute respiratory distress. In this study, the effects of PTX on RAW 264.7 cells were investigated to reveal the possibility of PTX as a therapeutic agent for ORN and COVID-19. To reveal PTX effects at the cellular level, protein expression profiles were analyzed in the PTX-treated RAW 264.7 cells by using immunoprecipitation high-performance liquid chromatography (IP-HPLC). PTX-treated RAW 264.7 cells showed increases in immunity- and osteogenesis-related proteins and concurrent decreases in proliferation-, matrix inflammation-, and cellular apoptosis-related proteins expressions. The IP-HPLC results indicate that PTX plays immunomodulatory roles in RAW 264.7 cells by regulating anti-inflammation-, proliferation-, immunity-, apoptosis-, and osteogenesis-related proteins. These results suggest that PTX may be used as supplement medications for ORN as well as for COVID-19.
Introduction
Pentoxifylline (PTX) is a methylxanthine derivative (1-(5-oxohexyl)-3,7-dimethylxanthine) that has been used for the past several decades to improve the blood rheological properties and treat symptoms associated with impaired microcirculation [1]. Other methylated xanthine compounds, which include caffeine, theophylline, aminophylline, and theobromine, are plant components and have similar major pharmacologic actions [2]. The major enzymatic action of cyclic nucleotide phosphodiesterases (PDEs) is the degradation of cyclic adenosine monophosphate (cAMP). PTX works as a nonselective inhibitor of PDEs, and therefore upregulates the effects of cAMP and adenosine-5 -triphosphate and increases erythrocyte distensibility. PTX reduces leukocyte adhesion to endothelial cells, enhances prostacyclin synthesis, and diminishes platelets aggregation. The accumulation of the above effects leads to capillary dilatation, a reduction in blood viscosity, and an improvement in peripheral microvasculature [3].
In recent studies, attention has been given to the possibility of treating PTX as an immunomodulatory agent. PTX has shown the ability to down-regulate pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 1 (IL-1). PTX inhibits TNF-α synthesis by blocking transcriptional activity [4,5]. A PTX-treated mice group showed significant TNF-α reduction in a radiation-induced lung injury model [6]. Zein et al. [7] performed a placebo-controlled randomized clinical trial in patients with nonalcoholic steatohepatitis. In comparison with the placebo-using group, PTX was reported to significantly reduce steatosis, lobular inflammation, and liver fibrosis. In a study of neonatal sepsis, PTX inhibited the production of inflammatory cytokines by microorganisms, particularly when used in combination with antimicrobial agents [8]. In a randomized controlled trial involving 120 newborns with late-onset sepsis and mean gestational age of 30 weeks, PTX administration decreased TNF-α and C-reactive protein (CRP) levels, shortened the respiratory period and short-term hospitalization, and decreased incidence of disseminated intravascular coagulopathy [9]. However, there was no difference in mortality and short-term morbidity between PTX treated and untreated newborn sepsis [9,10].
PTX has effects on inhibiting dermal fibroblast proliferation and extracellular matrix synthesis [11,12] and increasing the activity of tissue collagenase [13]. Previous studies have shown the ability of PTX to reduce the fibrosis progress induced by external stress such as irradiation. PTX inhibits intracellular signaling in response to transforming growth factor-β (TGF-β), and connective tissue growth factor (CTGF). Lin et al. [14] reported that PTX suppresses CTGF expression as well as the promoter effects of CTGF on kidney cells. However, in another study, no significant changes were found in TGF-β expression at either the mRNA or protein level in the irradiated rat heart model [15]. In postoperative peritoneal adhesion studies, PTX may reduce intraperitoneal adhesion formation by increasing peritoneal fibrinolysis activity and inhibiting angiogenesis and collagen synthesis [16]. Western blot analysis of radiation-induced pulmonary fibrosis models showed that PTX treatment suppressed the expression of plasminogen activator inhibitor-1 and fibronectin in irradiated lung tissues as well as in epithelial cells [17].
Osteoradionecrosis (ORN) is one of the significant side effects of radiotherapy; the pathogenesis of this disease remains unclear [18]. Delanian and Lefaix [19] argued that ORN progression is related to the activation and deregulation of fibroblast activity, and atrophic tissue is formed at the irradiation site. It has been reported that PTX is effective in bone regeneration of ORN when used together with tocopherol [20]. Delanian et al. conducted a study involving 22 breast cancer patients with radiation-induced fibrosis; significant surface regression was observed for the combination of PTX and vitamin E [21]. In a subsequent study, they reported that long-term treatment with PTX and tocopherol is effective and curative for refractory osteoradionecrosis [22]. Taken together, PTX has effects on maintaining mitochondrial viability, reducing the level of the TNF-α, IL-6, interferonγ, and IL-17 and upregulating the production of the anti-inflammatory cytokine IL-10, preserving microvasculature, preventing endothelial damage, and improving coagulation.
The world is facing a viral pandemic of coronavirus disease . The clinical symptoms of COVID-19 vary from asymptomatic or flu-like symptoms to serious conditions, including respiratory failure, acute respiratory distress syndrome, sepsis, or multiple organ dysfunction syndrome [23]. Methylxanthines have already shown improvement of the symptoms of adult respiratory distress syndrome, and PTX is a well-known anti-inflammatory and anti-oxidative molecule that has already been shown to suppress TNF-α as well as other inflammatory cytokines in pulmonary diseases, and this may be beneficial for better clinical outcomes in COVID-19 patients [24]. In the context of the COVID-19 pandemic, the diverse effect of PTX suggests that the drug can be used as an alternative treatment for patients with COVID-19 [25]. Even though there is no report about the antiviral activity against SARS-CoV-2, PTX is a potential treatment proposal for the SARS-CoV-2-induced complications including acute respiratory distress syndrome (ARDS) and dysregulated thrombosis [5,26]. In the treatment of ORN and COVID-19, control of the initial inflammatory response may affect disease severity and progression. In this regard, research on the cellular effects of PTX, one of the therapeutic agents for ORN, might be helpful in the treatment of COVID-19.
Immunoprecipitation high-performance liquid chromatography (IP-HPLC), a protein level detecting method, has been utilized in the determination of protein concentrations compared with reference controls. Protein samples and antibody-bound protein A/G agarose beads are mixed and incubated in a rotating stirrer. Then, the agarose beads are washed several times, and the target proteins are eluted by an IgG elution buffer and analyzed using UV spectroscopy in an automatic HPLC system. The advantage of IP-HPLC is that it is relatively accurate and reproducible among protein detection methods with a standard deviation of less than 5%. Simple experimental procedures can investigate the changes of expressions of multiple proteins in a relatively short time.
As mentioned earlier, there have been reports of using PTX for inflammation or infectious diseases, for example, in the treatment of osteoradionecrosis of the jaw [27]. Macrophages belong to the innate immune system representing the first line of defense against microorganisms, initiating a local inflammatory reaction. They are essential effector cells in inflammatory diseases and infectious diseases [28,29]. Therefore, in this study, RAW 264.7 cells derived from murine macrophages were used. To investigate the effects of PTX on RAW 264.7 cells, different protein (n = 132) expression profiles were analyzed using IP-HPLC.
IP-HPLC is a fast and accurate method (±5% standard deviation) for analyzing protein levels; brief procedures of IP-HPLC could be referenced [28][29][30]. In the IP-HPLC results, the sample protein peak areas (mAU*s) obtained from HPLC analysis in the negative control were used to eliminate the antibody peak area (mAU*s). Proportions (%) of experimental and control groups were plotted, and each IP-HPLC analysis was carried out two to six times to achieve a mean standard deviation within 5%, and the mean value was used for all IP-HPLC data. The average data were handled with SPSS (Version 25.0, SPSS Inc., Chicago, IL, USA) using the simple proportions and the Chi-square tests. The expression of housekeeping proteins, β-actin, α-tubulin, and GAPDH were used as internal controls. Expression changes of housekeeping proteins were adjusted to <±5% using a proportional baseline algorithm. The final data were graphed according to the properties of the protein groups [29][30][31]. We summarized these important steps in Figure S1.
Results
Expression of proliferation-related proteins. CDK4 expression was reduced to 92.32% of baseline in 24 hour-PTX-treated RAW 264.7 cells and elevated back to 99.91% of baseline in the 48 hour-treated sample. The expression level of Ki-67 was decreased to 92.0% of baseline in the 12-hour-treated sample and increased with time to 105.2%. Significantly lower expression of MPM2 (93.9%), proliferation-inhibiting proteins, p21 (90.2%), p27 (90.9%), and cyclin D2 (88.7%) was expressed than untreated controls. Changes of PCNA, PLK4, p14, and p16 were measured within ±5% in response to PTX, which were analogous with control housekeeping proteins ( Figure 1a). PTX inhibits the proliferation of RAW 264.7 cells. Expression of epigenetic modification-related proteins. Slightly increased expression of histone H1 (108.6%) and MBD4 (111.0%) but no significant change in the expression of KDM4D (95.1-103.6%) is found. A decrease in the expression of DMAP1 (88.0%), DNMT1 (94.5%), and HDAC-10 (94.9%) is also found. PTX may reduce DNA methylation and activate DNA transcription in RAW 264.7 cells. Therefore, PTX can be associated with epigenetic modifications and the regulation of gene expression. MBD4 may function to mediate the biological consequences of the methylation signal. MBD4 is similar in protein sequence to bacterial DNA repair enzymes and has been shown to perform some functions in DNA repair (Figure 2a). Expression of cellular differentiation-related proteins. Slightly reduced expression levels of the differentiation-related proteins including PLC-β2 (88.1%), TGase-2 (94.6%), HXK II (92.4%), Jagged-2 (93.8%), and GLI-1 (94.5%) is found. The expression of Notch-1 did not show significant changes, remaining similar to those of the housekeeping proteins. PTX treatment down-regulates cellular differentiation in RAW264.7 cells (Figure 2c).
Expression of RAS signaling proteins. PTX slightly reduced the expressions levels of V-Ki-ras2 KRAS (92.0%), NRAS (94.2%), and GTPase HRas (HRAS, 94.3%). MEKK (107.6%) expression was increased in the 12-hour-treated group but decreased with time similar to that of housekeeping proteins. The expression of ERK-1 (108.9%) increased in the 24-hour-treated group. The expression level of mTOR (94.0%) was decreased in the 12-hour-treated sample, while that of pAKT1/2/3 (89.1%) markedly decreased with time. Changes in JNK-1, Rab, and PKC expression were similar to housekeeping proteins. RAS signaling appeared to be reduced by PTX (Figure 3a).
Expression of NFkB signaling proteins.
A decrease in the expression of NFkB signaling proteins is shown. After PTX treatment, TNFα, GADD 153 decreased to a minimum of ±5%, but the expression levels of NFkB (84.9%), IKK (94.2%), pAKT (89.1%), PGC-1α (86.2%), and p38 (84.4%) were significantly reduced. RAW 264.7 cells treated with PTX showed a slight decrease in the expression of MDR (92.4%) and a slight rise in the expression of ERK1 (108.9%). The expression levels of GADD45 were slightly increased (105.0%) in the 12-hour-treated sample; however, levels decreased with time. NFκB is a key regulator of immune responses and inflammation. The NFκB signaling system is responsive to a number of stimuli. In our IP-HPLC result, protein expression related to NFκB signaling did not increase significantly.
Expression of immunity-related proteins. Significant increases in the expression of cluster of differentiation 4 (CD4, 112.1%) and CD20 (106.4%) were observed. The expression levels of CD31 and CD68 showed minimal changes of less than ±5%, which was analogous to that of the housekeeping proteins. The expression levels of CD3 and CD28 after PTX treatment were 87.4% and 91.6%, respectively. PTX is a phosphodiesterase inhibitor, the exact mechanism of various immunomodulatory functions has not been elucidated. Importantly, the expression levels of CD3 and CD28 were reduced by PTX, however, the exact mechanism could not be found. However, it is presumed that inhibition of these markers may reduce the T-cell mediated inflammatory response, thereby regulating the immune response. PTX can affect immunity-related proteins and, in turn, inhibit T-cellassociated immunity (Figure 4b).
Expression of F-mediated apoptosis-related proteins.
A decreased expression of FASL (90.9%), FADD (84.8%), and FLIP (88.7%) is shown. Caspase 8 expression levels were consistently down-regulated by PTX to 89.0% of baseline. The expression of FAS (95.6%) showed only a slight change but was within 5% of the control housekeeping proteins. The expression of PARP slightly decreased to 92.2% by PTX. These results suggested that PTX tends to inhibit FAS-mediated cell death (Figure 5b).
Expression of antioxidant-related proteins.
A slight increase in AMPK (106.6%) is found in samples treated for 24 h. The expression of LC3 (90.4%) and SOD-1 (89.2%) were decreased in the samples treated for 12 h and increased with time to 105.6% and 99.3% of baseline, respectively. GST (89.5%) was down-regulated by PTX treatment. However, the expression of NOS1 decreased to 88.0% in the sample treated for 48 h. These results suggest that PTX inhibits NO synthesis and the action of other antioxidant enzymes (Figure 6b).
Effects of PTX on the global protein expressions.
The results of this in vitro study are summarized in Figure 9. PTX was shown to inhibit cellular proliferation-, apoptosis-, and inflammation-related proteins in RAW 264.7 cells. Conversely, angiogenesis and antioxidant activities were not significantly affected. Although the origin of RAW 264.7 cells is a murine macrophage, PTX was expected to have an osteogenesis promoting effect (Table 2).
1, x FOR PEER REVIEW 13 of 19
Effects of PTX on the global protein expressions. The results of this in vitro study are summarized in Figure 9. PTX was shown to inhibit cellular proliferation-, apoptosis-, and inflammation-related proteins in RAW 264.7 cells. Conversely, angiogenesis and antioxidant activities were not significantly affected. Although the origin of RAW 264.7 cells is a murine macrophage, PTX was expected to have an osteogenesis promoting effect (Table 2).
Discussion
We treated RAW 264.7 cells (a murine macrophage cell line) with PTX and investigated global protein expressional changes in cells by IP-HPLC using 132 antisera. As PTX could be primarily engulfed by macrophages, after treating RAW 264.7 cells with PTX at 10 µg/mL for 12, 24, and 48 h, the RAW 264.7 cells were harvested with protein lysis buffer (0.3% SDS, 50 mM Tris-HCl pH 8.0, 0.3% β-mercaptoethanol, 1 mL PMSF, 1 mL EDTA) containing a protein inhibitor cocktail (Sigma, Chicago, IL, USA). Protein extracts were kept in a −70 • C deep freezer until required. In spite of there being a direct characterization between major inflammatory diseases such as COVID-19 and different protein expression profiles of unstimulated murine RAW 264.7 cells, we have tried to reveal and clarify the basic characteristics of PTX profiling. From these repeated and statistical data, the basic effects of PTX could be useful as antiviral and immunomodulatory functions, which will be appropriate for disease control, such as ORN and COVID-19.
In comparison with Western blot data of β-actin, a known cytoplasmic housekeeping protein, IP-HPLC exhibited a small error range of less than ±5.0% statistical significance [31][32][33]. In the IP-HPLC, housekeeping proteins, standard β-actin, α-tubulin, and GAPDH, were used as internal controls. Expression changes of housekeeping proteins were adjusted to ≤±5% using a proportional baseline algorithm [34]. PTX seemed to suppress proliferation-related proteins, up-regulated cMyc/MAX signaling in 12 and 24 h samples; however, decreased in the 48 h sample and is thought to promote cellular proliferation up to 24 h and suppress it more. These data suggest that PTX inhibited the p53/Rb/E2F signaling pathway which is associated with cellular proliferation. IP-HPLC may show a relative protein expression value compared to reference controls.
Methylxanthines have already shown improvement in the symptoms of adult respiratory distress syndrome, and PTX is a well-known anti-inflammatory and anti-oxidative molecule that has already been shown to suppress TNF-α as well as other inflammatory cytokines in pulmonary diseases, and this may be beneficial for better clinical outcomes in COVID-19 patients [24].
PTX reduced the expression of KRAS, NRAS, HRAS, and pAKT. The expression of ERK-1 was slightly up-regulated, while JNK-1, Rab, PKC levels were changed less than 5% in response to PTX. These results suggest that PTX slightly reduced RAS signaling compared to non-treated controls. PTX is known to inhibit TNF-α and the synthesis of other pro-inflammatory cytokines [35]. Inhibition of NFκB may be associated with the reduction of pro-inflammatory cytokines [36]. In this study, PTX showed gradual decreases in NFkB signaling-related proteins, however, TNF-α was not significantly affected by PTX treatment in this study. Inflammation-related protein, IL-1, IL-6, CD28, M-CSF, MCP-1, MMP-10, hepcidin, cathepsin K, LTA4H, CXCR4, and COX-2 were significantly downregulated. This feature is expected to reduce the cytokine storm that can occur in COVID-19 patients. PTX activates the adenosine A2A receptor, which is expressed by many types of immune cells, endothelial cells, and platelets, thereby up-regulating anti-inflammatory and anti-thrombotic molecules, and finally inhibiting the cytokine storm syndrome, which is believed to be the main pathogenic mechanism of COVID-19 s lethal complications [26].
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus genetically located within the genus Betacoronavirus that uses a glycoprotein (spike protein) to bind to the angiotensin-converting enzyme 2 (ACE2) receptor. After binding, the serine protease TGRBBS2 facilitates virus entry into the cell [37]. Currently, no therapeutic agent is effective in treating the SARS-CoV-2 infection, but the classes of drugs that are used include antiviral agents, neuraminidase inhibitors, and anti-inflammatory drugs [38]. As an antiviral agent, Lopinavir/ritonavir is an antiretroviral of the protease inhibitor class and is mainly used in COVID-19 patients with less severe symptoms and also in the early stage of the disease. Remdesivir, which belongs to the class of nucleotide analogs, was previously used in the Ebola virus epidemic in Africa and is currently used in moderate and severe COVID-19 [39].
As immunomodulatory drugs, an abnormal release of pro-inflammatory cytokines such as interleukin-6 (IL-6), interferon-gamma, and tumor necrosis factor-alpha was observed in moderate to severe stage COVID-19 patients. Anti-inflammatory drugs (particularly monoclonal antibodies) are used in COVID-19 patients. In this category, anti-IL6 (Tocilizumab), anti-IL-1 (Anakira), JAK inhibitors (Baricitinib), corticosteroids, antimalarials (Chloroquine/Hydroxychloroquine), heparins, and immunoglobulins. Antiviral agents are useful to inhibit the clinical progression and complications of COVID-19. Clinical and survival improvements were found in patients treated with plasma and hyperimmune immunoglobulins. Inflammation inhibitors are candidates for the treatment of advanced-stage COVID-19 [39].
Most patients with severe COVID-19 show elevated serum levels of pro-inflammatory cytokines including IL-6, IL-1β, IL-2, IL-8, IL-17, G-CSF, GM-CSF, MCP1, and TNF, characterized as cytokine storm [40]. A number of studies have trialed plans to reduce inflammatory responses. As already mentioned, PTX is derived from a methylxanthine derivative that inhibits phosphodiesterase-4 (PDE-4). PTX functions as a hemorrheologic agent and affects immunomodulation, inflammation, and oxidative stress [41]. In this study, the expression levels of IL-1, IL-6, M-CSF, MCP-1, MMP-10, and COX-2 were significantly down-regulated by PTX treatment in RAW 264 cells. Inhibition of proinflammatory cytokines by PTX treatment shares a context to other immunomodulatory agents used in COVID-19. The levels of CD4 and IL-10 were increased and PTX was found to increase immune function. In addition, the proliferation of macrophages is inhibited by PTX treatment, the macrophage-related acute immune response can be suppressed ( Figure S2).
The expressions of IGF1, HER1, and HER2 in growth factor-related protein expressions were slightly up-regulated. The expression levels of FGF-1 and FGF-2 were significantly reduced after PTX treatment. When PTX was applied to a renal fibroblast cell line, it inhibited fibroblast proliferation and suppressed FGF-2 synthesis [42]. Clinical studies on radiation-induced fibrosis have also shown that circulating the FGF-2 level was reduced after eight weeks of PTX treatment [43]. Tissue hypoxia can induce macrophage infiltration, which is a source of pro-fibrotic mediators, including TGF-β1 [44]. In this study, TGF-β1 expression was not significantly affected by PTX treatment.
The expression of CRP-1, CD4, IL-10, MMP-1, MMP-2, MMP-3, and MMP-9 was upregulated in inflammation-related protein profiles. The expression levels of IL-1, IL-6, CD3, CD28, M-CSF, MCP-1, MMP-10, hepcidin, cathepsin K, LTA4H, CXCR4, and COX-2 were all down-regulated by PTX treatment. In this study, immune-related markers were increased, and matrix inflammation-associated markers were decreased. The level of cytokines and T-lymphocytes in COVID-19 patients was studied, and it was suggested that a decrease in T cells in COVID-19 patients may be associated with a high serum concentration of TNF-α, IL-6 negatively regulating T cell survival or proliferation. [45] The severity of a cytokine storm and decreased T cells are associated with exacerbation of diseases such as pulmonary damage and respiratory distress [46,47]. The expression of CD4 is slightly increased by PTX treatment, in which it can be presumed that PTX contributes to macrophage immune function.
PTX downregulated the expression of p53, MDM2, and PARP. However, BAX expression was increased in the 12 and 24 h PTX treatment group and decreased slightly in the 48 h PTX treatment group to 98.8% of baseline. The expression of BAK was decreased in the 48 h sample, APAF-1 increased at 24 h and decreased at 48 h. The expression of caspase 9 decreased with time; however, the changes were not significant. PTX seems to down-regulate p53, but downstream proteins associated with the intrinsic apoptotic pathway require further study. PTX also down-regulated FAS-mediated apoptosis-related proteins, FASL, FAS, FADD, PARP, and caspase 8 were down-regulated by PTX treatment. PTX inhibited the extrinsic apoptotic pathway, which is in contrast to a previous study [48]. In this in vitro study, it was found that PTX suppressed the proliferation of RAW 264.7 cells and also suppressed the expression of proteins related to cellular apoptosis. However, we did not perform the cellular proliferation and apoptosis assay. In a previous animal study, an experiment irradiated the mandible of rats with radiation. It was shown that PTX had an effect of inhibiting cellular apoptosis [20]. Therefore, protein expressions related to proliferation and apoptosis were analyzed through the IP-HPLC in this study.
Referring to the antioxidant-related proteins, NOS1, SOD-1, GST, and LC3 were downregulated by PTX in contrast to the control group. This suggests that the antioxidant effect of PTX in RAW 264.7 cells is unclear. Luo et al. [49] studied the protective effects of Pentoxifylline on acute liver injury; the levels of SOD and glutathione (GSH) in liver tissue were elevated by PTX treatment compared to that of the control group. In a study of methotrexate-induced damage in the livers and kidneys of rats, immunostaining of iNOS and TNF-α was decreased in the PTX administered group, while SOD levels decreased in liver tissue. These results are similar to the result of our in vitro study [50].
The oncogenic protein expressions, TERT, and 14-3-3 were down-regulated; PTX did not elevate tumorigenic protein expression but actually showed some anti-cancer effects by up-regulating the expression of MBD4, which has some function in DNA repair. PTX not only has anti-tumor activity, but it also increases the susceptibility of cancer cells to radiation therapy [51].
Mild increases in cell protection-related proteins, such as HSP-70 (110.2%), AP-1 (111.3%), SP-1 (113.4%), and PKC (105.1%) also occurred. Most proteins associated with cellular protection increased in cells treated with PTX for 12 h and decreased for the next 24, and 48 h back to baseline, with the exception of the transcription factor. This means that the PTX has no deleterious effect on RAW 264.7 cells.
The expressions of OPG, osteopontin, and osterix, were slightly increased, and RANKL expression was markedly up-regulated by PTX treatment, which was not surprising since RAW 264.7 cells originate from murine macrophages and are likely to differentiate into osteoclasts. The advantage of IP-HPLC research is that it can investigate the expression of multiple proteins in one experiment. Therefore, when investigating osteogenesis, we tried to observe as many related proteins as possible. Very few studies have been published on PTX and osteogenesis; Horiuchi et al. [52] reported that PTX promotes rh-BMP-induced bone formation.
In the angiogenesis-related proteins, VEGF-A, vWF, ET-1, and CD31 did not show significant changes compared to the housekeeping proteins. The expression of HIF (108.6%), angiogenin (107.3%), and MMP-2 (109.1%) were slightly elevated by PTX treatment. PTX showed weak angiogenic effects. The effect of PTX on angiogenesis is controversial. In a study of segmental cortical bone defects of the radius in a rat model, PTX appeared to improve angiogenesis [53]. Conversely, PTX treatment significantly inhibited angiogenesis in a melanoma model [54].
The results of this study are summarized in Figure S3. PTX was shown to inhibit cellular proliferation, apoptosis, and inflammation-related proteins in RAW 264.7 cells. Conversely, angiogenesis and antioxidant activity were not significantly affected. PTX was expected to have an osteogenesis-promoting effect, which is expected to provide an advantage in the treatment of osteoradionecrosis with anti-inflammatory effects.
Moreover, having a potential role in suppressing inflammation and immune modulation, PTX has the potential to serve as an adjuvant therapeutic agent in COVID-19 pandemic situations, but additional preclinical and clinical studies are needed to prove the effect of PTX.
Besides, it may be possible to glimpse the potential as an adjuvant treatment in COVID-19 pandemic situations, but additional preclinical and clinical studies are needed.
Conclusions
PTX showed decreased expressions of proliferation-, extrinsic apoptosis-, and inflammation-related proteins and increased expressions of osteogenesis-related proteins in this in vitro study ( Figure S3). The anti-inflammatory effect, cytoprotective effect, and osteogenesis-promoting effect are thought to be helpful in the treatment of osteoradionecrosis. In addition, the results showed the possibility of using PTX as a therapeutic adjuvant for COVID-19. Additional animal studies or clinical studies are needed to verify these results.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/app11178273/s1. Figure S1. Schematic flows of IP-HPLC procedures with data analysis. Figure S2. Our suggested summary of PTX as the immunomodulatory drugs in the COVID-19 management. Figure S3. Summary of differential protein expressions in RAW 264.7 cells treated with PTX focused on the proliferation, inflammation, angiogenesis, and osteogenesis. Funding: There is no funding related to this article.
Informed Consent Statement: Written informed consent was obtained from patient's legal guardian for publication of this case report and accompanying images. Data Availability Statement: Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study. | v3-fos-license |
2020-12-03T09:03:33.097Z | 2020-11-28T00:00:00.000 | 229332123 | {
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} | pes2o/s2orc | Glycocalyx sialic acids regulate Nrf2-mediated signaling by fluid shear stress in human endothelial cells
Activation of the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is critical for vascular endothelial redox homeostasis in regions of high, unidirectional shear stress (USS), however the underlying mechanosensitive mediators are not fully understood. The endothelial glycocalyx is disrupted in arterial areas exposed to disturbed blood flow that also exhibit enhanced oxidative stress leading to atherogenesis. We investigated the contribution of glycocalyx sialic acids (SIA) to Nrf2 signaling in human endothelial cells (EC) exposed to atheroprotective USS or atherogenic low oscillatory shear stress (OSS). Cells exposed to USS exhibited a thicker glycocalyx and enhanced turnover of SIA which was reduced in cells cultured under OSS. Physiological USS, but not disturbed OSS, enhanced Nrf2-mediated expression of antioxidant enzymes, which was attenuated following SIA cleavage with exogenous neuraminidase. SIA removal disrupted kinase signaling involved in the nuclear accumulation of Nrf2 elicited by USS and promoted mitochondrial reactive oxygen species accumulation. Notably, knockdown of the endogenous sialidase NEU1 potentiated Nrf2 target gene expression, directly implicating SIA in regulation of Nrf2 signaling by USS. In the absence of SIA, deficits in Nrf2 responses to physiological flow were also associated with a pro-inflammatory EC phenotype. This study demonstrates that the glycocalyx modulates endothelial redox state in response to shear stress and provides the first evidence of an atheroprotective synergism between SIA and Nrf2 antioxidant signaling. The endothelial glycocalyx therefore represents a potential therapeutic target against EC dysfunction in cardiovascular disease and redox dyshomeostasis in ageing.
Introduction
Vascular endothelial cell defences against oxidative stress are coordinated by the transcription factor Nrf2, which modulates antioxidant gene expression through binding to DNA sequences termed antioxidant response elements (ARE) [1]. Under basal conditions, proteasomal degradation of constitutively synthesised Nrf2 is mediated by its cytosolic redox-sensitive partner Kelch-like ECH-associated protein 1 (Keap-1) [2]. Cytotoxic insults such as electrophiles and xenobiotics disrupt this interaction [3,4], allowing Nrf2 to accumulate in the nucleus where it promotes the transcription of genes encoding antioxidant, phase II detoxifying and glutathione synthesising enzymes to restore redox balance [5].
As reviewed previously [6], endothelial Nrf2 signaling is promoted by high unidirectional shear stress (USS) [7], whereas arterial regions exposed to low oscillatory shear stress (OSS) are prone to atherogenesis, partly due to diminished endothelial nitric oxide synthase (eNOS) expression [8] and attenuated antioxidant and anti-inflammatory properties of Nrf2 activation [9]. Exposure of EC to USS has been shown to promote Nrf2-dependent induction of cytoprotective genes [10], due to oxidation of thiol groups on Keap-1 [11] by cellular sources of reactive oxygen species (ROS) [12,13]. Activation of the Nrf2 pathway by USS can also be mediated by kinase signaling events [14,15] and is primed by shear-sensitive expression of Krüppel-like factor 2 (Klf2) [16], responsible for transcriptional programing of endothelial atheroprotection [17]. In contrast, Nrf2 stabilization and nuclear translocation in response to OSS does not promote ARE-dependent gene transcription [12] due to additional epigenetic regulation by histone deacetylases and mechano-sensitive microRNAs [6].
Despite the pivotal role of shear-sensitive Nrf2 regulation in determining susceptibility to vascular disease, the biomechanical mediators of this effect remain to be fully elucidated. Various plasma membrane molecules, microdomains and cytoskeletal components participate in shear stress mechano-sensation and transduction [18]. In particular, the glycocalyx (GCX), comprised of glycoproteins, proteoglycans, glycosaminoglycans (GAG) and glycolipids, has dimensions and biochemical composition that dependent on the dynamic equilibrium between its biosynthesis, degradation and local shear stress profiles [19]. The GCX contributes to the regulation of vascular tone via its mechanotransduction properties and is critical for blood rheology in the microcirculation, molecular filtration across the vascular wall, as well as thromboresistance and immuno-modulation [20]. Sialic acid (SIA) monosaccharides occupy the terminal branches of glycan chains within the GCX of EC, blood cells and common pathogens [21]. Arterial segments exposed to disturbed shear stress exhibit SIA deterioration, which predisposes them to atherogenesis [22]. Diminished SIA in the endothelial GCX is also associated with an enhanced risk of vascular dysfunction in diabetes [23] and is observed in rodent models of ageing [24]. Notably, EC desialylation by exogenous sialidases has been shown to impair NO-dependent vasodilatation by shear stress [25] due to enhanced ROS generation [26]; however the contribution of SIA in shear mediated induction of endogenous antioxidant defences remains to be elucidated.
In this study, we report the first evidence that fluid shear stress regulates EC redox signaling via alterations in the SIA component of the GCX. Using primary human EC, we demonstrate differential SIA expression and Nrf2-mediated antioxidant responses to USS and OSS. Furthermore, cleavage of SIA by exogenous neuraminidase led to diminished USS-mediated Nrf2 activation and an enhanced proinflammatory EC phenotype. In contrast, silencing of endogenous sialidase NEU1 enhanced Nrf2 responses to flow, highlighting the shearsensitive crosstalk between SIA and endogenous antioxidant defences. Our findings demonstrate that OSS-mediated SIA modifications lead to diminished activation of atheroprotective Nrf2 signaling, suggesting that GCX could be a key therapeutic target not only for age-related cardiovascular disease (CVD) but also infectious diseases, cancer and diabetes.
Endothelial cell isolation and culture
Umbilical cords from healthy, full-term pregnancies were obtained from the Maternity Unit at St. Thomas were isolated within 2 days of delivery using collagenase digestion as previously described [27]. Cells were cultured in gelatin-coated flasks in Medium 199 (M199) containing 10% (v/v) fetal and 10% (v/v) neonatal calf serum, NaHCO 3 (18 mmol L -1 ), penicillin/streptomycin (119 U ml -1 /120 μg ml -1 ), L-glutamine (5 mmol L -1 ) and endothelial cell growth supplement (ECGS, 5 ng ml -1 ) in a 5% CO 2 /95% humidified air incubator at 37 • C. EC monolayers were passaged with trypsin and all experiments were performed at passage 3. The HUVEC-derived endothelial cell line EA. hy926 (gifted by Unilever UK) [28] was used for infection with lentiviral vectors and maintained under the same conditions as HUVEC.
Fluid shear stress application
The Ibidi parallel-plate flow system (Ibidi GmbH, Germany) was used to recapitulate the laminar and oscillatory shear stress profiles associated with anti-and pro-atherogenic EC phenotypes, respectively. As the endothelial glycocalyx is established upon reaching quiescence [29], EC seeded in μ-I 0.6 Luer slides were maintained in static culture for 48 h to allow sufficient GCX growth. Cell monolayers were then exposed to shear stress (τ, dynes cm -2 ), calculated using the formula τ = μ 60.1 Φ. τ is proportional to the dynamic viscosity of the medium μ (0.00782 dyn s cm -2 for M199 at 37 • C [30]) and the flow rate Φ (ml min -1 ) generated by the air pressure pump. Cells were preconditioned to two consecutive 30 min cycles of 2 and 5 dyn cm -2 of unidirectional flow (USS), followed by either disturbed flow of ±5 dyn cm -2 where the direction reverses periodically (OSS, 1Hz oscillations) or USS of 15 dyn cm -2 , each for the indicated experimental periods. All cells were incubated in M199 without ECGS (basal M199) for 12 h before and during shear stress application.
Transmission electron microscopy (TEM)
Changes in GCX size and organisation were assessed by TEM at the Centre for Ultrastructural Imaging (King's College London). To preserve GCX integrity HUVEC were cultured on the detachable bottom of an Ibidi sticky slide μ-I 0.6 Luer and were perfused with two lysine-acetate solutions; the first containing 2% glutaraldehyde and 0.08% Alcian Krüppel-like factor NEU1
Abbreviations
Neuraminidase-1 NF-κB Nuclear transcription factor-κB NQO-1 NAD(P)H quinone oxidoreductase-1 Nrf2 Nuclear factor erythroid 2-related factor 2 OSS oscillatory shear stress ROS reactive oxygen species SIA sialic acid USS unidirectional shear stress VCAM-1 Vascular cell adhesion molecule-1 Blue (AB), followed by one containing 2% paraformaldehyde, 2.5% glutaraldehyde and 0.075% Ruthenium Red (RR). AB, RR and lysine are cationic reagents with high affinity for the negatively charged GCX [31]. Samples were osmicated (1% OsO 4 ) and dehydrated in a graded ethanol series before embedding in epoxy resin. Ultrathin (70-90 nm) sagittal sections obtained with a Leica UC7 ultramicrotome were mounted on 150 μm mesh copper grids and double-contrasted in UranyLess and 3% lead citrate (Electron Microscopy Sciences, UK) before examination under a JEM-1400Plus microscope (JEOL). High power electron micrographs of at least 10 different cells per condition were analysed using FIJI software [32]. Twenty measurements were collected from each cell, at points where both phospholipid bilayers were visible to ensure that luminal GCX depth was measured perpendicular to the plasma membrane [33]. GCX thickness was determined as half the maximal pixel intensity of the distance between the luminal edge and the lipid bilayer. Occasionally strands extending up to 200 nm were visible that were not included in the quantification process.
WGA images (2048 × 2048 pixels) were acquired with LSM-780 confocal laser scanning microscope (AxioObserver.z1, Carl Zeiss GmbH, Germany) using an oil immersion objective (Zeiss, Plan-Apochromat ×40/1.3 NA). WGA-CF TM 448A and DAPI were excited with Argon (458/488/514 nm, 25 mW) and diode (405 nm, 30 mW) lasers, respectively. For some experiments, planar sections were obtained along the z-axis (0.1 μm apart) and reconstructed into orthogonal views with FIJI software which was also used for false coloring and image analysis. For some experiments, SIA and DAPI were visualized with an epifluorescence microscope as described below. The mean fluorescence intensity of background subtracted fields of view (FOV) was normalized to the respective number of cell nuclei and expressed as mean cell intensity (MCI) of at least 300 cells per experimental condition.
Determination of free SIA
The total amount of free SIA was determined enzymatically using the NANA Assay kit (Abcam, Cambridge, UK) according to the manufacturer's protocol. Briefly, conditioned cell culture medium was centrifuged (1500 rpm, 5 min) and equal volumes of sample or assay standard were allowed to react (RT, 30 min) with the Oxi-Red probe that relies on free SIA oxidation to give fluorescence at excitation/emission:535/587 nm that was measured with a plate reader (ClarioStar; BMG Labtech, Germany). The concentration of free SIA was normalized to the original sample volume.
RT-qPCR
The mirVana™ miRNA isolation kit (Ambion, Thermo Fisher Scientific) was used to extract total RNA which was reverse transcribed in equal amounts (300 ng) with the high capacity cDNA kit (Applied Bio-systems™, Thermo Fisher Scientific). Gene expression was determined with SYBR® green I (Sensimix™ No-ROX kit, Bioline) using specific primer pairs (Table 1) and amplified by Rotor-Gene™ 6000 thermal cycler (Corbett Research, UK). Target gene levels were interpolated by a standard curve of known copy number concentrations and normalized to the geometric mean of five reference genes using geNorm algorithm [35] ( Table 1).
Immunoblotting
Whole-cell protein was extracted with a sodium dodecyl sulfate lysis buffer (2% w/v) containing protease and phosphatase inhibitors. Total protein content was determined with the bicinchoninic acid assay (Pierce, Thermo Fisher Scientific) before separation of equal amounts of denatured protein by gel electrophoresis and transfer to PVDF membranes. Non-specific binding sites were blocked with 5% w/v skimmed milk before overnight incubation (4 • C) with primary antibodies raised against proteins of interest or β-actin that was used as a loading control. Protein expression was detected with horseradish peroxidaseconjugated secondary antibodies and countered by ECL reagents. Immunoblots were visualized with the G-box gel documentation system (Syngene Bioimaging) and band densitometric analysis was carried out with FIJI software.
Lentiviral gene transfection
EA.hy926 cells were infected at multiplicity of infection of 10 (48 h) with lentiviral particles containing anti-Nrf2, anti-NEU1 or non-target (scrambled) shRNA and puromycin resistance genes (Santa Cruz Biotechnology). Transfection efficiency was enhanced with of polybrene Table 1 List of primer sequences.
Abbreviations
Antibiotic-resistant cell populations were expanded over two weeks in the continuous presence of puromycin, which was removed 48 h before experimentation.
Nrf2 and NFkB immunocytochemistry
Fixed cells were permeabilized with TritonX-100 (0.1%, 10 min) and blocked with 4% BSA. Cellular localization of Nrf2 or the p65 (Rel-A) subunit of NFkB was examined by specific primary (overnight, 4 • C) and AlexaFluor® 488 and 555 secondary antibodies (1 h at RT), respectively. Immunofluorescence images were acquired with a water immersion objective (Olympus, LUMPlanFL ×40/0.8 NA) of an inverted epifluorescence microscope (Nikon Diaphot) fitted with a Nikon DXM1200F digital camera. Nuclear and cytoplasmic fluorescence intensity were quantified and background corrected using FIJI software.
Detection of mitochondrial ROS
At the end of each experimental protocol, cells were loaded with the dihydroethidium-conjugated fluorogenic probe MitoSOX TM Red (excitation/emission: 510/580 nm, Invitrogen) prepared in serum-free M199 (5 μМ). Incubation for 30 min under static conditions was followed by fixation with 4% PFA and nuclei staining with DAPI before imaging with an inverted epifluorescence microscope as described above. FIJI software was used to quantify and background correct the mean fluorescence intensity that was normalized to the number of cell nuclei per FOV.
Statistics
Data denote mean ± S.E.M. from experiments of at least 3 different HUVEC donors or 3 independent EA. hy926 cultures, unless otherwise stated. Statistical comparisons between two independent groups were performed with unpaired Student's t-test while one-or two-way ANOVA with Tukey or Bonferroni post hoc tests were used to evaluate statistical differences between more than two conditions. P values < 0.05 were considered statistically significant.
Laminar flow enhances luminal GCX expression in vitro
Although HUVEC maintained in culture have diminished GCX thickness compared to the umbilical vein in vivo [36], multiple studies have demonstrated the vasoprotective properties of the GCX in vitro. Since static culture does not represent the dynamic conditions developed in the vasculature, we exposed HUVEC to laminar flow to more accurately recapitulate the physiological GCX environment. As revealed by TEM, HUVEC maintained in static culture have a rudimentary GCX, which becomes more uniform and thicker after prolonged exposure to USS (Fig. 1A). SIA is a major component of the vascular GCX and is markedly reduced in atheroprone regions of the vasculature exposed to disturbed shear stress profiles [37]. Given the abundance of SIA in the human umbilical vein [38], we next assessed its expression using WGA lectin labelling. Consistent with previous studies, static cells had abundant SIA expression at different passages (p0 to p3, data not shown) and while application of USS enhanced SIA levels (Fig. 1B), WGA intensity was diminished in cells exposed to OSS. In line with the TEM findings above, the 3-dimensional volume views of the confocal reconstructions, demonstrated enhanced apical localization of WGA staining in response to laminar flow (Fig. S1), however, that was not observed under static or OSS conditions.
To ascertain whether shear stress-modulation of WGA binding is due to altered SIA expression and not a consequence of stereochemical changes affecting their interaction [39], the free SIA content of the conditioned culture medium was investigated. Free SIA was accumulated in the culture medium of cells exposed to USS relative to OSS or static culture (Fig. 1C). Because glycan sialylation depends on the balance between SIA biosynthesis and the opposing actions of endogenous sialyltransferases and sialidases, the transcription of genes encoding enzymes involved in SIA metabolism was also assessed (Fig. 1D). mRNA levels of the rate-limiting SIA biosynthetic enzyme glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase 1 (GNE) [40] paralleled the differential SIA expression in response to USS and OSS. In contrast, only OSS enhanced mRNA expression of the nuclear enzyme cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) which generates SIA and nucleotide sugar pairs [41]. These are subsequently transported into the Golgi via the SLC35A1 antiporter [42] that was here reduced at mRNA level in response to OSS. Consistent with previous reports [43], the endogenous sialidase NEU1, which hydrolyses terminal SIA from the adjacent glycans, was the most abundant isoform expressed in HUVEC (data not shown). NEU1 mRNA was enhanced under flow conditions (Fig. 1D), but to a significantly greater extent by disturbed rather than laminar flow. These observations highlight that SIA remodelling is particularly susceptible to variations in shear stress and thus may regulate mechano-sensitive signaling.
OSS and SIA disruption attenuate antioxidant Nrf2 signaling activation by physiological flow
One of the most characterized Nrf2 targets is heme oxygenase-1 (HO-1), a stress response enzyme transcriptionally upregulated to confer protection against oxidative damage [5]. Physiological shear stress is a potent inducer of HO-1 expression, which is diminished in vascular areas susceptible to atherosclerosis and cells exposed to disturbed flow [7,9]. In line with these studies, HO-1 was upregulated in HUVEC exposed to flow, but the response to physiological USS was significantly greater (Fig. 2A). Under these conditions, similar results were obtained for the Nrf2-regulated detoxifying enzyme NAD(P)H quinone 1 oxidoreductase 1 (NQO1) and the modifier subunit of glutamate cysteine ligase (GCLM) required for biosynthesis of glutathione (Fig. S2A). Flow-mediated changes in antioxidant enzyme expression were also associated with increased Nrf2 nuclear localization in response to USS compared to OSS and static culture (Fig. S2B). To confirm that upregulation of these antioxidant enzymes is Nrf2 dependent, lentiviral silencing of Nrf2 was achieved in the shear-responsive EA. hy926 endothelial cell line which abolished USS upregulation of total Nrf2 protein levels (Fig. S2C). USS-mediated induction of HO-1 protein expression was also abrogated in Nrf2 knockdown cells (Fig. 2B), and this was replicated for NQO1 and GCLM expression (Fig. S2C) and with shorter exposure to USS (data not shown). Our study established a similar temporal regulation of the Nrf2/ARE pathway and SIA expression within the same 48-h period of shear stress conditioning. As enzymatic removal of SIA enhances ROS production [26], we next investigated whether this is due to mechanosensitive modulation of the Nrf2/ARE pathway. To selectively remove the SIA component of the GCX, cells were treated with 2U ml -1 neuraminidase ( Fig. 2C and Fig. S3) for a short period of time (30 min) to avoid the observed time-and dose-dependent decline in cell viability (data not shown). As GCX regrowth is a dynamic process with distinct recovery timeline for different components [44] and is enhanced in response to physiological flow [45], control and neuraminidase-treated cultures were subjected to USS for different times. Cell exposure to USS enhanced WGA staining at 24 h compared to 8 h in both treatment conditions (Fig. 2C). Neuraminidase significantly decreased WGA staining for at least 8 h post treatment, but significant SIA restoration was observed after 24 h (Fig. 2C). To avoid cytotoxicity caused by continuous exposure to neuraminidase, we used the acute SIA recovery period (<8 h) to assess its role in Nrf2 signaling. SIA removal with neuraminidase attenuated the induction of HO-1, NQO1 and GCLM protein levels in response to 6 h of USS (Fig. 2D). After 24 h, however, the induction of these enzymes by laminar shear stress was restored, concomitant with regrowth of SIA. Notably, treatment with neuraminidase under static conditions did not affect antioxidant enzyme expression at similar time points assessed (data not shown).
SIA removal impairs Nrf2 nuclear accumulation in response to physiological flow
We next investigated the mechanisms by which SIA regulate Nrf2 signaling. USS stabilises newly synthesised Nrf2 protein and enhances its nuclear accumulation [10,13], which we observed after 4 h of USS application (Fig. 3A). SIA removal with neuraminidase significantly reduced nuclear Nrf2 levels in cells exposed to USS (Fig. 3A). Moreover, neuraminidase attenuated USS-induced phosphorylation of Nrf2-Ser40 (Fig. 3B), previously shown to reduce Nrf2 association with Keap-1 in cells maintained in static culture [46]. In response to physiological flow the latter mechanism is mediated by protein kinase B (Akt) activity [7]. Indeed, acute exposure of HUVEC to USS stimulated Akt phosphorylation, which was significantly reduced following SIA cleavage with neuraminidase (Fig. 3C). Activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway can further promote Nrf2 activity via inhibition of glycogen synthase kinase 3β (GSK3β) [47]. When HUVEC were treated with neuraminidase prior to USS exposure, phosphorylation of GSK3β at Tyr216 was increased (Fig. 3D).
NEU1 knockdown promotes Nrf2 activation by physiological flow
To further investigate the role of SIA in shear stress mediated modulation of Nrf2 signaling, we silenced the endogenous sialidase NEU1. In EA. hy926 cells transduced with control shRNA, OSS enhanced NEU1 protein expression to a greater extent than USS, while flow conditioning of NEU1 knockdown cells did not alter sialidase levels compared to static culture (Fig. 4A). As NEU1 expression was previously associated with cell surface desialylation [48], we next assessed the effects of NEU1 silencing on SIA levels. NEU1 knockdown significantly enhanced SIA immunofluorescence in cells exposed to USS (Fig. 4B) and SIA levels were attenuated by disturbed compared to laminar flow. Moreover, enhanced SIA expression in the absence of NEU1 was associated with upregulation of Nrf2 target enzymes HO-1, NQO1 and GCLM in response to USS (Fig. 4C). Taken together, our findings suggest that SIA disruption reduces mechanosensitive activation of endogenous antioxidant systems by Nrf2 which is key for EC adaptation to oxidative stress in regions of high shear stress and may thus elicit dysfunctional EC phenotypes, investigated next.
Disturbed flow and SIA cleavage enhance mitochondrial ROS levels
Mitochondrial free radicals are important mechanosensitive secondary messengers responsible for inducible expression of Nrf2 targets in response to physiological flow [13,49]. However, mitochondrial ROS generation in response to disturbed flow is pro-apoptotic [50] and may contribute to EC-originated atherogenesis. Here, we observed enhanced levels of mitochondrial ROS in HUVEC exposed to prolonged OSS compared to laminar flow or with culture under static conditions (Fig. 5A). SIA removal with neuraminidase increased MitoSOX Red fluorescence in response to acute USS exposure (Fig. 5B) and a similar, albeit not statistically significant trend was observed in static cultures.
SIA removal promotes a pro-atherogenic EC phenotype in response to physiological flow
Cleavage of SIA from the endothelial GCX also reduces flowmediated NO bioavailability [51], therefore we next examined eNOS expression and phosphorylation following SIA removal. Neuraminidase did not affect total eNOS protein levels, however, in cells exposed to USS, SIA cleavage reduced phosphorylation of eNOS stimulatory sites Ser 1177 and 633 ( Fig. 6A and B) which are critical for NO output [52,53]. Moreover, physiological EC function is disrupted in arterial regions susceptible to atherogenesis due to the diminished expression of transcription factors Klf2 and Klf4 [54,55]. The atheroprotective properties of Klf2 stimulation are partly mediated by induction of Nrf2 targets [16], therefore we assessed the effects of SIA disruption on shear-sensitive Klf2 and Klf4 regulation. Prolonged (24 h) EC exposure to OSS reduced Klf2 and Klf4 mRNA levels relative to USS (data not shown). Similarly, SIA cleavage with neuraminidase attenuated early induction of both Klf2 and Klf4 by USS which was restored following SIA re-growth at 24 h (Fig. S4).
Deficits in Klf2 and Klf4 expression promote atherosusceptible EC phenotypes partly via upregulation of vascular cell adhesion molecule 1 (VCAM-1) [55,56], and suppression of Nrf2 signaling by disturbed shear stress elicits vascular inflammation through similar mechanisms in vivo [9]. As SIA cleavage with neuraminidase attenuated USS-mediated Nrf2 responses and protein expression of Klf2 and Klf4, we next investigated whether it is also a pro-inflammatory EC stimulus. Prolonged (48 h) HUVEC exposure to physiological USS significantly reduced VCAM-1 protein levels compared to OSS and static culture (data not shown). Notably, SIA cleavage with neuraminidase enhanced VCAM-1 expression under both static and acute USS conditions (Fig. 6C). Nuclear transcription factor-κB (NF-κB) is a key mediator of flow-sensitive VCAM-1 expression [57] and exhibits a biphasic pattern of initial activation and subsequent inhibition by prolonged laminar flow [58]. In agreement with these studies, acute cell exposure to USS increased nuclear translocation of the NF-κB p65 subunit, while SIA removal with neuraminidase further enhanced this effect (Fig. 6D) and promoted nuclear translocation of p65 under static conditions.
Discussion
This study is the first to demonstrate that the SIA component of the glycocalyx acts as a novel shear-sensitive regulator of endogenous Nrf2mediated antioxidant defences in human EC. Moreover, we provide novel evidence that in the absence of SIA, EC cultured under USS have impaired eNOS phosphorylation, enhanced levels of mitochondrial ROS and the proinflammatory marker VCAM-1, thus phenotypically resembling EC exposed to pro-atherogenic OSS. Diminished SIA at arterial branch points exposed to disturbed flow [37] and, in combination with systemic stressors or risk factors such as age [24], predisposes these sites to atherogenesis. To recapitulate the endothelial desialylation that occurs in response to OSS, we removed SIA with exogenous neuraminidase, which has been shown to promote neointimal thickening and oxidized low density lipoprotein (ox LDL) accumulation in vivo [59], elicit pro-inflammatory responses [60] and enhance vascular permeability [61]. Given the wide range of EC homeostatic functions mediated by Nrf2 targeted transcription, here we describe a new, potentially anti-atherogenic role for SIA via Nrf2 signaling.
Using TEM [62], we demonstrated that USS enhanced the thickness of endothelial GCX in vitro, and although additional GCX components may contribute to this effect [63], our findings correlated with increased immunofluorescence of surface SIA and transcript levels of its biosynthetic enzyme GNE [40]. Studies in animal models have previously shown using TEM a reduced anatomical GCX depth at arterial regions exposed to disturbed blood flow patterns [64,65], which also exhibit reduced WGA staining [37,39]. This is consistent with our finding of reduced SIA immunofluorescence in cells exposed to OSS, possibly due to deficits in GNE and SLC35A1 transcription afforded by USS, but also via upregulation of CMAS, which generates cytosolic SIA-nucleotide donors [66]. The epimerase activity of GNE is tightly inhibited by cytosolic levels of the SIA-nucleotide pairs which are normally concentrated into the trans-Golgi by the SLC35A1 antiporter [42]. Enhanced CMAS transcripts and reduced SLC35A1 expression in EC exposed to OSS may therefore rise the cytosolic SIA-nucleotide donor concentration and further impede GNE function. Upregulation of DNA-methyltransferases by disturbed flow [67] may also reduce GNE transcription via promoter CpG islet hypermethylation [68]. Notably, tissue hyposialylation due to deficits in GNE activity leads to age-related neuronal loss [69] and enhances oxidative stress in the skeletal muscle of patients with GNE myopathy [70]. Maintenance of surface SIA by shear-sensitive regulation of its biosynthesis and degradation is therefore crucial for the regulation of vascular homeostasis.
Moreover, the enhanced levels of free SIA we measured in the USSconditioned culture medium suggested increased SIA biosynthesis and turnover as shown previously for the GCX component HS [45]. Serum total SIA is elevated following acute myocardial infarction and in patients with type II diabetes and correlates with the severity of atherosclerosis [71] and circulating markers of oxidative stress [72]. Notably, SIA is susceptible to oxidative cleavage by ROS [73] and oxidative desialylation of plasma proteins such as LDL also contributes to the circulating SIA pool [74]. It is possible that enhanced generation or diminished scavenging of ROS observed in response to OSS further contributed to reduced expression of SIA. Taken together, our results suggest a multifaceted dysregulation of biosynthesis and surface retention of SIA in response to OSS.
The decline in cellular SIA in EC exposed to oscillatory flow was also associated with enhanced expression of NEU1. The endogenous sialidase NEU1 resides in two subcellular compartments with distinct homeostatic functions; lysosomal NEU1 regulates cytosolic SIA levels by recycling sialoconjugates [75], whereas in the plasma membrane it initiates inflammatory cascades via desialylation of surface molecules such as ICAM-1 [76] and TLR4 [77]. NEU1-mediated SIA cleavage also inhibits Akt signaling downstream of integrin α5β1 [78] and the latter mediates proinflammatory NF-kB activation in response to OSS [79]. In the present study, NEU1 silencing enhanced SIA expression in cells exposed to USS but had little effect on SIA levels following exposure to OSS, likely due to the sustained deficit in SIA biosynthesis described above. Importantly, enhanced SIA expression as a result of NEU1 knockdown upregulated the expression of Nrf2 target antioxidant enzymes in response to USS. Pharmacologic or genetic inhibition of NEU1 activity has been shown to reduce serum cholesterol and alleviate vascular dysfunction that underlies atherogenesis in the ApoE− /− mouse [48,80]. This suggests that shear-sensitive NEU1 expression may determine vascular sites of atherogenesis, and based on our findings, we propose that this is partly mediated via Nrf2-dependent regulation of endothelial redox signaling.
As reported previously [10], USS enhanced the expression of endogenous antioxidant defenses via Nrf2 signaling but only in the presence of intact SIA. Functional nuclear accumulation of Nrf2 in response to USS is mediated by PI3K and downstream Akt and protein kinase C activity [7,14], thus reduced Akt activation following SIA removal possibly attenuated Nrf2 responses to USS. Akt signaling also represses GSK3β activity [47], which is known to promote Nrf2 nuclear export [81] and its Keap-1 independent cytosolic degradation [82]. This may contribute to the diminished induction of Nrf2 nuclear accumulation by USS following SIA cleavage. Moreover, direct tethering of the Keap-1/Nrf2 complex on the outer mitochondrial membrane is postulated to maintain mitochondrial redox homeostasis [83], thus impaired Nrf2 activation in the absence of SIA has likely enhanced mitochondrial ROS accumulation in response to USS. In agreement with our findings, vascular ROS accumulation is observed in porcine femoral arteries perfused with neuraminidase [26] and extracellular ROS levels are upregulated when EC are exposed to neuraminidase in the presence of a phagocytic stimulus [84]. Based on evidence that SIA can directly interact with H 2 O 2 and •OH [85,86], it is possible that the antioxidant defences conferred by SIA-mediated scavenging were diminished after removal with neuraminidase. Although neuraminidase does not disrupt the extracellular superoxide dismutase (ecSOD) [26] which is bound to the HS component of the GCX [87], in the latter study, ecSOD did not provide sufficient antioxidant defence in the absence of SIA. Our finding of diminished Nrf2-mediated antioxidant responses therefore represents a novel mechanistic link between SIA disruption and vascular redox imbalance.
Enhanced oxidative stress as a result of endothelial desialylation with neuraminidase has profound implications for vascular tone regulation as it contributes to impaired NO-mediated vasodilation [26]. Although the pro-oxidant environment directly decreases NO bioavailability [88], SIA removal with neuraminidase also inhibits flow-mediated NO production [51] and reduces soluble guanylate cyclase activity ex vivo [25]. Our finding of reduced eNOS phosphorylation in EC treated with neuraminidase therefore implicates SIA in mechanotransduction of USS for NO-mediated vasomotor control. This is in line with previous reports of reduced eNOS Ser 1177 [89] and Ser 633 [90] phosphorylation following disruption of other key GCX components. In this study, neuraminidase did not affect the expression of the major GCX glycosaminoglycan HS, however removal of SIA can decrease the negative surface charge that, in turn, distorts GCX structure and possibly alters mechanical force transmission for intracellular signaling throughout the GCX layer [20].
We also demonstrated for the first time that SIA cleavage with neuraminidase upregulates endothelial VCAM-1 expression. As attenuated Nrf2 signaling has been directly implicated in the pathogenesis of vascular inflammation, SIA disruption may additionally initiate proinflammatory events via redox dysregulation. SIA deterioration due to systemic inflammation [91] or cleavage by neuraminidase [60] promotes inflammatory cell trafficking on healthy vessels and desialylation of VCAM-1 enhances EC adhesiveness under laminar flow [92]. Additionally, Nrf2 knockout mice exhibit enhanced VCAM-1 expression in normally atheroprotected aortic regions [9] and VCAM-1 levels are enhanced by Nrf2 silencing in EC exposed to shear stress in vitro [93]. In the latter study this is partly alleviated by antioxidant treatment, therefore enhanced mitochondrial ROS observed in our study in the absence of SIA is likely to have contributed to NF-kB activation [94]. Enhanced VCAM-1 expression has been observed following endothelial Klf4 depletion [55] whereas overexpression of Klf2 and Klf4 attenuates NF-kB assembly and VCAM-1 promoter activation [55,56]. Therefore, the interactions between SIA and the mechanosensitive transcription factors Klf2 and Klf4 are likely to protect against proinflammatory changes in arterial regions exposed to USS.
Perturbations in GCX underly EC dysfunction in vascular pathologies associated with oxidative stress such as diabetes, stroke, hypertension and atherosclerosis [95]. Moreover, the age-related decline in adaptive cellular responses to oxidative stress, especially blunting of vascular Nrf2 antioxidant signaling, plays a key role in the accumulation of oxidative modifications that contribute to macromolecular damage and inflammation in CVD [96]. Microvascular dysfunction has also been linked to age-related GCX decline [97], therefore therapeutic strategies that mitigate GCX deterioration are likely to reduce the risk and severity of CVD in ageing [98]. Notably, experimental restoration of SIA has been shown to be efficacious against atherosclerosis [99], obesity-related hypertension [100] and age-related renal microvascular dysfunction [101]. Furthermore, it was recently reported that GCX enhancement by the GAG supplement sulodexide activates Nrf2 signaling to confer cytoprotection against ischaemia-reperfusion injury [102]. Further studies are thus warranted to further elucidate interactions between GCX components in coordinating Nrf2-regulated antioxidant defences. In summary, our findings provide a novel insight into the molecular mechanisms by which the endothelial GCX maintains Nrf2-mediated redox homeostasis and highlights the therapeutic potential of targeting SIA metabolism to ameliorate vascular dysfunction in atherogenesis and age-related CVD.
Authors contributions
R.C.S. and M.F. conceptualized the study and were awarded the grant funding; P-M. P. developed the methodology and performed the experiments; P.K. assisted with the collection of umbilical cords and performed some of the experiments; G.V. and R.F. assisted with the TEM analyses of the glycocalyx; P-M.P., R.C.S., S.J.C. and G.E.M drafted the manuscript which was reviewed by all authors. R.C.S. is the guarantor of this study, with responsibility for the integrity of the data and accuracy of the data analysis.
Declaration of competing interest
Authors declare no conflicts of interest. | v3-fos-license |
2021-01-07T09:06:39.843Z | 2021-01-06T00:00:00.000 | 234276050 | {
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} | pes2o/s2orc | Nutraceuticals Obtained by SFE-CO 2 from Cladodes of Two Opuntia ficus-indica (L.) Mill Wild in Calabria
: The aim of the present study was to evaluate the possibility to extract, by supercritical fluids, nutraceuticals as polyphenolic compounds, able in the prevention and in the treatment of a series of chronic-degenerative diseases, from plant matrices like the cactus pear. Supercritical fluid technology is an innovative method to extract nutraceuticals from natural matrices. This method offers numerous advantages that include the use of moderate temperatures, solvents with good transport properties (high diffusivity and low viscosity), and cheap and nontoxic fluids. Fresh cladodes from two different wild ecotypes of Opuntia ficus-indica (L.) Mill. were extracted both with methanol and with SFE-CO 2 using different samples preparations, to maximize the % yields and the selectivity of extraction of polyphenols. The biggest contents of phenolics, evaluated by Folin-Ciocalteu assay, has been observed with the sample dehydrated of O. ficus-indica cultivar that shows, as well, the best yield % (m/m) of extraction with both methanol and SFE-CO 2 . Better results were obtained with the samples of O. ficus-indica cult. ( OFI cult .), in spite of the O. ficus-indica s.l. ( OFI s.l .); the two different ecotypes of OFI showed dissimilar phytochemicals profile. We noticed that the reduction of both quantity and quality of polyphenols was drastic with the increase of pressure at 250 bar; this shows that high pressures result in a loss of bioactive principles, like polyphenols. By changing the variables of extraction processes with SFE-CO 2 and by varying the preventive treatments of the natural matrices, it was possible to increase the selectivity and the purity of the products. Thus, the optimization of this useful and green technique allowed us to increase the value of the Opuntia cladodes, a by-product very diffused in Calabria, which is an extraordinary source of nutraceuticals. These extracts could be used directly as functional foods or as starting material in the pharmaceutical, nutraceutical or cosmetic companies; they are safe and without any solvents traces and it is possible to obtain it in a few hours respect to the conventional extraction that requires longer extraction time.
Introduction
Nowadays, the consumption of natural extracts from plants and seeds present in nature and the discovery of the many benefits connected to their intake, have favoured and promoted research in the field of extraction and use.
For this purpose, in recent years, extract of plant parts (seeds, cladodes, stem, fruits, etc.) of the family Opuntiaceae have been investigated, as for instance: Opuntia joconostle seeds for cholesterol-lowering properties [1]; Opuntia humifusa cladodes, for cytotoxic activity against the human breast cancer cell lines MCF-7 and human colon SW-480 cells, and stem and fruits able to inhibit the growth of U87MG glioblastoma cells [2]; Opuntia Rutin is found in many fruits and vegetables and has been used in over 130 thera peutic medicinal preparations that have been registered as drugs worldwide [21]. In liter ature, it has been found that Rutin improves the memory of mice in Alzheimer's as i reduces the levels of Aβ-oligomer and attenuates oxidative stress and neuroinflammation [22].
Isoquercitrin, as glycosylated flavonoid, is rapidly absorbed and transformed, in th gastro-intestinal tract, in glucuronidated Quercetin [23,24] that was found to be the majo form in plasma after oral administration of Isoquercitrin in rats [25]. Nicotiflorin, as wel shows protective effects on memory dysfunction in multi-infarct dementia model rat [26]. Narcissin is known for its biological proprieties, such as hepatoprotective, antioxi dant, anti α-glucosidase, and cytotoxicity against human myelogenous erythroleukemi cells [27].
In general, flavonoids can be found in many varieties of fruit, vegetables and cereal [28][29][30][31]. They play a very important role in a series of pathologies, both in their contro and in their prevention, such as in Alzheimer's, cancer, etc., perhaps because they ac eliminating free radicals. [32][33][34][35][36].
Conventionally the used techniques to obtain polyphenols extracts, such as Soxhle maceration [37], organic solvent extraction [38][39][40], autoclave treatment and microwav [37] or ultrasound-assisted extraction [39][40][41][42][43][44], usually require several hours or days and spending a large volume of solvents, frequently toxics, and with problematic garbage dis posal. The correct separation between solute and solvent is important, as a possible deg radation of the thermolabile components would compromise the benefits of the extract Furthermore, any residual solvent could lower the quality and quantitative yield of th extraction. Conventionally the used techniques to obtain polyphenols extracts, such as Soxhlet, maceration [37], organic solvent extraction [38][39][40], autoclave treatment and microwave [37] or ultrasound-assisted extraction [39][40][41][42][43][44], usually require several hours or days and spending a large volume of solvents, frequently toxics, and with problematic garbage disposal. The correct separation between solute and solvent is important, as a possible degradation of the thermolabile components would compromise the benefits of the extract. Furthermore, any residual solvent could lower the quality and quantitative yield of the extraction.
Instead, for the extraction of polyphenols from cladodes, in this work it was used a green extraction technique: the supercritical extraction (SFE) with CO 2 [37], generally used to obtain oils from natural products [45][46][47][48].
This method has been used for the purpose of investigating the possibility of phytochemicals extraction without any solvent and to optimize the selectivity between phenolics and other substances in the cladodes of two ecotypes of Opuntia ficus-indica wild in Calabria, O. ficus-indica cult. (OFI cult.) and O. ficus-indica s.l. (OFI s.l.).
Supercritical fluid technology is the most innovative method for preparing bioactive products from plants used as supplements for functional foods [49] and it results as promising technology both in food farming and pharmaceutical industry. Supercritical fluids have high solvation capabilities, which are similar to those of liquids, as well as diffusion properties similar to those of gases. For these reasons, the extraction using supercritical fluids is particularly suitable for the extraction of biocompounds from plant matrices [50].
Although there are several usable supercritical fluids, supercritical CO 2 is the most commonly used. This application success is due to its low critical constants (Tc = 31.1 • C, Pc = 7.38 MPa), to the fact that it is non-toxic and non-flammable and because it is available at high purity at low cost. Moreover, other advantages are a high diffusion coefficient and low viscosity; being gas at atmospheric condition, the CO 2 immediately seeps out when brought to the environment, so the products obtained are free from the "extraction solvent" and thermal degradation compounds.
The extracts obtained by SFE-CO 2 are also considered as Generally Recognized As Safe (GRAS) for the American Food and Drug Administration, being possible to add them to all food without undesirable effects for health. Some data have also been reported on the application of SFE-CO 2 from vegetable by-products [51].
Today, new chemistry knowledge at the molecular level regarding the functional and structural properties of active principles may allow a better selection of the products and extracts that can satisfy the request of the market, also according to the specific needs of both food and pharmaceutical industries [52]. Thus, with this innovative and advantageous technique, we extracted the Rutin, Isoquercitrin, Nicotiflorin and Narcissin from hard to manipulate plant matrices, namely the cladodes, with the aim to use them as safe supplements in the food and pharmaceuticals companies.
Plant Material
Two cladodes ecotypes of Opuntia ficus-indica (L.) Mill. (Figure 2) were analysed in this study: one supposed to be a cultivar with the hybrid origin, which often escapes, from cultivation and behave like an invasive species, it is almost spineless and it was accepted with the name Opuntia ficus-indica cult. (OFI cult.); and the second one with white and hard spines, long about 3-4 cm, sharing some characters with Opuntia amyclaea Ten., O. maxima Miller and O. ficus-barbarica. Hereafter we will use for the spiny ecotype the name The cladodes from both O ficus-indica were collected between June-August 2012 in Calabria (South of Italy). All the cladodes, covered by spines and multicellular hairs or trichomes, were manually cleaned, cut into small pieces and then homogenized ( Figure 3) with a TYPE HR 2064 PHILIPS-600 W and frozen until analysis. The cladodes from both O ficus-indica were collected between June-August 2012 in Calabria (South of Italy). All the cladodes, covered by spines and multicellular hairs or trichomes, were manually cleaned, cut into small pieces and then homogenized ( Figure 3) with a TYPE HR 2064 PHILIPS-600 W and frozen until analysis. The cladodes from both O ficus-indica were collected between June-August 2012 in Calabria (South of Italy). All the cladodes, covered by spines and multicellular hairs or trichomes, were manually cleaned, cut into small pieces and then homogenized ( Figure 3) with a TYPE HR 2064 PHILIPS-600 W and frozen until analysis.
Chemicals
Solvents as methanol (analytical grade and for HPLC), trifluoroacetic acid, water (analytical grade and for HPLC) and reagents such as Folin-Ciocalteu and Chlorogenic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Preliminary Analysis
The pH value of the homogenized system (pH 700, Eutech Instruments, Germany), the activity level (Novasina AW Sprint-TH 500, Switzerland), the Brix degrees (ATAGO, Hand Refractometer N Type Series, Japan) and humidity (Mettler Toledo Moisture Analyzer HB43-S, Switzerland) were measured. The analyses were repeated in triplicate and the results are reported as mean value and standard deviation.
Chemicals
Solvents as methanol (analytical grade and for HPLC), trifluoroacetic acid, water (analytical grade and for HPLC) and reagents such as Folin-Ciocalteu and Chlorogenic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Preliminary Analysis
The pH value of the homogenized system (pH 700, Eutech Instruments, Germany), the activity level (Novasina AW Sprint-TH 500, Switzerland), the Brix degrees (ATAGO, Hand Refractometer N Type Series, Japan) and humidity (Mettler Toledo Moisture Analyzer HB43-S, Switzerland) were measured. The analyses were repeated in triplicate and the results are reported as mean value and standard deviation.
Exhaustive Extraction and Total Phenolics Content
The fresh cladodes of Opuntia ficus-indica both ecotypes were extracted with methanol (48 h × 3 times) at 4 • C. The same procedures were followed for a sample of OFI cult. and OFI s.l. Moreover, the exhaustive analysis was performed on samples dried in the oven for 360 min at 32 ± 1 • C; removing the 30% of the weight.
The extraction solutions were filtered in synthetic cloth, concentrated and dried under vacuum at 35 ± 1 • C for the thermolability of the polyphenols compounds.
The total phenolic content of the cladodes extracts was quantified using Folin-Ciocalteu reagent and chlorogenic acid used as standards. The absorbance was measured at 726 nm (Perkin Elmer Lambda 40 UV/VIS spectrophotometer) and the total content was expressed (mean ± S.D. of three determinations) as mg of chlorogenic acid equivalents on 100 g of fresh raw material (Singleton Rossi, 1965).
Extraction of Polyphenols with Supercritical Fluids
The supercritical extractions by CO 2 were performed on a Spe-ed SFE 4 extractor (Applied Separation, Allentown, PA, USA) and following the necessary steps: loading in the 50 mL stainless steel vessel, pressurization with CO 2 and waiting for a transitory state of temperature, which is kept constant by an oven module. After the reaching of target temperature and pressurization, in order to guarantee the intimate contact between the CO 2 and the matrix, the system was kept closed for 20 min (static phase), and only after The different methods to prepare the samples and pressure are listed in Ta All samples were cleaned, homogenized and frozen after collection, to quently analysed.
Some samples analysed were centrifuged after defrosting at 3500 rpm for 1 only the precipitated part was used to extract the polyphenols while other sam partially dried at 32 °C in the oven to reduce the amount of water.
The extraction time was 1 h. The final extracts were collected in glass tube with an aluminium foil and frozen until analysis. Each extraction was carried o The extract left the vessel through a valve, which was in advance thermostated to avoid CO 2 solidification, due to the expansion. Experiments were carried out at 40 • C, 110 bar and 250 bar by varying the preventive sample preparation with PSE accessories, as Ottawa Sand and an Spe-ed TM PSE Matrix (Hydroscopic Samples Dispersing Agent). All the extracts were stored at −18 • C before HPLC analysis.
This extraction method has always attracted because it is safe and eco friendly. In addition, the supercritical fluid, in the specific case CO 2 , has properties similar to those of gas in the supercritical state so that it can extract in a very short time compared to the classic extraction types, returning an extract already separated from the solvent after depressurization. Supercritical fluids have a high diffusion coefficient and a low viscosity which favours their intimate contact with the matrix. On the other hand, however, this method has limits, especially in relation to the use of the supercritical solvent because CO 2 has low polarity. Therefore this type of extraction technique is generally used for the extraction of oils, fats or in any case polar substances. Only the use of other solvents, such as ethanol or water, increases the solvent power of CO 2 in the supercritical state.
The different methods to prepare the samples and pressure are listed in Table 1. All samples were cleaned, homogenized and frozen after collection, to be subsequently analysed.
Some samples analysed were centrifuged after defrosting at 3500 rpm for 10 min and only the precipitated part was used to extract the polyphenols while other samples were partially dried at 32 • C in the oven to reduce the amount of water.
The extraction time was 1 h. The final extracts were collected in glass tubes covered with an aluminium foil and frozen until analysis. Each extraction was carried out in duplicate.
HPLC Analysis
The analysis of all the extracts, both from solvent extraction and belonged from SFE, was carried out by means of HPLC using a Smartline HPLC system (Knauer, Germany). Chromatographic separation was carried out using a 2.0 mm ID × 150 mL, with precolumn, C-18 TSKgel ODS-100 V, 21810 (TOSOH BIOSCIENCE), both thermostatically at 40 • C. The operative conditions: mobile phase, flow rate and gradient of elution utilized are reported in Table 2. For the mobile phase, methanol and water with trifluoroacetic acid (TFA) were used. Absorbance spectra were recorded every 2 s, between 200 and 450 nm, with a bandwidth of 4 nm, and chromatograms were acquired at 254 and 280 nm. HPLC analysis was performed in duplicate.
A wavelength of 280 nm was used for quantification [18], while the calibration line was obtained from the integration of the absorption peaks obtained from a series of dilutions of Rutin, Isoquercitrin, Nicotiflorine and Narcissin.
Preliminary Analysis
Cladodes by Opuntia genus showed a weakly acid pH and this allows easier conservation of the homogenized system. The results are shown in Table 3. The higher amount of saccharides was found in the sample of Opuntia ficus-indica s.l., than that one of Opuntia ficus indica cult., and this is possible due to different factors such as the variety and age of the plant, soil, and climate. The pH was measured, because it can influence the viscosity of the mucilage and it could hamper the extraction, and it is similar for the two types of plant. The water activity and humidity values are quite similar, guarantying the same times of exposition to airflow and heat during the dehydration.
Exhaustive Extraction, Total Phenolics Content and HPLC Analysis
The high content of total phenolics was observed with the Folin-Ciocalteu assay in the samples dried in the oven, Cult_AD and Sl_AD; these show as well the better quantitative yield of extraction, calculated using the following equation [45]: where W d.e. is the weight of the dry extract obtained and W m the mass of the plant macerated. The data for the yield of extraction at different sample preparation and the total phenolics content are shown in Table 4. In Table 4 it is possible to point out that samples dried give a higher yield of extraction and total phenolics content with respect to the fresh macerated ones, showing that the total polyphenol contents vary depending on the type of treatment and extraction.
Comparing the same fresh sample collected in two different months, it is possible to observe that the extraction yield was slightly higher in the sample Cult_J with respect to the Fresh frozen macerated of August, but the total phenolic content is almost unchanged between samples of Cult_J and Cult_AF. This indicates that the different maturation period does not influence the content of secondary metabolites, such as phenols.
Moreover, the total phenolic content is higher in the dried samples Cult_AD and Sl_AD, probably because drying process is designed to dehydrate the matrix in order to stop the common enzymatic processes; the aqueous environment of the cytoplasm of plant cells could damage the active compounds [52].
The highest content of total phenols was found in the sample of Sl_AD, perhaps because being a wild ecotype is less affected by climate change, more adaptable than a cultivated plant.
In Figure 5 are reported the data, obtained by the HPLC analysis, of extractions with solvent.
Rutin is one of the polyphenols presents in greater amount in the analysed species. Moreover, it is possible to observe that by dring the sample it is possible to obtain extracts with a greater amount of some polyphenols (Cult_AD and Sl_AD).
As reported in literature rutin is already described to be present in cladode extracts of different Opuntia species [8,20].
Isoquercitrin is another bioactive compound present in our extract in similar quantity in the two species analysed while the quantity of Nicotiflorin and Narcissin are very low in the extracts. The different exposure to the sun or climate change can cause the secondary metabolism of the plant. As a consequence, the antioxidant profile can be influenced.
Sl_AD, probably because drying process is designed to dehydrate the matrix in order to stop the common enzymatic processes; the aqueous environment of the cytoplasm of plant cells could damage the active compounds [52].
The highest content of total phenols was found in the sample of Sl_AD, perhaps because being a wild ecotype is less affected by climate change, more adaptable than a cultivated plant.
In Figure 5 are reported the data, obtained by the HPLC analysis, of extractions with solvent. Figure 5. Quantification in mg of standards in 100 g of dry extract. Figure 5. Quantification in mg of standards in 100 g of dry extract.
Extraction with Supercritical Fluids and HPLC Analysis
The yields extractions are calculated by using the following equation according to Yieddes et al. [45]: where W extract is the weight of the extract obtained, which is the difference between the mass of glass trap with extract and the mass of empty glass trap and W load is the mass of sample load in the column before the extraction. The results are reported in Table 5; instead, the results of HPLC quantification on mg of the compound for 100 g of material loaded (mean ± S.D. of two determinations) for OFI cult. are shown in Figure 6, instead of OFI s.l. in Figure 7. The yields of extraction with SFE-CO2 are lower than that one with solvent as well as the amount of polyphenols, but the resulting extracts do not need to be separated from solvent, they are purer and cleaner.
It is possible to observe that the better results were obtained with the samples of OFI cult. despite the OFI s.l. In particular, the best yield of extraction was 2.14% ± 0.35, obtained with the sample CULT_30E_20O of OFI cult dehydrated at 30% and added with 20% (of the total weight loaded for the extraction) of Ottawa Sand at 110 bar. Given the results obtained from the extractions, it is possible to assume that Ottawa sands, hydrophobic natural silica particles, guarantee a better dispersion than diatomaceous earth, which also The yields of extraction with SFE-CO2 are lower than that one with solvent as well as the amount of polyphenols, but the resulting extracts do not need to be separated from solvent, they are purer and cleaner.
It is possible to observe that the better results were obtained with the samples of OFI cult. despite the OFI s.l. In particular, the best yield of extraction was 2.14% ± 0.35, obtained with the sample CULT_30E_20O of OFI cult dehydrated at 30% and added with 20% (of the total weight loaded for the extraction) of Ottawa Sand at 110 bar. Given the results obtained from the extractions, it is possible to assume that Ottawa sands, hydrophobic natural silica particles, guarantee a better dispersion than diatomaceous earth, which also The yields of extraction with SFE-CO 2 are lower than that one with solvent as well as the amount of polyphenols, but the resulting extracts do not need to be separated from solvent, they are purer and cleaner.
It is possible to observe that the better results were obtained with the samples of OFI cult. despite the OFI s.l. In particular, the best yield of extraction was 2.14% ± 0.35, obtained with the sample CULT_30E_20O of OFI cult dehydrated at 30% and added with 20% (of the total weight loaded for the extraction) of Ottawa Sand at 110 bar. Given the results obtained from the extractions, it is possible to assume that Ottawa sands, hydrophobic natural silica particles, guarantee a better dispersion than diatomaceous earth, which also acts as a drying agent, being hydrophilic. The Ottawa sands, therefore, by favoring a homogeneous dispersion, under the same initial conditions (type of sample and initial drying conditions), guarantee a tighter contact between the matrix and the supercritical fluid. At the increase of the dehydration to 90% for the sample CULT_90E_20O, a decrease in the yields' % follows, but an improvement is obtained concerning the selective extraction of polyphenols. In this sample, it is possible to observe the highest quantities of Rutin, Narcissin and Nicotiflorin, in accordance with the extractions by maceration, where the greatest quantity of polyphenols was obtained for the samples first dried and then macerated in methanol ( Table 4).
The treatment of OFI cult. with 20% of Diatomee Sand (sample CULT_20D) implies a reduction of both quantity and selectivity of extraction of polyphenols, perhaps because the Diatomee Sand is hygroscopic. This sand absorbs water from the surrounding environment reducing the function of co-solvent of water naturally present in the matrix.
Being polyphenols polar compounds, the pressure was then raised to 250 bar for the sample CULT_20P, maintaining the same sand, trying to increase the polarity of CO 2 , but the effect of the sand was stronger. It was obtained a reduction of both yield % and selectivity of polyphenols; this shows that high pressures result in a loss of bioactive principles sensitive as polyphenols.
When the sample is centrifuged (sample CULT_C_20D) to remove water, it was obtained a good increase of the yield % of extraction and a little improvement on the selectivity of polyphenols.
The yields % of extractions of the OFI s.l. were very low, ranging between 0.02 and 0.25%; but the selectivity and purity of the extracts derived therefrom are very high, as can be seen in Figure 8 for the sample SL_30E_20D. acts as a drying agent, being hydrophilic. The Ottawa sands, therefore, by favoring a homogeneous dispersion, under the same initial conditions (type of sample and initial drying conditions), guarantee a tighter contact between the matrix and the supercritical fluid. At the increase of the dehydration to 90% for the sample CULT_90E_20O, a decrease in the yields' % follows, but an improvement is obtained concerning the selective extraction of polyphenols. In this sample, it is possible to observe the highest quantities of Rutin, Narcissin and Nicotiflorin, in accordance with the extractions by maceration, where the greatest quantity of polyphenols was obtained for the samples first dried and then macerated in methanol ( Table 4). The treatment of OFI cult. with 20% of Diatomee Sand (sample CULT_20D) implies a reduction of both quantity and selectivity of extraction of polyphenols, perhaps because the Diatomee Sand is hygroscopic. This sand absorbs water from the surrounding environment reducing the function of co-solvent of water naturally present in the matrix.
Being polyphenols polar compounds, the pressure was then raised to 250 bar for the sample CULT_20P, maintaining the same sand, trying to increase the polarity of CO2, but the effect of the sand was stronger. It was obtained a reduction of both yield % and selectivity of polyphenols; this shows that high pressures result in a loss of bioactive principles sensitive as polyphenols.
When the sample is centrifuged (sample CULT_C_20D) to remove water, it was obtained a good increase of the yield % of extraction and a little improvement on the selectivity of polyphenols.
The yields % of extractions of the OFI s.l. were very low, ranging between 0.02 and 0.25%; but the selectivity and purity of the extracts derived therefrom are very high, as can be seen in Figure 8 for the sample SL_30E_20D.
Conclusions
There is a global trend toward the use of natural flavonoids present in fruits, vegetables, oilseeds and herbs as antioxidants or functional foods.
In the present work, two different ecotypes of O. ficus-indica, showing dissimilar phytochemicals profile, were studied. The total phenolic content in the extracts with solvents is almost unchanged between the fresh samples collected in June and in August. This indicates that the different maturation period, if the plant still did not produce the fruit,
Conclusions
There is a global trend toward the use of natural flavonoids present in fruits, vegetables, oilseeds and herbs as antioxidants or functional foods.
In the present work, two different ecotypes of O. ficus-indica, showing dissimilar phytochemicals profile, were studied. The total phenolic content in the extracts with solvents is almost unchanged between the fresh samples collected in June and in August. This indicates that the different maturation period, if the plant still did not produce the fruit, does not interfere with the content of secondary metabolites, such as phenols, that is almost the same. The polyphenol present in greater amount in all species is Rutin; the dried samples (Cult_AD and Sl_AD) contain a greater amount than the fresh.
Being polyphenols polar compounds, the yields % quantitative of extraction with SFE-CO 2 are not high even if the extractions are very selective and the extracts nearly pure.
Concerning the SFE-CO 2 better results were obtained with the samples of OFI cult. in spite of the OFI s.l., probably because the ecotype OFI s.l. was much mucilaginous, impeding to complete some extractions.
The better SFE-CO 2 extraction results were obtained with samples preventively dried, as the sample CULT_90E_20O. Thus, the reduction of water, responsible for the degradation of active compounds, in presence of the Ottawa sand, produces highly selective extracts.
The treatment with Diatomee Sand implies a reduction of both quantity and selectivity of extraction of polyphenols, perhaps because the Diatomee Sand is hygroscopic and it adsorbs water from the surrounding environment, reducing the function of co-solvent of the water naturally present in the matrix. By changing the % hydration, it is possible to modulate the selectivity of extraction.
The polyphenols are polar compounds, therefore the pressure was increased to 250 bar to increase the polarity of CO 2 . Nevertheless, a reduction of both the % yield quantitative and selectivity of extraction was obtained, demonstrating that the high pressures result in a loss of bioactive principles, like polyphenols. Our results evidenced as, for the first time, by improving the parameters of SFE-CO 2 , which is an innovative, safe and cheap technique, it is possible to extract qualitatively and quantitatively the polyphenolic fraction from plant matrices hard to treat, namely the Opuntia cladodes. These results increase their value as good nutraceuticals sources, instead to be considered only a by-product from prickly pear cultivation in Calabria.
Finally, the cladodes extracts could be used as food supplements or starting materials in the pharmaceuticals industries. | v3-fos-license |
2018-12-02T16:55:10.311Z | 2018-10-01T00:00:00.000 | 53741087 | {
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} | pes2o/s2orc | Nutritional composition and total collagen content of two commercially important edible bivalve molluscs from the Sea of Japan coast
The study aimed to evaluate the chemical composition and nutraceutical potential of two commercially significant edible bivalve mollusc species (Anadara broughtonii and Mactra chinensis). The edible parts (motor muscle, mantle and adductor) of these molluscs were analyzed for their proximate composition, collagen content, amino acid profile, chemical score and elemental constituents. Both molluscs had low fat content (2.43–6.91 g/100 g dry weight), and protein (55.36–68.01 g/100 g dry weight) and carbohydrates (11.36–20.37 g/100 g dry weight) were their main components. Total collagen content of the edible bivalve molluscs varied from 30.5 to 39 mg/g wet weight, accounting for approximately half of their total protein content. Among amino acids, glycine, glutamate, aspartic acid, alanine, leucine, lysine and arginine were present at high levels in the edible parts of both bivalve molluscs, while the major elements present were sodium, potassium, magnesium, calcium, iron, zinc and nickel. Having high-quality protein content, edible bivalve molluscs could be excellent sources of nutritive ingredients and, after further study, may find applications in nutricosmetics and functional foods.
Introduction
Bivalves have always been an important fishery commodity and a part of a global multi-million dollar business. They are commercially valuable products because of their high biological and nutritional value, which is associated with the presence of specific proteins and vitamins as well as their mineral composition (España et al. 2007; Karakoltsidis et al. 1995).
Bivalve molluscs are consumed as traditional food in many countries and are considered delicious and nutritious. The consumption of bivalve molluscs in some countries has increased in recent years in response to the higher market availability of aquaculture products. Various species of bivalve molluscs consumed in Russia can be produced through aquaculture or by harvesting natural resources.
Industrial-scale fishing of bivalve molluscs has developed rapidly in recent years, notably in the area of the Sea of Japan near Russia. The most important species that can be found there are Anadara broughtonii, Spisula sachalinensis, Mactra chinensis, and others.
A. broughtonii belongs to the family Arcidae and is a rather common type of Mollusca, in the Bivalvia class. It is found in the upper-sublittoral in subtropical areas of the Asian Pacific, with commercial stocks at depths of 2-15 m. The maximum lifespan of A. broughtonii is 65 years, the total mass of individuals varies from 80 to 380 g, and shell length is 65-80 mm. A. broughtonii inhabits muddy and sandy bottoms, burrowing to depths of 10-25 cm. It is found in the Yellow Sea and the Sea of Japan (Rakov 2002). M. chinensis from the family Mactridae-with Pacific, Asian, subtropical, and upper-sublittoral typesinhabits shallow, sandy waters (1-12 m) of the East China, Yellow, Japan and Okhotsk seas. The maximum age of this clam is up to 12 years. The total mass of individuals varies from 10 to 71 g, and shell length is 30-80 mm. The commercial size of its shell is 45 mm (Arzamastsev et al. 2001). The key macronutrients present in or seafood in general are proteins and carbohydrates. A particularly low fat content and the presence of high-quality proteins constitute nutritional properties characteristic of mollusc meat. Protein fractions and their amino acid composition are one of the most important indicators of the nutritive quality of the protein.
Despite the fact that clams have a long history of being a part of many traditional diets, data on the chemical composition of their edible parts is scarce and mostly concern only the foot, whilst the mantle and adductor can also be good sources of nutrients (Miletic et al. 1991;Orban et al. 2007;Chen et al. 2012).
The aim of this work was a comparative study of the nutritional quality of two commercially important, edible bivalve molluscs harvested on the coast of the Sea of Japan, in the Primorsky region of Russia, focusing on the total collagen content in different parts of the mollusc (muscle, mantle and adductor).
Sample and preparation
The clam A. broughtonii ranges from 65 to 80 mm in length, and its mass varies from 80 to 200 g. The clam M. chinensis has a length of 45-50 mm and a mass varying from 40 to 60 g. Both mollusc species were collected once every month from Amur Bay (43°06 0 N and 131°44 0 E), Sea of Japan, in the Primorsky region in Russia in June, September and November 2013, and in February and April 2014 (all analyses were done using pooled molluscsseasonal changes were not taken into consideration in this study). Live bivalve molluscs (about 5 kg of each species) were transported under refrigeration (? 6°C) to the laboratory within 3 h and sampled randomly for this study. Upon arrival, the clams were manually shucked by cutting the adductor muscle with a knife. The clam juice was removed and the edible portion, constituting approximately 10.07-12.36% of total weight for A. broughtonii, and 15.25-18.10% of total weight for M. chinensis, was collected. The edible portion was then dissected into 4 parts: muscle, mantle, adductor and viscera, which were 31. 78-39.50%, 22.45-25.09%, 11.93-14.63% and 16.75-20.78% of the edible portion of A. broughtonii, and 27.12-30.97%, 18.68-27.16%, 9.54-12.70% and 14.80-17.35% of the edible portion of M. chinensis, respectively. All portions were powdered using a blender (Phillips, Guangzhou, China) in the presence of liquid nitrogen. The samples were packed in a polyethylene bag, sealed and stored at -20°C until use. The storage time was no longer than 1 month.
Proximate composition analysis
The moisture content was measured according to methodology described by the Association of Official Analytical Chemists (AOAC) (2000). Samples were dried in an oven at 105°C until a constant weight was obtained. Crude protein, fat and ash content was measured in accordance with AOAC Method 970.42 (2000). A conversion factor of 5.8 was used for determination of total crude protein in jellyfish (Doyle et al. 2006). Fat was determined according to AOAC Method 960.39 (2000). Ash was determined according to AOAC Method 920.153 (2000) by incinerating the sample in a muffle furnace at 550°C for 12 h. Total carbohydrate was calculated by difference [100 -(moisture ? protein ? fat ? ash)]. The obtained concentrations were expressed on a wet and dry weight basis.
Fractionation of the edible parts of the clams
The muscle, mantle and adductor of the bivalve molluscs were fractionated in accordance with the method of Hashimoto et al. (1979). The temperature during analysis was maintained at 4°C. The samples (20 g) were transferred to 200 mL of deionized water, and stirred using a mechanical stirrer (RZR 1 Heidolph Instruments Gmbh & Co. Schwabach, Germany) at a speed of 400 rpm for 6 h. The extract was centrifuged at 3000g using a centrifuge (Epp 5418R, Eppendorf AG, Germany) for 10 min. The process was repeated three times and the supernatants were combined. TCA was added to the solution to obtain a final concentration of 5% (weight/volume). The mixture was centrifuged at 3000 g for 10 min. The resulting precipitate was regarded as the sarcoplasmic protein fraction, and the remaining supernatant was considered as the non-protein nitrogenous compounds (NPN) fraction.
The residue from the previous extraction was stirred with 10 volumes of 0.5 M KCl (one part of residue and 10 parts of KCl solution) for 6 h, followed by centrifugation at 3000g for 10 min. The extraction was performed three times. The supernatants were combined and regarded as the myofibrillar protein fraction. The residue was combined with 10 volumes of 0.1 M NaOH (one part of residue and 10 parts of NaOH solution), and the mixture was stirred continuously for 6 h. The mixture was then centrifuged at 3000g for 10 min. The extraction was performed three times. The supernatants were combined and regarded as the alkali-soluble protein fraction. The final residue was regarded as the stroma protein fraction. The content of total nitrogen in all fractions was determined by the Kjeldahl method, AOAC Method 981.10 (2000), and after that the nitrogen distribution was calculated. The purpose of the nitrogen distribution analysis was to estimate the content of various protein fractions in different parts of bivalve molluscs, in order to further develop the technology of their use in food, and also to determine the digestibility of clam proteins. All protein fractions were also subjected to determination of protein patterns.
Amino acid analysis
The frozen portions of the clams were hydrolyzed for 22 h at 110 ± 1°C with 6 M HCl in sealed glass tubes filled with nitrogen. Following hydrolysis, 1 mL of hydrolyzate was withdrawn and evaporated to dryness under vacuum at 45°C to remove HCl. The hydrolyzate was dissolved in 1 mL of sodium citrate buffer (pH 2.2), and then the samples were analyzed by a Hitachi L8800 Automatic Amino Acid Analyzer (Hitachi, Tokyo, Japan). The identity and quantity of each amino acid was assessed by comparison with the retention time and peak area of a standard (Sigma).
The tryptophan content was determined in a separate analysis. The weighed samples were hydrolyzed in 5 N NaOH containing 5% SnCl2 (w/v) for 20 h at 110°C (Hugli and Moore 1972). After hydrolysis, the hydrolyzate was neutralized with 6 N HCl and centrifuged, and then the supernatant was analyzed by a Hitachi L8800 Automatic Amino Acid Analyzer. The identity and quantity of tryptophan was assessed by comparison with the retention time and peak area of a standard (Sigma). All determinations were performed in triplicate. Amino acid ((threonine (Thr), tryptophan (Trp), cysteine ? methionine (Cys ? Met), valine (Val), phenylalanine ? tryptophan (Phe ? Tyr), isoleucine (Ile), leucine (Leu), and lysine (Lys)) contents were expressed as mg/g wet weight.
Amino acid scores
The essential amino acid score was calculated in relation to the FAO/WHO reference amino acid requirement pattern for preschool and school children (3-10 years old) and adults (WHO/FAO/UNU Expert Consultation 2007): Amino acid score ¼ Amino acid in a sample/reference amino acid à 100
Total collagen content
Total collagen content can be extrapolated by multiplying the amount of total hydroxyproline content in each sample by a factor of 8. The concentration of hydroxyproline was determined after acid hydrolysis of samples using 6 M HCl for 6 h at 105°C. The hydroxyproline (Hyp) content was quantified using a commercial Hyp detection kit. The OD values of the samples were measured at 550 nm using a hydroxyproline colorimetric assay kit (BioVision, BioVision Inc, CA, USA) (Woessner 1961).
Total collagen content in samples expressed in g/100 g dry weight was estimated as follow: where H = total hydroxyproline content (g/100 g dry weight), CF = conversion factor.
Meanwhile, the ratio of total collagen to total protein content was estimated as follows: Total collagen ð%Þ ¼ Estimated collagen content/Total protein content  100 where, Estimated collagen content g/1000 g ð Þ ¼ Hyp contentðg/1000 gÞ Â 8 Total protein content ¼ amino acids content including hydroxyproline ð Þ g/1000 g ð Þ The conversion factor used to convert Hyp content for the estimation of total collagen content in this study was 8 as collagen has been reported to contain 12.5%-13.5% Hyp, depending on the conversion factor used in the conversion of nitrogen content to protein (Ignateva et al. 2007;Kolar 1990;Mazorra-Manzano et al. 2012). Previously, in the case of animal skins, e.g. fish, which contained high protein, a conversion factor of 7.5 or 7.7 was applied (bigeye snapper skin) (Kittiphattanabawon et al. 2005); 13.5 (murine lungs) (Kliment et al. 2011), and in some cases 14.7 (Baltic cod) (Sadowska et al. 2003) for the estimation of collagen based on Hyp content.
Determination of elements
Mineral contents in the dried clam portions were determined by atomic absorption spectrometry using an AA-7000 spectrometer (Shimadzu, Japan) with the system of double atomization (flame and electrothermal). Iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), nickel (Ni), molybdenum (Mo), aluminium (Al), cadmium (Cd), lead (Pb) and chromium (Cr) were determined using a graphite cuvette and deuterium background corrector lamp according to the AOAC method (2000).
Determination of sodium (Na), potassium (K), calcium (Ca) and magnesium (Mg) in the dried clam portions was performed using an air-acetylene flame at a fuel flow rate of 50 L h -1 according to AOAC Method 999.11 (2000). The burner height was 5-12, 4-10 and 4-10 mm; slit width was 0.2, 1.0, 0.5 and 0.5 nm; the wavelength was 589.0, 766.5, 422.7 and 258.2 nm; and the lamp current was 6.0, 4.0, 5.0 and 5.0 mA for Na, K, Ca and Mg, respectively. Five specimens, which were previously dried at 80°C, from five different molluscs were combined into one sample after digestion with nitric acid. Limits of detection and quantification for the investigated elements are shown in Table 1.
The limits of detection (LOD) (calculated as 3 s/a, where 'a' is the slope of the calibration curve and 's' is the standard deviation of 10 consecutive measurements of the blank solution) were between 0.01 and 7 lg g -1 . The limits of quantification (LOQ) (calculated as 10 s/a, where 'a' is the slope of the calibration curve and 's' is the standard deviation of 10 consecutive measurements of the blank solution) were between 0.05 and 20.55 lg g -1 .
Statistical analysis
The results were expressed as mean values with standard deviation (n = 3). The differences between the mean values for the three parts of each bivalve mollusc were calculated using one-way analysis of variance (ANOVA), and statistically significant differences were reported at p \ 0.05. Data analysis was carried out using SPSS 16 software (SPSS Inc., Chicago, IL, USA).
Results and discussion
Proximate composition of the edible parts of the molluscs In general, the edible parts of the bivalve molluscs M. chinensis and A. broughtonii were rich in protein and carbohydrates, and had high moisture levels ( Table 2).
Such composition is typical of other bivalve molluscs (Karnjanapratum et al. 2013). It was established that the moisture content of the clams' tissues changed from 81 g/ 100 g wet weight to 13.42 g/100 g dry weight. Similar values were reported by Oliveira et al. (2011).
The edible parts of M. chinensis were characterized by a higher moisture content than those of A. broughtonii. The mantle of A. broughtonii and M. chinensis had the highest level of moisture in comparison to other parts. The content of basic components in the raw edible parts was identical for both species and decreased in the following order: The content of proteins, carbohydrates and ash was higher in the parts of M. chinensis in comparison to A. broughtoni.
The protein content in various parts of the molluscs decreased in the following order: muscle [ adductor [ mantle. Differences in the total protein content between the muscle and the mantle was 19.3% (wet weight) for M. chinensis, and 25.6% (wet weight) for A. broughtonii. These findings were consistent with the results reported by other researchers for protein and moisture content for bivalve molluscs. For instance, the moisture of the Asian hard clams Meretrix meretrix (Xie et al. 2012) The distribution of total minerals content in the edible parts of the mollusc species was different. The highest content of ash was observed in the muscle of M. chinensis, while in the case of A. broughtonii-in the mantle. Moreover, the quantitative differences in the level of elements between A. broughtonii and M. chinensis reached statistical significance (p \ 0.05) and were equal to 36.8% for the mantle, 62.9% for muscle and 69.5% for adductor. The lowest ash content was observed for adductor of A. broughtonii. It was established that all edible parts of the clams, regardless of the species, contained a small amount of lipids not exceeding 1.37%, which was in accordance with findings from previous studies on the bivalve molluscs (Chakraborty et al. 2002;Fuentes et al. 2009;Karnjanapratum et al. 2013). It was found that the tissues of M. chinensis had less fat content than those of A. broughtonii (p \ 0.05). The distribution of fat between the individual edible parts was similar in both clam species, and its level decreased in the following order: adductor [ muscle [ mantle. The fat content in the A. broughtonii adductor was almost two times higher than in the same part of M. chinensis. It is well known that the fat of marine organisms, including bivalve molluscs, is rich in biologically active polyunsaturated fatty acids-eicosapentaenoic acid (C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA), which makes the lipids in the molluscs (despite their low content) valuable for the food industry The chemical composition of the organs and tissues of the molluscs depends on age, sex, sex maturity ratings, water temperature, stomach fullness, stress level and other environmental factors (Kasyanov 1989). The variety of functions of individual parts and tissues may also be linked to significant differences in their mineral content (Gosling 2003).
A. broughtonii and M. chinensis
The fractional protein composition of the edible parts of the molluscs (Table 3) was significantly different.
In general, proteins were characterized by high content of sarcoplasmic and alkali fractions (p \ 0.05).
More myofibrillar proteins were observed for A. broughtonii than M. chinensis. Myofibrillar proteins in the edible parts of the molluscs changed in the following, order: adductor [ mantle [ muscle. There were no statistically significant difference in the content of stroma proteins between the two mollusc species and their individual parts (p [ 0.05). Stroma proteins were the predominant proteins in the muscle and mantle of the molluscs, sarcoplasmic proteins were prevalent in the adductor of M. chinensis, while myofibrillar proteins were predominant in the adductor of A. broughtonii. The content of myofibrillar proteins varied greatly between the mollusc species-from 23.5% in the muscle of M. chinensis to 36.8% in the adductor of A. broughtonii. Sarcoplasmic proteins in fish and molluscs are mainly myoglobin, enzymes and other albumins (Hui et al. 2012).
Amino acid composition
All edible parts of the molluscs showed high content of amino acids. Regardless of the body part, the major amino acids were aspartic acid, glutamic acid, glycine, alanine and leucine. In general, the adductor was characterized by higher content of all amino acids than other edible parts of the clams (p \ 0.05).
The total amino acid content in the mantle (89.03 mg/g wet weight of M. chinensis and 75.63 mg/g wet weight of A. broughtonii) differed significantly between the two species and was equal to 17.7%. There were no statistical differences in the total content of amino acids in the muscle (88.31 mg/g wet weight of M. chinensis and 87.11 mg/g wet weight of A. broughtonii) between the two clam species (p [ 0.05). The content of cysteine, methionine, alanine and glutamic acid in the muscle of the molluscs was significantly different when comparing the two clam species. The differences were bigger in the mantle and related to the content of aspartic and glutamic acids, threonine, serine, proline, glycine, cysteine, lysine and arginine. The mantle of M. chinensis contained much more of these amino acids than that of A. broughtonii. Differences in the content of amino acids in the adductor between the two mollusc species were statistically insignificant and concerned only serine, isoleucine, and tryptophan.
The proportions of the major groups of amino acidsessential (isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, valine, and tryptophan), conditionally essential (histidine, arginine, glycine, proline, and serine) and non-essential (alanine, glutamic and aspartic acid)-varied between the two investigated clam species. The level of essential amino acids ranged from 32.1% of the total content of amino acids in the mantle of M. chinensis to 41.8% in the muscle of A. broughtonii (Fig. 1). The adductor of the molluscs was characterized by an identical level of essential amino acids. The contents of conditionally essential and non-essential amino acids were not significantly different (p [ 0.05). The content of essential acids in all parts of the investigated mollusks (except for the mantle of M. chinensis) was high. Overall, the differences in the level of non-essential amino acids between the two clam species were not very high.
Amino acid scores
Amino acid scores and the first limiting amino acid are established methods of assessing the quality of proteins, which are widely used in studies on the nutritive value of proteins (Friedman 1996; WHO/FAO/UNU Expert Consultation 2007).
The results of the evaluation of amino acid scores for various edible parts of molluscs are presented in Table 4.
The total essential amino acids content in the individual parts of the clams was higher than the recommended daily intake (262 mg/g total protein) and varied from 310 mg/g total protein for adductor to 366 mg/g total protein for mantle. The muscle and mantle of A. broughtonii were characterized by higher essential amino acids than the same parts of M. chinensis. A comparison of the amino acid score of proteins in M. chinensis and A. broughtonii showed that there was a distinct difference between them. The proteins in the edible parts of M. chinensis were characterized by 3 limiting amino acids, while the proteins in A. broughtonii had 2 limiting amino acids.
Then, comparing our results with the amino acid composition of the reference protein recommended by FAO/ WHO/UNU, it was found that almost all the main indicators of the amino acids in the parts of the analysed clams were higher than 100%, with the exception of cysteine ? methionine and valine. Therefore, cysteine ? methionine and valine are the limiting amino acids. Although direct hydrolysis may have resulted in about 10% loss of methionine and 55% loss of cysteine (Xie et al. 2012;Spindler et al. 1984), the content of the sulphur-containing amino acids (cysteine and methionine) exceeded 100 in all the samples. Thus, the proteins of the molluscs A. broughtonii and M. chinensis are rich in the S-containing amino acids. Total hydroxyproline and collagen content The evaluation of the collagen content was carried out on the basis of analysing the hydroxyproline content. All parts of the investigated mollusks were characterized by a high content of hydroxyproline, and the highest value was observed for M. chinensis. The mantle of M. chinensis (98.8 mg/g dry weight) and A. broughtonii (90.0 mg/g dry weight) contained more hydroxyproline (p \ 0.05) than the muscle (87.5 mg/g and 85.0 mg/g dry weight, respectively) and adductor (83.8 mg/g and 76.3 mg/g dry weight, respectively). The hydroxyproline content of the edible parts of molluscs was comparable to level for jellyfish reported earlier (Khong et al. 2016). The conversion factor used to convert the hydroxyproline content for the estimation of total collagen content in this study was 8.07, as collagen has been reported to contain about 12.5%-13.5% hydroxyproline, depending on the conversion factor used in the conversion of nitrogen content to protein content (Ignateva et al. 2007;Kolar 1990;Mazorra-Manzano et al. 2012). Overall, the trend for estimated total collagen in the molluscs was the same as for hydroxyproline content. Among the edible parts, the highest content of total collagen was found in the mantle (p \ 0.05), and it was statistically higher in M. chinensis than in A. broughtonii. Similarly in adductor concentration of total collagen was statistically higher in M. chinensis than in A. broughtonii. The collagen level in the muscle of both of the species of molluscs varied insignificantly (p [ 0.05), but the muscle was characterized by a higher content than the adductor. The ratio of collagen to total protein content (collagen content/crude protein) was not significantly different between the investigated clam species as well as between their individual edible parts (p [ 0.05). The proteins in the adductor of M. chinensis contained less collagen (30.5%) than the proteins in the mantle of A. broughtonii (38.8%). Collagen is therefore the major protein in the edible parts of the analysed molluscs. Table 5 shows the level of elements in the tissues of M. chinensis and A. broughtonii. Na (1134-1527 mg 100 g -1 ) and K (1088-2293 mg 100 g -1 ) were the main macroelements in all of the clam parts, and the highest content of Na was observed for muscle of A. broughtonii and adductor of M. chinensis, while the highest content of K was observed for muscle of A. broughtonii and M. chinensis. The highest Ca content was observed for muscle of A. broughtonii and for adductor of M. chinensis. Ca and K content were significantly different for two species of the molluscs (p \ 0.05). Data are mean ± standard deviation (n = 3)
Mineral content
The major microelements present in the tissues of the clams were Fe, Zn, Cr, Ni and Mn. Other detected elements were Cu, Cr, Mo and Al, of which Cu and Al showed the highest concentrations. The tissues of M. chinensis were characterized by the highest content of Fe, Ni, Al and Mn, while the tissues of A. broughtonii contained more Zn, Cu, Cr, and Mo.
Conclusion
The edible molluscs contained high amount of water, whereas the dry mass was rich in protein and minerals, but low in fats. The tissues of A. broughtonii contained more protein and fat than those of M. chinensis, whereas ash content was significantly higher in the tissues of M. chinensis than those of A. broughtonii. The major elements present in the mollusc tissues were sodium, potassium, magnesium, calcium, iron and zinc. Collagen was found to be the major protein in edible molluscs, and aspartic acid, glutamic acid, glycine, alanine and leucine were found to be the dominant amino acids. Protein was found to be significantly higher in the mantle compared to the muscle and adductor, whereas ash content was significantly higher in the muscle than the mantle and adductor Na, K, Mg Ca, Fe and Z were major elements in mollus. | v3-fos-license |
2018-04-03T00:28:22.588Z | 2017-06-19T00:00:00.000 | 12275544 | {
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} | pes2o/s2orc | Activation of D1R/PKA/mTOR signaling cascade in medial prefrontal cortex underlying the antidepressant effects of l-SPD
Major depressive disorder (MDD) is a common neuropsychiatric disorder characterized by diverse symptoms. Although several antidepressants can influence dopamine system in the medial prefrontal cortex (mPFC), but the role of D1R or D2R subtypes of dopamine receptor during anti-depression process is still vague in PFC region. To address this question, we investigate the antidepressant effect of levo-stepholidine (l-SPD), an antipsychotic medication with unique pharmacological profile of D1R agonism and D2R antagonism, and clarified its molecular mechanisms in the mPFC. Our results showed that l-SPD exerted antidepressant-like effects on the Sprague-Dawley rat CMS model of depression. Mechanism studies revealed that l-SPD worked as a specific D1R agonist, rather than D2 antagonist, to activate downstream signaling of PKA/mTOR pathway, which resulted in increasing synaptogenesis-related proteins, such as PSD 95 and synapsin I. In addition, l-SPD triggered long-term synaptic potentiation (LTP) in the mPFC, which was blocked by the inhibition of D1R, PKA, and mTOR, supporting that selective activation of D1R enhanced excitatory synaptic transduction in PFC. Our findings suggest a critical role of D1R/PKA/mTOR signaling cascade in the mPFC during the l-SPD mediated antidepressant process, which may also provide new insights into the role of mesocortical dopaminergic system in antidepressant effects.
cycle with 5 mice in one cage under specific pathogen free (SPF) condition. All procedures were carried out in accordance with the EU Directive 2010/63/EU on the protection of animals used for scientific purpose and the protocols were approved by the Animal Care Committees of Shanghai Institute of Materia Medica, Chinese Academy of Science. The naï ve C57BL/6 mice were divided into four groups (9-10 mice per group): 1) vehicle, 2) 5 mg· kg -1 l-SPD, 3) 10 mg· kg -1 l-SPD and 4) 10 mg· kg -1 fluoxetine administration (i.p.). All drugs were administrated once daily at 9:00 am for 10 days. The injected volume was 10 ml/kg
Tail suspension test (TST)
Tail suspension test (TST) is the most commonly used experiments to assess depressive-like behavior 1 . In this experiment, naï ve C57BL/6 mice were moved to the testing room 2 h before the behavioral tests started. The mice were suspended by fixing their tail on a TST apparatus. The test lasted 6 min, and the process was monitored online. Due to the most mice were very active at the beginning of the test and the potential effects of the treatment can be obscured during the first two minutes, the immobility of the last 4 min were measured.
Elevated plus maze (EPM)
The EPM apparatus for C57BL/6 mice consisted of two enclosed (30 x 5 x 30 cm) and two open arms (30 x 5 cm) with a height of 60 cm to the floor. The same type of arm was opposite to each other and connected by a central area (5 x 5 cm). At the beginning of the EPM test, the mice were comforted and placed in the center of maze with the head facing an enclosed arm. Each animal was allowed to perform for 6 minutes, and the activity was monitored online. Behaviors scores were calculated as the percent of the distance moved into the open arms. Arm entries were defined as entry of all four paws into the arm.
Immunohistochemistry (IHC)
The C57BL/6 mice were deeply anesthetized and perfused with 0.9% physiological saline. Next, the mice were perfused with 4% paraformaldehyde and immersed into 4% paraformaldehyde for 24 hours. The brain was dehydrated in 30% sucrose for 3 days. For the location of the PFC, we referred to the mouse brain atlas and the thickness of brain slice was 30 µm. Brain slices were treated by 0.3% Triton X-100 and 0.3% H2O2 and then blocked in 10% goat serum (Gibco) and incubated in primary antibody (anti-pmTOR (Ser 2448), 1:50, Cell Signaling; anti-PSD 95, 1:50, Invitrogen) at 4°C overnight. Brain slices were washed and incubated in secondary antibody (goat pAb to Rb IgG, 1:200, Abcam) for 2 hours. Then, the brain slices were incubated in streptavidin biotin peroxidase complex (SABC) for 1 hour and then washed and transferred to microscope slides. After being dried, the brain slices were stained using DAB substrate solution for 5 min.
Brain slices were dehydrated in gradient alcohol (70%, 95%, 100% and 100%) and cleared by xylene. Mounted brain slice by mounting solution and observed under a microscope.
Statistical analysis
The data are expressed as the mean ± SEM. The results of behavioral tests including TST and EPM in C57BL/6 mice were analyzed using one-way ANOVA, and post hoc LSD tests were performed. The western blot results were analyzed using one-way ANOVA, and post hoc LSD tests were performed.
Figure S1
Antidepressant-like and anxiolytic-like effects of l-SPD in C57BL/6 mice. (A) The schematic representation for experimental procedures. The C57BL/6 mice were treated with vehicle, l-SPD (5 mg· kg -1 or 10 mg· kg -1 ) and fluoxetine (10 mg· kg -1 ) daily for 10 days, the drugs were administrated at 9:00 am every day. Behavioral tests were performed 24 hours after the last drug injection. The brain tissues were obtained 24 hours after the last drug administration. Figure S2A. In Figure 3A, only red dashed boxes part were displayed. | v3-fos-license |
2017-02-17T08:44:35.884Z | 2017-02-01T00:00:00.000 | 18009304 | {
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} | pes2o/s2orc | Adsorptive Desulfurization of Model Gasoline by Using Different Zn Sources Exchanged NaY Zeolites
A series of Zn-modified NaY zeolites were prepared by the liquid-phase ion-exchange method with different Zn sources, including Zn(NO3)2, Zn(Ac)2 and ZnSO4. The samples were tested as adsorbents for removing an organic sulfur compound from a model gasoline fuel containing 1000 ppmw sulfur. Zn(Ac)2-Y exhibited the best performance for the desulfurization of gasoline at ambient conditions. Combined with the adsorbents’ characterization results, the higher adsorption capacity of Zn(Ac)2-Y is associated with a higher ion-exchange degree. Further, the results demonstrated that the addition of 5 wt % toluene or 1-hexene to the diluted thiophene (TP) solution in cyclohexane caused a large decrease in the removal of TP from the model gasoline fuel. This provides evidence about the competition through the π-complexation between TP and toluene for adsorption on the active sites. The acid-catalyzed alkylation by 1-hexene of TP and the generated complex mixture of bulky alkylthiophenes would adsorb on the surface active sites of the adsorbent and block the pores. The regenerated Zn(Ac)2-Y adsorbent afforded 84.42% and 66.10% of the initial adsorption capacity after the first two regeneration cycles.
Introduction
The sulfur compounds of the exhaust emissions from gasoline and diesel fuels are the main impurities in air pollutants, which are very harmful to the environment and human health [1,2]. Moreover, sulfur components poison catalysts in the exhaust gas converter for reducing CO and NO x emissions [3,4]. The desulfurization of transportation fuels such as diesel and gasoline is also important from the point of application in fuel cells, as liquid hydrocarbons such as gasoline can be used conveniently as a feed in the fuel cells [5]. The fuel cells, however, require more stringent conditions on sulfur levels which are of the order of 1 ppmw, and preferably 0.1-0.2 ppmw, to avoid poisoning of the catalyst due to sulfur [6,7]. Therefore, sulfur contents in transportation fuels should be limited to a very low level with a stringent fuel specification [8,9]. According to the recently announced Euro V norms, the fuel sulfur contents will have to be reduced to as low as 10 ppmw in the near future [1].
Various methods to remove the sulfur in oil distillates have been proposed in many developed countries, such as hydro desulfurization (HDS) [10,11], adsorptive desulfurization (ADS) [12,13], oxidative desulfurization [14,15], biological desulfurization [16,17], etc. The traditional HDS process is widely employed on an industrial scale. Typically, the HDS technology is usually operated at a temperature in the range of 300-450 • C, and at a H 2 pressure of 3.0-5.0 MPa, with CoMo/Al 2 O 3 or NiMo/Al 2 O 3 catalysts [18,19]. Industrially, the HDS process involves catalytic treatment with H 2 to convert the various sulfur compounds to hydrogen sulfide and requires severe operating conditions [1]. The olefins in feedstock will react with H 2 to form alkanes under such conditions, resulting in significant loss of the octane number [20]. Moreover, some sulfides, especially aromatic thiophenes and TP derivatives, are hard to remove by HDS [12]. In order to achieve deep desulfurization, there is a need to enlarge the reactor size and consume more energy [21]. Therefore, to overcome these drawbacks, significant efforts have been shifted to ADS. ADS is considered a promising approach with several advantages, including ambient operating conditions and selective removal of refractory thiophenic compounds.
To develop a proper adsorbent, many studies have been performed using metal oxides [22,23], carbon-based materials [24][25][26] as well as zeolites and their metal-loaded derivatives [27,28]. Among these materials, Y-type zeolites have been investigated widely due to their high surface area, size-selective adsorption capacity, high ion-exchange capacity, and good thermal stabilities [29,30]. Yang and his co-workers [12,13,28,31,32] explored Ag-, Cu-, Ni-, and Zn-exchanged Y zeolites, which exhibited a high sulfur adsorption capacity for thiophenic compounds. They proposed the π-complexation interaction mechanism of thiophenic compounds with metal-exchanged zeolites. In addition, they also noted that the adsorption performance of the Cu(I)-Y zeolites would decrease when aromatics were present in the fuel, and the decline may be attributed to the competitive adsorption of sulfur compounds and aromatics via π-complexation. Velu et al. [33] reported that ion-exchanged NH 4 -Y zeolites with transition metals such as Cu, Ni, Zn, Pd, and Ce exhibited selective adsorption of organic sulfides. Ce-exchanged NH 4 -Y zeolites exhibited a higher selectivity for the adsorption of sulfur compounds compared to the selectivity of aromatics than the other ion-exchanged zeolites. They indicated the removal of the sulfur compounds by a direct sulfur-metal (S-M) interaction rather than by π-complexation. M. Oliveira et al. The authors of [34] investigated the adsorption capacity of TP and toluene on NaY zeolites exchanged with transition metals (5 wt % Ni, Zn and Ag) and their competitive adsorption behavior systematically. The obtained results indicated the importance of inserting transitions metals in the zeolites' structure to enhance the adsorption of both aromatic and sulfur compounds in organic liquid mixtures. Zhang, Z.Y. [35] synthesized Cu-, Zn-, Ag-exchanged and binary metal-exchanged NaY adsorbents with excellent performances and proposed a synergistic effect between different metal cations. Though progress has been made, it is still a challenge to easily synthesize such a NaY-based adsorbent with a good performance.
Zinc was also the main component of a Ni/ZnO sorbent for the adsorptive HDS of kerosene for fuel-cell applications, where ZnO in this system acts as an acceptor of sulfur that is released during the regeneration of the sulfided nickel surface species [36]. Several Zn-based adsorbents, pure metal oxide (ZnO) and mixed oxides (zinc ferrite and zinc titanate) have been attractive for gas-phase high-temperature desulfurization because of their favorable sulfidation thermodynamics, high H 2 S removal efficiency, good sulfur-loading capacity, high regenerability, and sufficient strength [37,38]. In addition, zinc-based nanocrystalline aluminum oxide has been used in the application of adsorptive desulfurization of transportation fuels [39].
Zn 2+ has the electronic configuration 3d 10 4s 0 . It indicates that there is some donation of the electron charge transfer from the π orbital of TP to the vacant s orbital of Zn 2+ , simultaneously with the back-donation of electron charge transfer from the d orbitals of Zn 2+ to the π* orbital of TP. Therefore, ZnY can adsorb TP. Many people have studied the adsorption desulfurization performance of ZnY zeolites. Most of them use Zn(NO 3 ) 2 as the Zn source for the ion exchange. We believe that the use of different sources of Zn can obtain different adsorption desulfurization properties. At present, very few people are engaged in this research. So in this work, cyclohexane and TP were used as a model fuel and model sulfur compound, respectively. A series of Zn-modified NaY zeolites were prepared by a liquid-phase ion-exchange method with different Zn sources and tested by adsorption of TP from model gasoline fuels. The main propose of this study was to examine and compare the desulfurization performance of ion-exchanged NaY zeolites from different Zn sources.
Moreover, to uncover the adsorption mechanism, toluene and 1-hexene were added to the solution to test the selectivity. Finally, the regeneration ability and reuse of the spent adsorbents was investigated. The model gasoline fuels tested in this work used gas chromatography-flame ionization detector (GC-FID).
Characterization Results
XRD analysis was performed to check the crystallinity of the ZnY zeolite adsorbents and to detect the formation of metal oxides after the liquid-phase ion exchange. As illustrated in Figure 1, the powder XRD spectra of all samples exhibited a typical FAU (faujasite) framework and no significant diffraction lines associated with Zn-related compounds were identified. It is evident that the modified samples kept the original framework structure of the NaY zeolites and no new phases emerged. The results confirmed that the structures of the zeolites were not disturbed during preparation. However, their relative crystallinity decreased slightly after calcination. The relative crystallinity of Zn(Ac) 2 -Y, Zn(NO 3 ) 2 -Y and ZnSO 4 -Y (10 h, 60 • C) dropped to 84.5%, 85.0% and 90.1%, respectively, which can be attributed to the crystal lattice collapse that occurred during the calcination process [40]. selectivity. Finally, the regeneration ability and reuse of the spent adsorbents was investigated. The model gasoline fuels tested in this work used gas chromatography-flame ionization detector (GC-FID).
Characterization Results
XRD analysis was performed to check the crystallinity of the ZnY zeolite adsorbents and to detect the formation of metal oxides after the liquid-phase ion exchange. As illustrated in Figure 1, the powder XRD spectra of all samples exhibited a typical FAU (faujasite) framework and no significant diffraction lines associated with Zn-related compounds were identified. It is evident that the modified samples kept the original framework structure of the NaY zeolites and no new phases emerged. The results confirmed that the structures of the zeolites were not disturbed during preparation. However, their relative crystallinity decreased slightly after calcination. The relative crystallinity of Zn(Ac)2-Y, Zn(NO3)2-Y and ZnSO4-Y (10 h, 60 °C) dropped to 84.5%, 85.0% and 90.1%, respectively, which can be attributed to the crystal lattice collapse that occurred during the calcination process [40]. Table 1. It can be observed that the ratio of the Si/Al of NaY zeolite was 2.341. The Si/Al ratios of the Zn(NO3)2-Y, Zn(Ac)2-Y and ZnSO4-Y zeolites were close to the parent NaY, suggesting that the dissolution of Al 3+ rarely occurs during the exchange process. For the samples of Zn(NO3)2-Y, Zn(Ac)2-Y and ZnSO4-Y (10 h, 60 °C), the Na exchange degrees were 56.70%, 59.47%, 43.17%, respectively. Upon ion exchange, Zn(Ac)2-Y (10 h, 60 °C) exhibited the highest degree, which means it has the largest amount of adsorptive active sites. As the time went on, the ion exchange degree increased simultaneously. The chemical compositions determined by ICP-AES are summarized in Table 1. It can be observed that the ratio of the Si/Al of NaY zeolite was 2.341. The Si/Al ratios of the Zn(NO 3 ) 2 -Y, Zn(Ac) 2 -Y and ZnSO 4 -Y zeolites were close to the parent NaY, suggesting that the dissolution of Al 3+ rarely occurs during the exchange process. For the samples of Zn(NO 3 ) 2 -Y, Zn(Ac) 2 -Y and ZnSO 4 -Y (10 h, 60 • C), the Na exchange degrees were 56.70%, 59.47%, 43.17%, respectively. Upon ion exchange, Zn(Ac) 2 -Y (10 h, 60 • C) exhibited the highest degree, which means it has the largest amount of adsorptive active sites. As the time went on, the ion exchange degree increased simultaneously. The morphologies of the adsorbent by SEM are illustrated in Figure 2. With a suitable ion exchange time (10 h), the ZnY crystal particle distribution was uniform, and there was no occurrence of the phenomena of powder and aggregation (Figure 2b-d).
Molecules 2017, 22, 305 4 of 12 The morphologies of the adsorbent by SEM are illustrated in Figure 2. With a suitable ion exchange time (10 h), the ZnY crystal particle distribution was uniform, and there was no occurrence of the phenomena of powder and aggregation (Figure 2b-d). To compare the Zn particle distributions over NaY, TEM micrographs of the samples are presented in Figure 3. The result shows that the Zn particles were dispersed well and confined to the channels of NaY, and large metallic Zn particles are not observed in the Figure 3a. In contrast, in Figure 3b, the ion-exchange time was 14 h, and the Zn species (dark dot-like objects in Figure 3b) in the channels were prone to aggregate and agglomerate on the outer surface of NaY. To compare the Zn particle distributions over NaY, TEM micrographs of the samples are presented in Figure 3. The result shows that the Zn particles were dispersed well and confined to the channels of NaY, and large metallic Zn particles are not observed in the Figure 3a. In contrast, in Figure 3b, the ion-exchange time was 14 h, and the Zn species (dark dot-like objects in Figure 3b) in the channels were prone to aggregate and agglomerate on the outer surface of NaY. The morphologies of the adsorbent by SEM are illustrated in Figure 2. With a suitable ion exchange time (10 h), the ZnY crystal particle distribution was uniform, and there was no occurrence of the phenomena of powder and aggregation (Figure 2b-d). To compare the Zn particle distributions over NaY, TEM micrographs of the samples are presented in Figure 3. The result shows that the Zn particles were dispersed well and confined to the channels of NaY, and large metallic Zn particles are not observed in the Figure 3a. In contrast, in Figure 3b, the ion-exchange time was 14 h, and the Zn species (dark dot-like objects in Figure 3b) in the channels were prone to aggregate and agglomerate on the outer surface of NaY.
Effect of Adsorbent Preparation Conditions
To obtain Zn-exchanged NaY samples with different Zn exchange degrees, three types of Zn sources were employed. The adsorptive desulfurization results are displayed in Figure 4. Notably, Zn(Ac) 2 -Y (10 h, 60 • C) showed the highest sulfur adsorption capacity of 17.21 mg of S/g. The samples of the Zn(NO 3 ) 2 -Y (10 h, 60 • C) and ZnSO 4 -Y (10 h, 60 • C) adsorbents had sulfur adsorption capacities of 14.68 and 8.93 mgS/g. Further, the sulfur removals (R%) of Zn(Ac) 2 -Y, Zn(NO 3 ) 2 -Y and ZnSO 4 -Y (10 h, 60 • C) were 44.21%, 39.58% and 20.29%, respectively. The best performance was observed with the Zn(Ac) 2 -Y (10 h, 60 • C) adsorbent owing to it having the highest Zn exchange degree (59.47%) and the most adsorption activity sites. To elaborate, a higher Zn exchange degree (%) is favorable for a higher sulfur adsorption capacity.
Effect of Adsorbent Preparation Conditions
To obtain Zn-exchanged NaY samples with different Zn exchange degrees, three types of Zn sources were employed. The adsorptive desulfurization results are displayed in Figure 4. Notably, Zn(Ac)2-Y (10 h, 60 °C) showed the highest sulfur adsorption capacity of 17.21 mg of S/g. The samples of the Zn(NO3)2-Y (10 h, 60 °C) and ZnSO4-Y (10 h, 60 °C) adsorbents had sulfur adsorption capacities of 14.68 and 8.93 mgS/g. Further, the sulfur removals (R%) of Zn(Ac)2-Y, Zn(NO3)2-Y and ZnSO4-Y (10 h, 60 °C) were 44.21%, 39.58% and 20.29%, respectively. The best performance was observed with the Zn(Ac)2-Y (10 h, 60 °C) adsorbent owing to it having the highest Zn exchange degree (59.47%) and the most adsorption activity sites. To elaborate, a higher Zn exchange degree (%) is favorable for a higher sulfur adsorption capacity. The effect of the treatment time of the ion-exchange time on the sulfur adsorption activity by using model gasoline FM-1 at 40 °C is shown in Figure 5a. The sulfur adsorption capacity increased primarily from 17.21 to 19.32 mgS/g when the Zn 2+ ion-exchange time increased from 6 h to 10 h, and it then dropped quickly to 14.45 mgS/g. As the ion-exchange time prolonged to 14 h, the sulfur adsorption capacity (19.32 mgS/g) and the sulfur removal (52.09%) reached the maximum values. When the ion-exchange equilibrium is reached, increasing the Zn 2+ ion-exchange time may cause Zn 2+ clusters to aggregate and agglomerate on the outer surface of NaY, thus gradually decreasing the sulfur adsorption capacity.
The effect of the ion-exchange temperature on the sulfur adsorption capacity was also studied and the results are shown in Figure 5b. It is shown that the sulfur adsorption capacity apparently increased and then decreased with the increase of the ion-exchange temperature, and reached a maximum (19.32 mgS/g) at 60 °C. The content of Zn 2+ increased as the temperature increased. The increase of the ion-exchange temperature promotes the Na + to get enough energy from the outside to break away from the framework force and form the cation vacancies. Meanwhile, the diffusion rate of Zn 2+ is accelerates to enter the framework cation vacancies to compensate for the charge. However, further increasing the temperature from 60 to 90 °C caused the Zn 2+ exchange rate to rise too fast, and this may cause pore blockage, which is detrimental to the sulfur adsorption capacity. The effect of the treatment time of the ion-exchange time on the sulfur adsorption activity by using model gasoline FM-1 at 40 • C is shown in Figure 5a. The sulfur adsorption capacity increased primarily from 17.21 to 19.32 mgS/g when the Zn 2+ ion-exchange time increased from 6 h to 10 h, and it then dropped quickly to 14.45 mgS/g. As the ion-exchange time prolonged to 14 h, the sulfur adsorption capacity (19.32 mgS/g) and the sulfur removal (52.09%) reached the maximum values. When the ion-exchange equilibrium is reached, increasing the Zn 2+ ion-exchange time may cause Zn 2+ clusters to aggregate and agglomerate on the outer surface of NaY, thus gradually decreasing the sulfur adsorption capacity.
The effect of the ion-exchange temperature on the sulfur adsorption capacity was also studied and the results are shown in Figure 5b. It is shown that the sulfur adsorption capacity apparently increased and then decreased with the increase of the ion-exchange temperature, and reached a maximum (19.32 mgS/g) at 60 • C. The content of Zn 2+ increased as the temperature increased. The increase of the ion-exchange temperature promotes the Na + to get enough energy from the outside to break away from the framework force and form the cation vacancies. Meanwhile, the diffusion rate of Zn 2+ is accelerates to enter the framework cation vacancies to compensate for the charge. However, further increasing the temperature from 60 to 90 • C caused the Zn 2+ exchange rate to rise too fast, and this may cause pore blockage, which is detrimental to the sulfur adsorption capacity.
Effect of Adsorption Conditions
The effect of the adsorption temperature on the sulfur adsorption capacity was investigated in the range of 20 to 60 °C over Zn(Ac)2-Y zeolites (10 h, 60 °C), and the results are presented in Figure 6. It was observed that the sulfur adsorption capacity (21.12 mgS/g) and the sulfur removal (56.01%) apparently increased and then decreased with the increase in the temperature and reached a maximum at 30 °C. The reason may be that increasing the system temperature will greatly increase the diffusion rate of the TP, owing to the decrease in the viscosity of the solution. However, when the temperature is >50 °C, the desorption rate will increase sharply, which causes the sulfur adsorption capacity to decrease [41][42][43]. Gasoline is a complex mixture of hydrocarbons composed mainly of paraffins and aromatics. TP should have a great affinity toward toluene because both are similar species and, as a result, will compete against the zeolite adsorbents for interactions. To clarify the influence of co-existing toluene on TP adsorption, the adsorptive removal of TP on Zn(NO3)2-Y, Zn(Ac)2-Y, ZnSO4-Y (10 h, 60 °C) by using MF-2 was investigated. Figure 7a depicts that the desulfurization performance of Zn(Ac)2-Y (10 h, 60 °C) was significantly higher than that of Zn(NO3)2-Y (10 h, 60 °C) and ZnSO4-Y (10 h, 60 °C). It also can be seen that in the case of 5 wt % toluene, the sulfur adsorption capacity decreased from
Effect of Adsorption Conditions
The effect of the adsorption temperature on the sulfur adsorption capacity was investigated in the range of 20 to 60 • C over Zn(Ac) 2 -Y zeolites (10 h, 60 • C), and the results are presented in Figure 6. It was observed that the sulfur adsorption capacity (21.12 mgS/g) and the sulfur removal (56.01%) apparently increased and then decreased with the increase in the temperature and reached a maximum at 30 • C. The reason may be that increasing the system temperature will greatly increase the diffusion rate of the TP, owing to the decrease in the viscosity of the solution. However, when the temperature is >50 • C, the desorption rate will increase sharply, which causes the sulfur adsorption capacity to decrease [41][42][43].
Effect of Adsorption Conditions
The effect of the adsorption temperature on the sulfur adsorption capacity was investigated in the range of 20 to 60 °C over Zn(Ac)2-Y zeolites (10 h, 60 °C), and the results are presented in Figure 6. It was observed that the sulfur adsorption capacity (21.12 mgS/g) and the sulfur removal (56.01%) apparently increased and then decreased with the increase in the temperature and reached a maximum at 30 °C. The reason may be that increasing the system temperature will greatly increase the diffusion rate of the TP, owing to the decrease in the viscosity of the solution. However, when the temperature is >50 °C, the desorption rate will increase sharply, which causes the sulfur adsorption capacity to decrease [41][42][43]. Gasoline is a complex mixture of hydrocarbons composed mainly of paraffins and aromatics. TP should have a great affinity toward toluene because both are similar species and, as a result, will compete against the zeolite adsorbents for interactions. To clarify the influence of co-existing toluene on TP adsorption, the adsorptive removal of TP on Zn(NO3)2-Y, Zn(Ac)2-Y, ZnSO4-Y (10 h, 60 °C) by using MF-2 was investigated. Figure 7a depicts that the desulfurization performance of Zn(Ac)2-Y (10 h, 60 °C) was significantly higher than that of Zn(NO3)2-Y (10 h, 60 °C) and ZnSO4-Y (10 h, 60 °C). It also can be seen that in the case of 5 wt % toluene, the sulfur adsorption capacity decreased from Gasoline is a complex mixture of hydrocarbons composed mainly of paraffins and aromatics. TP should have a great affinity toward toluene because both are similar species and, as a result, will compete against the zeolite adsorbents for interactions. To clarify the influence of co-existing toluene on TP adsorption, the adsorptive removal of TP on Zn(NO 3 ) 2 -Y, Zn(Ac) 2 -Y, ZnSO 4 -Y (10 h, 60 • C) by using MF-2 was investigated. Figure 7a depicts that the desulfurization performance of Zn(Ac) 2 -Y (10 h, 60 • C) was significantly higher than that of Zn(NO 3 ) 2 -Y (10 h, 60 • C) and ZnSO 4 -Y (10 h, 60 • C). It also can be seen that in the case of 5 wt % toluene, the sulfur adsorption capacity decreased from 21.12 mgS/g without toluene to 3.44 mgS/g on Zn(Ac) 2 -Y (10 h, 60 • C), which means that the sulfur adsorption capacity declined 83.71%. The results demonstrated that strong competitive adsorption takes place between TP and toluene. Zn 2+ has the electronic configuration 3d 10 4s 0 . It indicates that there is some donation of the electron charge transfer from the π orbitals of TP to the vacant s orbitals of Zn 2+ , simultaneously with the back-donation of the electron charge transfer from the d orbitals of Zn 2+ to the π* orbitals of TP. The toluene contains a benzene ring structure, which forms the competitive adsorption with TP, leading to a sharp drop in the sulfur adsorption capacity [12,31].
(10 h, 60 °C) was significantly higher than that of Zn(NO3)2-Y (10 h, 60 °C) and ZnSO4-Y (10 h, 60 °C). It also can be seen that in the case of 5 wt % toluene, the sulfur adsorption capacity decreased from 21.12 mgS/g without toluene to 3.44 mgS/g on Zn(Ac)2-Y (10 h, 60 °C), which means that the sulfur adsorption capacity declined 83.71%. The results demonstrated that strong competitive adsorption takes place between TP and toluene. Zn 2+ has the electronic configuration 3d 10 4s 0 . It indicates that there is some donation of the electron charge transfer from the π orbitals of TP to the vacant s orbitals of Zn 2+ , simultaneously with the back-donation of the electron charge transfer from the d orbitals of Zn 2+ to the π* orbitals of TP. The toluene contains a benzene ring structure, which forms the competitive adsorption with TP, leading to a sharp drop in the sulfur adsorption capacity [12,31]. In addition to the aromatics, the olefins in the gasoline fuel also have a negative effect on TP removal. To test the influence of co-existing olefin on TP adsorption, the experiments were carried out using Zn(NO3)2-Y, Zn(Ac)2-Y, and ZnSO4-Y (10 h, 60 °C) as the adsorbents. It can be seen from Figure 7b that the sulfur adsorption capacity decreased from 21.12 to 17.23 mgS/g. For the samples of ZnSO4-Y, the Na exchange degree was 43.17%. Upon ion exchange, ZnSO4-Y had the lowest adsorptive active sites. When the adsorption experiments were carried out, the surface of the adsorbents could be clearly observed to generate a little light pink substance. This may be due to the alkylation reaction between olefin and TP, which generates a complex mixture of bulky alkylthiophenes [43]. At the same time, the alkylthiophenes adsorb on the surface active sites of the adsorbent, and block the pores of the adsorbent. Thus, it will restrict TP from entering the super cage and cause the sulfur adsorption capacity to decrease.
Adsorbent Regeneration
As illustrated in Figure 8, the adsorbent regenerated by the thermal treatment afforded 84.42%, 66.10%, 58.37% and 51.97% of the initial adsorption capacity for the second, third, fourth and fifth adsorption runs. The sulfur adsorption capacity decreased with the increase of the adsorptionregeneration cycle. This could be due to strong interactions of the Zn 2+ with the adsorbed TP or, more likely, the low zeolite framework stability of the Zn 2+ . The X-ray diffraction analysis of regenerated Zn(Ac)2-Y (10 h, 60 °C) is shown in Figure 9 which indicates that parts of the zeolites' structure experience crystal lattice collapse and lost crystallinity after the calcination processes. In addition to the aromatics, the olefins in the gasoline fuel also have a negative effect on TP removal. To test the influence of co-existing olefin on TP adsorption, the experiments were carried out using Zn(NO 3 ) 2 -Y, Zn(Ac) 2 -Y, and ZnSO 4 -Y (10 h, 60 • C) as the adsorbents. It can be seen from Figure 7b that the sulfur adsorption capacity decreased from 21.12 to 17.23 mgS/g. For the samples of ZnSO 4 -Y, the Na exchange degree was 43.17%. Upon ion exchange, ZnSO 4 -Y had the lowest adsorptive active sites. When the adsorption experiments were carried out, the surface of the adsorbents could be clearly observed to generate a little light pink substance. This may be due to the alkylation reaction between olefin and TP, which generates a complex mixture of bulky alkylthiophenes [43]. At the same time, the alkylthiophenes adsorb on the surface active sites of the adsorbent, and block the pores of the adsorbent. Thus, it will restrict TP from entering the super cage and cause the sulfur adsorption capacity to decrease.
Adsorbent Regeneration
As illustrated in Figure 8, the adsorbent regenerated by the thermal treatment afforded 84.42%, 66.10%, 58.37% and 51.97% of the initial adsorption capacity for the second, third, fourth and fifth adsorption runs. The sulfur adsorption capacity decreased with the increase of the adsorption-regeneration cycle. This could be due to strong interactions of the Zn 2+ with the adsorbed TP or, more likely, the low zeolite framework stability of the Zn 2+ . The X-ray diffraction analysis of regenerated Zn(Ac) 2 -Y (10 h, 60 • C) is shown in Figure 9 which indicates that parts of the zeolites' structure experience crystal lattice collapse and lost crystallinity after the calcination processes.
Ion-exchange method was used for the preparation of Zn modified Y zeolites. First, 5 g NaY zeolites were treated with 100 mL of 0.1 mol Zn(NO3)2, ZnSO4 and Zn(Ac)2 solution, respectively. As Zn(Ac)2 is easily to hydrolysis in aqueous solution, therefore, 0.2 mL HAc was added to prevent hydrolysis. Following that, the ion-exchanged zeolites were filtered, washed thoroughly with deionized water to constant pH, and subsequently drying at 100 °C for 6 h. Finally, the samples were calcined at 500 °C for 6 h in air atmosphere. The obtained samples were denoted as Zn(Ac)2-Y, Zn(NO3)2-Y and ZnSO4-Y according to different Zn sources.
Experimental Section
Model gasoline fuels were prepared by dissolving appropriate amounts of TP into cyclohexane and denoted as MF-1. To obtain more information about S adsorption capacity, 5 wt % of toluene and 5 wt % of 1-hexene were also added and labeled as MF-2 and MF-3, respectively.
Characterizations
The chemical composition of all samples was measured by ICP-AES with a Perkin-Elmer Spectrometer. X-ray diffraction (XRD) patterns of the samples were recorded on a MiniFlex600 X-ray diffract meter (Tokyo, Japan) with Cu-Kβ radiation (λ = 1.54056 Å) operating at 40 KV, 30 mA. Data were collected in the range of 2θ = 5 • -40 • at a scanning speed of 4 • /min. The morphologies of the adsorbents were characterized by SEM (S-4800, Hitachi, Tokyo, Japan) with applied potential of 5 kV. Transmission electron micrographs of the samples were recorded by a JEOL JEM-2100F transmission electron microscope (Tokyo, Japan) with an acceleration voltage of 200 kV.
Adsorption Experiments
The desulfurization from the fuels was conducted by a static adsorption experiments operated at ambient temperature and pressure. Before adsorption, zeolite adsorbents were degassed 6 h at 120 • C. In a typical run, both 25 mL of the model fuel and 0.5 g of the adsorbents were added to a Three-necked flask. The mixtures were treated for 2 h under stirring, afterwards the solution was separated from the adsorbents with a syringe filter. And all the samples collected during the experiments were analyzed using a GC (GC-6820, Santa Clara, CA, USA) equipped with an capillary column and a flame ionization detector (FID).
The sulfur adsorption capacity (q t ) is calculated using the following equation: The sulfur removal (R%) is calculated according to the formula: where c 0 and c t (ppmw) are the initial and t (min) concentration of sulfur in the model fuels, V (mL) is the volume of the model fuels, ρ (g/mL) is the density of the model fuels, and m (g) is the mass of adsorbent, respectively.
Regeneration Experiments
Safety, high efficiency and availability need to be considered for the regeneration of saturated adsorbents. Heating the spent adsorbents using air is an effective choice for the regeneration of saturated adsorbents. In this work, the feasibility for regeneration of the zeolite adsorbents were investigated by heating the spent zeolite adsorbents. The saturated sample (1.0 g) by MF-1 was dried at 110 • C for 6 h and then calcined at 500 • C for 6 h. To evaluate the reusability of adsorbents, the experiments were carried out for three times at the same conditions.
Conclusions
A detailed investigation on various Zn 2+ ion-exchanged NaY zeolite adsorbents confirmed their high capacity for sulfur removal. The sulfur adsorption capacity of ZnY zeolites is dependent on the Zn sources, ion-exchange time and temperature, adsorption temperature and whether they contains aromatics and olefins. A sample of Zn(Ac) 2 -Y, prepared by an ion-exchange method with Zn(Ac) 2 ·2H 2 O as a precursor, exhibited the highest capacity for sulfur removal. The excellent performance was related to the higher exchange degree which is controlled by Zn sources. Additionally, there is a suitable temperature and ion-exchange time for the synthesis process.
On the other hand, for the aromatics and olefins that exist (the experiment with Zn(Ac) 2 -Y (10 h, 60 • C)) in the model gasoline, the sulfur adsorption capacity will drop. It is proposed that the Zn 2+ of Zn(Ac) 2 -Y (10 h, 60 • C) mainly adsorbs TP by π-complexation when the model gasoline fuel contains toluene. The competitive adsorption of toluene and TP has an obvious negative effect on the sulfur adsorption capacity. The 1-hexene in the model gasoline fuel is detrimental to TP removal. This may be due to the alkylation reaction between 1-hexene and TP, which generates a complex mixture of bulky alkylthiophenes that occupy active sites of the adsorbents.
Thermal treatment was used to regenerate the Zn(Ac) 2 -Y (10 h, 60 • C) adsorbent spent by MF-1, and the regenerated Zn(Ac) 2 -Y (10 h, 60 • C) samples were characterized. The X-ray diffraction analyses indicated that parts of the zeolites' structure experienced crystal lattice collapse and lost crystallinity after the calcination processes. The sulfur adsorption capacity decreases with the increase of the adsorption-regeneration cycle. This could be due to the low zeolite framework stability of the Zn cations. | v3-fos-license |
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} | pes2o/s2orc | The M-phase regulatory phosphatase PP2A-B55δ opposes protein kinase A on Arpp19 to initiate meiotic division
Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.
Supplementary Figure 1
Legend. GST-Arpp19 is truncated in C-terminus during expression and purification from bacteria. (a) GST-Arpp19 coupled to sepharose GSH-beads was phosphorylated or not in metaphase II (MII) extracts. Proteins bound to beads were subjected to a 10% SDS gel electrophoresis. After Coomassie staining, two major bands were detected, the upper and most abundant one corresponding to the molecular weight expected for full-length GST-Arpp19 (FL-gst Arpp19) and a lower one that could correspond to a truncated form of Arpp19 (T-gst Arpp19). The preparative stained gel was prepared one time prior its analysis. kDa: kiloDalton. (b) Each band was cut out from the polyacrylamide gel and analyzed by LC-MS/MS.
The sequences of peptides identified by LC-MS/MS analysis from either FL-gst Arpp19 or T-gst Arpp19 are aligned with Xenopus Arpp19 sequence. The C-terminal peptide, PSLVASKLAG, is never recovered from T-gst Arpp19. When GST-Arpp19 has been phosphorylated in MII extracts, phosphorylated S109 (in red and underlined) is detected by LC-MS/MS in some peptides generated by FL-gst Arpp19 but never in the peptides generated by T-gst Arpp19. In contrast, phosphorylated S67 (in blue and underlined) is detected in peptides generated by both FL-gst Arpp19 and T-gst Arpp19. Source data are provided as a Source Data file. Method. Bacterially expressed GST-Arpp19 was bound to Sepharose GSH-beads 1 . GST-Arpp19 coupled to beads was incubated or not in metaphase II extracts to generate the double S167-S109 phosphorylated form of Arpp19. (https://www.ebi.ac.uk/pride/profile/reviewer_pxd022739).
Supplementary Figure 2
Legend. Biochemical isolation of S109-phosphatase from prophase extracts -Separation of fraction 6 from the Mono Q column with Phenyl-Superose and Superose 12 columns. (a) Prophase oocytes were lysed and centrifuged at 15,000 xg. The supernatant was then ultracentrifuged or not at 100,000 xg. S109-phosphatase activity was assayed in the 15,000 xg and 100,000 xg supernatants supplemented or not with PKI using pS109-GST-Arpp19 as a substrate (pS for phosphorylated substrate). S109 phosphorylation of GST-Arpp19 (pS109) and total GST-Arpp19 ( gst Arpp19) were western blotted using respectively phospho-S109-Arpp19 and GST antibodies. GST-Arpp19 coupled to beads was doubly phosphorylated and then used as a substrate of S109-and S67-phosphatase activities in prophase extracts as described in the Method section.
In experiment 3, S109-phosphatase activity was recovered in a single fraction after the Mono Q column, Q8, which was further loaded on the Phenyl-Superose column. S109-phosphatase activity was recovered in PS11 after the Phenyl-Superose column. PS11 was loaded on the Superose 12 column. The phosphatase content was estimated by LC-MS/MS sequencing in fractions from Superose 12 column. S109-phosphatase activity is indicated with "+" or "-". Data availability: The data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022739 experiment 3 -Analysis of PS11 and Superose fractions (input: Q8 > PS11). S109phosphatase activity was recovered in a single fraction after the Mono Q column, Q8, which was further loaded on the Phenyl-Superose column. S109-phosphatase activity was recovered in PS11 after the Phenyl-Superose column. PS11 was loaded on the Superose 12 column.
The phosphatase content was estimated by LC-MS/MS sequencing in PS11 and in fractions from Superose 12 column. S109-phosphatase activity is indicated with "+" or "-". Data availability: The data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022739 | v3-fos-license |
2017-07-10T19:42:43.848Z | 2014-06-26T00:00:00.000 | 9899795 | {
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} | pes2o/s2orc | Ammonium Production off Central Chile (36°S) by Photodegradation of Phytoplankton-Derived and Marine Dissolved Organic Matter
We investigated the production of ammonium by the photodegradation of dissolved organic matter (DOM) in the coastal upwelling system off central Chile (36°S). The mean penetration of solar radiation (Z1%) between April 2011 and February 2012 was 9.4 m, 4.4 m and 3.2 m for Photosynthetically Active Radiation (PAR; 400–700 nm), UV-A (320–400 nm) and UV-B (280–320 nm), respectively. Ammonium photoproduction experiments were carried out using exudates of DOM obtained from cultured diatom species (Chaetoceros muelleri and Thalassiosira minuscule) as well as natural marine DOM. Diatom exudates showed net photoproduction of ammonium under exposure to UVR with a mean rate of 0.56±0.4 µmol L−1 h−1 and a maximum rate of 1.49 µmol L−1 h−1. Results from natural marine DOM showed net photoproduction of ammonium under exposure to PAR+UVR ranging between 0.06 and 0.2 µmol L−1 h−1. We estimated the potential contribution of photochemical ammonium production for phytoplankton ammonium demand. Photoammonification of diatom exudates could support between 117 and 453% of spring-summer NH4 + assimilation, while rates obtained from natural samples could contribute to 50–178% of spring-summer phytoplankton NH4 + requirements. These results have implications for local N budgets, as photochemical ammonium production can occur year-round in the first meters of the euphotic zone that are impacted by full sunlight.
Introduction
Since the discovery of decreasing concentrations of stratospheric ozone over the Antarctic, high levels of incident harmful solar ultraviolet radiation (specifically UV-B) have been a constant feature over the southern hemisphere, mainly during spring. The size of the ozone hole reached a historical maximum in 2006 while unprecedented low levels have also been reported over the arctic [1]. In mid-latitudes, ozone concentrations are currently 6% lower than the long term average for the area [2].
The impact of the different solar spectra in the ocean can lead to deleterious effects on plankton communities [3][4][5][6][7]. However, it is also possible to detect ''positive'' effects of exposure to solar radiation. For instance, the photo-dissociation of dissolved organic matter (DOM) may increase the bioavailability of dissolved organic carbon (DOC) for bacterial growth, potentially stimulating carbon transfer towards higher trophic levels via the microbial loop [8][9][10][11]. Previous studies on the photochemical production of organic and inorganic compounds of low molecular weight via exposure of DOM to UV radiation (UVR) showed the importance of this mechanism for marine microbial activity [8,9,[12][13][14]. It is now known that the effect of UVR on DOM can generate, among others, compounds such as carbon monoxide [15], ammonium (NH 4 + ) [12], amino acids (such as glutamine and alanine), nitrite (NO 2 2 ) and urea [12,13,[16][17][18][19]. However, the potential contribution of photoammonification could vary significantly among marine biomes [12,14,18,[20][21][22]. UVR could increase by 20% the availability of dissolved inorganic nitrogen (DIN) via ammonium production (photoammonification) in rivers in the southeastern continental shelf of the United States [12]. Additionally, other studies estimated that photoammonification can represent 50% of phytoplankton demand on the Orinoco River plume [22] while meeting 12% of the estimated annual phytoplankton demand (in terms of new N) in the oligotrophic Eastern Mediterranean Sea [14]. The upwelling-system off central Chile (36uS; 73uW) in the Humboldt Current System (HCS) is one of the most productive areas of the world ocean [23]. The observed biological production in this system is supported by the assimilation of new nitrogen (as nitrate injected into the euphotic zone by mixing and vertical advection during seasonal upwelling events) and regenerated nitrogen (derived from in situ remineralization of organic matter that results in NH 4 + release [24,25]). Additionally, ammonium assimilation by phytoplankton is persistent throughout the year representing almost half of nitrate uptake in active upwelling conditions [26]. Recurrently high concentrations of ammonium off central Chile are also thought to sustain intense chemosynthetic activity via nitrification [27], particularly within the euphotic zone and oxycline [26].
The aim of this study was to evaluate the potential effect of solar radiation, both Photosynthetically Active Radiation (PAR; 400-700 nm) and UV spectra (280-400 nm) in the coastal zone off central Chile (36uS) by quantifying the production of ammonium via photodegradation of marine DOM using exudates two cultured diatom species as well as natural DOM samples.
Methods
During this study we focused on two sites located in an active coastal upwelling system off central Chile (36uS). Ammonium photoproduction experiments were performed with sea water collected from station 18 of the COPAS Time Series program. However, schedule restrictions prevented the use of radiometers for incident solar radiation profiling at this station. Station 18 (36u 30.8009S, 73u 7.7509W; Fig. 1) has been studied by the time series program of the COPAS center since 2000 [28]. Samplings for this study were performed in the frame of this time series program with official support of the Chilean Navy (Directemar). Atmospheric measurements of incident solar radiation were carried out in the Concepción area (36u 49.6789S 73u 21.7849W; Fig. 1) while estimations of the depth of solar penetration in the water column were carried out via underwater measurements at Coliumo Bay off the upwelling system (36u 49.6699S 73u 02.1629W; Fig. 1). Although field samplings involved plankton and water chemistry only, this study did not involve endangered or protected species.
a) Incident Solar Radiation measurements
We carried out atmospheric measurements of incident solar radiation twice per month at noon between April 2011 and February 2012 using a portable radiometer (RM-21 Dr. Gröbel, Germany) equipped with sensors for three spectral ranges: UV-B (defined hereafter as 280-320 nm), UV-A (defined hereafter as 320-400 nm) and PAR (defined hereafter as 400-700 nm). Values of PAR, UV-A and UV-B radiation are expressed in Wm 22 .
The same instrument was used for estimating the penetration of solar radiation in the water column. Measurements were done under calm weather and low wind conditions every 3 weeks. The depth of penetration of the three spectra was estimated by measuring each spectrum at two depths: 0 m (immediately below the surface) and at 0.3 m depth. The coefficient of vertical light attenuation (Kd in m 21 ) for all spectra was calculated according to Eq. (1): Where Ed (Z) is the irradiance at depth z and Ed (0) is the irradiance just below the surface of the water column [29]. The depth of 1% penetration of incident surface irradiance (Z1%) was calculated for all wavelength spectra (PAR, UVR) by 2.3/Kd. Integrated values of PAR, UV-A and UV-B were calculated by numerically integrating (trapezoidal method) radiation values between the surface and Z1% and will be expressed in Wm 21 .
b) Photoammonification from diatom-derived and marine DOM
We performed experiments in order to evaluate the production of ammonium via photo-transformation of DOM using representative diatom cultures with different cell densities (Table 1).
Experiments were carried out between September 2011 and April 2014 (Table 1 and 2). DOM was obtained from exudates of two cultured species dominant in the study area, Chaetoceros muelleri (Lemmermann, 1898) and Thalassiosira minuscule (Krasske, 1841). Cultures were maintained in Walne + Si media and filtered through precombusted GF/F filters (0.7 mm, Millipore; 450uC for 6 h). Filtrates containing diatom exudates were diluted previous to incubation using milli-Q water (Table 1) in order to obtain the volume required for performing the experiment (see Table 2). Samples were then distributed in 500 mL quartz bottles (mean transmittance of 70% between 280 and 700 nm) and irradiated with either full solar radiation (PAR+UVR) or UVR only (including UV-A+ UV-B; Table 1). Dark control samples were incubated in darkened 500 mL Duran Schott bottles.
All experiments were performed using an irradiation chamber (UV Chamber B-03, Dr Gröbel, Germany) equipped with either PAR+UV (280-700 nm) or UV-A and UV-B lamps (280-400 nm). This instrument is equipped with an internal temperature control system allowing low temperature variations during the experiments. Table 1 summarizes the doses received by all samples. During all incubations, doses of PAR, UVA and UVB were generally within ranges of previously reported winter solar radiation for the study area [30]. We however acknowledge that spring and summer irradiance may be underestimated.
Samples for ammonium determination (20 mL) were taken in triplicate and stored in the dark at room temperature after addition of 5 mL of phthaldialdehyde for fluorometry (OPA). Samples were analyzed by the fluorometric method [31] using a Turner Design fluorometer.
Another set of experiments was designed to evaluate the production of ammonium from marine DOM using natural seawater samples. Seawater (25 L) was taken at 5 m depth at the COPAS time series station 18 (R/V Kay Kay II, Table 2 and Fig. 1). During each sampling, a conductivity temperature depth cast was done (CTD; SeaBird) in order to determine the structure of the water column. The depth of the mixed layer (MLD) was estimated using the thermal criterion (0.2uC) [32].
Experiments were carried out during austral spring and summer (September 2011 -January 2012). Water samples were filtered through pre-combusted GF/F filters (0.7 mm; 450uC, 6 h) using a peristaltic pump. Filtrates were distributed in autoclaved 500 mL glass bottles (Duran Schott for dark control) or 500 mL quartz bottles (UV-A+UV-B treatment). The time of exposure and doses received by all samples are summarized in Table 2.
Samples for ammonium (in triplicate) and bacterioplankton abundance were taken before incubation (T0) and every 2 h. Ammonium determination was carried out as described in the previous section. Determination of nitrite (NO 2 2 ) and nitrate (NO 3 2 ) was made in duplicate in 10 mL samples, which were frozen until laboratory analysis using a standard colorimetric automatic technique (Bran Luebbe autoanalyzer). Bacterioplankton abundance was determined by flow cytometry according to Marie et al (2000) [33]. Samples (1350 mL) were taken in duplicate in sterile cryovials, fixed with glutaraldehyde (at 0.1% final concentration) and stored at 280uC until laboratory analysis at PROFC laboratory at University of Concepcion, Chile.
During all incubations, doses of PAR, UVA and UVB were generally within ranges of previously reported winter solar radiation for the study area [30]. We therefore acknowledge that spring and summer irradiance may be underestimated.
c) Quantifying net ammonium photoproduction
In order to accurately estimate ammonium photoproduction, we took into consideration the presence of bacterioplankton in samples filtered through 0.7 mm. We therefore assumed that in situ regeneration of ammonium can occur during the incubation, which was estimated for samples incubated in dark conditions. Consequently, we established the following assumptions: 1) The exposure of DOM samples to UVR or PAR+UV radiation always results in ammonium production (other labile N compounds are not taken into account) 2) Photolysis of DOM only occurred under exposure to UVR and was absent in dark controls or PAR exposed samples 3) Complete degradation of DOM leading to limitation does not occur during the experiments 4) Bacterial ammonium ''regeneration'' is constant during the incubation. Based on these assumptions we propose Eq. (2) for estimating the ammonium production by photolysis of the DOM. Equation (2) evaluates the net change in the ammonium concentration through exposure to PAR+UV/UVR, while taking into account the simultaneous ammonium production that takes place via remineralization of DOM by bacterioplankton activity or ammonium consumption.
The term [NH 4 + ] T0 represents the ammonium concentration at the beginning of the incubation. The term [NH 4 + ] T1 represents the ammonium concentration at the end of the incubation. The term [NH 4 + ] Total represents total ammonium production via photolysis. [NH 4 + ] Total divided by the time of incubation give us the rate of ammonium photoproduction. The sub-indexes ''Exposure'' and ''Dark'' identify exposed samples from the dark controls.
Data of ammonium concentration during photodegradation of DOM for each exposure treatment and dark control were analyzed by a paired t-test.
Data of ammonium concentration and bacterioplankton abundance of natural DOM experiments were analyzed by a one-way ANOVA after checking for normality assumption (Kolmogorov-Smirnov test, a = 0.05) and homoscedasticity (Cochran test, a = 0.05). Pairwise multiple comparison were performed using the Tukey test as an a posteriori test (a = 0.05).
a) Incident solar radiation in the study area
The mean value per month of incident atmospheric PAR (700-400 nm), UV-A (400-320 nm) and UV-B (320-280 nm) radiation measured in the Concepcion area (36uS) is shown in Fig The penetration of solar radiation in the water column (Z1%) at Coliumo Bay is shown in Fig. 2D. As expected, the monthly mean value of Z1% during the entire study period was higher for PAR radiation than for UV-A and UV-B (9.4 m, 4.4 m and 3.2 m respectively). For PAR, the maximum penetration was found in winter 2011 (29.7 m in May). This can be explained by the influence of freshwater from river discharge or precipitation that determines salinity conditions in the first 20 m of the water column during winter time [34]. Photoproduction of Ammonium off Central Chile PLOS ONE | www.plosone.org b) Photoammonification rates from diatom-derived and marine DOM Figure 3 shows the ammonium evolution during photoammonification experiments in cultures of C. muelleri. Except for exudates derived from the lowest cell density, ammonium production was detected in all cases after exposure to PAR+UV or UV radiation only ( Fig. 3A-F). Maximum ammonium concentrations were reached after 5 h of exposure and were always higher than dark control values (t-test, P = 0.001).
There was variability in ammonium concentrations at T0 due to differences in cell density at the moment of filtration. For instance, initial ammonium concentration for the experiments carried out in 23/04/2014 were 16.5560.31 and 13.9361.00 mmol L 21 for cell densities of 2.49E+06 and 3.66E+06 cell mL 21 respectively. The experiment carried out the day after 24/04/2014 showed an initial concentration of 3.5660.21 mmol L 21 for cell density of 4.07E+06 cell mL 21 . In spite of such variability, calculations of net ammonium photoproduction over time should not be affected by T0 concentrations.
For exudates from culture of diatom C. muelleri at density of 2.49E+06 cell mL 21 (April 2014; Fig. 1A) ammonium concentrations increased after exposure to PAR+UV radiation, both PAR+ UV treatment and dark control. Although ammonium concentrations were higher in the dark control compared to PAR+UV treatment (t-test, P = 0.011), ammonium was produced in both cases at rates of 0.66 mmol L 21 h 21 and 0.46 mmol L 21 h 21 for dark and PAR+UV treatments, respectively.
Exposing exudates of C. muelleri from a higher cell density (February 2011; 2.52E+06 cell mL 21 ), to PAR+UV radiation ( Fig. 3B) resulted in high ammonium concentrations compared to the dark control (t-test paired, P = 0.036). However, ammonium concentrations decreased during the incubation at a rate of 0.01 mmol L 21 h 21 and 0.4 mmol L 21 h 21 for PAR+UV-exposed and dark samples respectively (i.e. a 40 fold difference between both treatments). Ammonium photoproduction generated 0.12 mmol of NH 4 + at a rate of 0.38 mmol L 21 h 21 according to Eq. (2).
Exudates of C. muelleri obtained from cultures with higher cell density (April 2014; 3.66E+06 cell mL 21 ) showed high ammonium concentrations after exposure to PAR+UV radiation (Fig. 3C). Ammonium increased in the PAR+UV treatment at a rate of 0.62 mmol L 21 h 21 , higher than the rate obtained for the dark control (0.26 mmol L 21 h 21 ). However, we did not find significant differences between PAR+UV treatment and dark control (paired t-test, P = 0.543). Ammonium photoproduction after 5 h of incubation could be estimated according to Eq. (2) as 0.36 mmol L 21 h 21 . In this case we estimated that 0.77 mmol of NH 4 + were produced during the incubation.
High ammonium concentrations were also obtained using exudates of C. muelleri with higher cell densities (September 2011; 3.97E+06 cell mL 21 ). After exposure to PAR+UV radiation ( Fig. 3D), ammonium concentrations were higher in samples exposed to PAR+UV compared to dark samples (paired t-test, P = 0.001). Ammonium was also produced in dark samples via microbial remineralization at a rate of 0.01 mmol L 21 h 21 . The estimated rate of ammonium photoproduction according to Eq. (2) reached 0.51 mmol L 21 h 21 . The amount of ammonium generated by photoproduction during the incubation reached 0.26 mmol.
In the case of exudates of C. muelleri at 4.07E+06 cell mL 21 (April 2014), exposure to UV radiation only produced high ammonium concentrations compared to the dark control (P = 0.010). Ammonium increased at rates of 1.09 and 0.43 mmol L 21 h 21 for in both UV exposed samples and dark control, respectively. Ammonium photoproduction after 5 h of incubation was estimated according to Eq. (2) as 0.61 mmol L 21 h 21 .
At higher cell densities (4.44E+06 cell mL 21 ) ammonium production experiments (November 2011; Fig. 1F) resulted in higher concentrations in the treatment exposed to UVR than dark control (paired t-test, P = 0.004). Ammonium levels increased during the incubation in samples exposed to UVR at a rate of 1.23 mmol L 21 h 21 , while decreasing in the dark control at a rate of 0.15 mmol L 21 h 21 . The estimated rates of ammonium photoproduction of ammonium after 5 h of incubation according to Eq. (2) reached 1.49 mmol L 21 h 21 . The total amount of ammonium generated via photoammonification reached 2.24 mmol for this experiment.
The variation of ammonium concentrations during exposure of exudates of T. minuscule coming from cultures with different cell densities to PAR+UV radiation is shown in Fig. 4. Higher ammonium concentrations were found in the exposed samples (DOM from cultures at 2.82E+06 cell mL 21 ) compared to dark conditions (paired t-test, P = 0.025). The observed ammonium production rate reached 0.08 mmol L 21 h 21 in the treatment exposed to PAR+UV radiation, while in the dark control ammonium was consumed at a rate of 0.02 mmol L 21 h 21 . The estimated ammonium photoproduction rate after 5 h of incubation according to Eq. (2) was 0.13 mmol L 21 h 21 and resulted in the generation of 0.19 mmol of NH 4 + . Exposure of exudates of T. minuscule (3.41E+06 cell mL 21 ) to UVR led to higher ammonium concentrations compared to initial conditions (t-test, P,0.001, Fig. 4B). Ammonium production rates reached 1.31 mmol L 21 h 21 and were lower in the dark control (0.75 mmol L 21 h 21 ; Fig. 4B). Estimated ammonium photoproduction according to Eq. (2) reached 0.56 mmol L 21 h 21 after 4 h of incubation. Ammonium generated by photoproduction was quantified as 0.67 mmol for this experiment.
Ammonium production was also observed in exudates of T. minuscule at higher cell densities (3.65E+06 cell mL 21 ) exposed only to UVR as shown in Fig. 4C. Ammonium concentrations were higher in the treatment exposed to UVR compared to dark conditions (paired t-test, P = 0.0242). The rate of ammonium production was 0.99 mmol L 21 h 21 which is higher than the rate obtained for dark samples (0.29 mmol L 21 h 21 ). Therefore, the estimated ammonium photoproduction (Eq. (2) We evaluated ammonium production via photodegradation of marine DOM by exposure to UVR (UV-A+UV-B) during spring (November 2011; Fig. 6). Ammonium concentrations decreased during the first 2 h of exposure to UVR at a rate of 0.05 mmol L 21 h 21 and reached concentrations of 4.7860.06 mmol L 21 (Fig. 6A). After 4 h of exposure, concentrations continued to decrease and reached values close to 4.2360.33 mmol L 21 . Ammonium concentrations in dark samples decreased during the first 2 h of exposure but increased towards the end of the incubation period (4.5460.19 mmol L 21 after 2 h and 6.3360.23 mmol L 21 after 4 h). Interestingly, bacterioplankton abundance (Fig. 6B) was significantly lower in samples exposed to UVR compared to samples incubated in the dark (paired t-test, P = 0.002). Cell abundance after 2 h of exposure decreased in the samples exposed to UVR (1.8660, 06610 5 cell mL 21 ), whereas dark samples showed no change compared to initial values (3.1460.12610 5 cell mL 21 ). After 4 h of exposure, cell abundance increased in the dark control while decreasing in the sample exposed to UVR (3.3760.08610 5 cell mL 21 (Fig. 6C). Nitrite also showed an increase over time (Fig. 6D), but only for irradiated while remaining unchanged in dark samples. Values at T0 (0.25 mmol L 21 ) increased 2 fold after 4 h of incubation (0.5 mmol L 21 ). This suggests that ammonium consumption can be due to Another set of experiments was carried out in spring (September 2011) and summer (January 2012; Fig. 7). Samples were exposed to total sunlight (PAR+UVR), PAR only or dark conditions. Results for September 2011 showed increasing ammonium concentrations increased in the samples exposed to PAR+UVR (3.3560.41 mmol L 21 ) compared to samples exposed to PAR radiation only (2.1960.12 mmol L 21 ; Tukey test, P,0.05). Following Eq. (2), we estimated the rate of ammonium photoproduction in the treatment exposed to PAR+UVR as 0.2 mmol L 21 h 21 . Bacterioplankton abundances in samples exposed to PAR radiation increased from 3.1860.16610 5 cell mL 21 to 3.9861.41610 5 cell mL 21 after 4 h of incubation. On the contrary, samples exposed to PAR+UVR as well as the dark control showed a decrease in cell counts over time (3.1660.1610 5 cell mL 21 and 2.9660.15610 5 cell mL 21 , respectively), although no significant difference was found among all treatments (ANOVA one way, P = 0.7638).
The experiments carried out in summer (January 2012; Fig. 7B) also showed increasing ammonium concentrations in samples exposed to PAR+UV compared to initial conditions (0.9460.04 mmol L 21 vs 0.7560.003 mmol L 21 ) as well as PAR radiation only and dark conditions (Tukey test, P,0.05). Also, and as observed in our spring experiments described above, samples exposed to PAR radiation showed the lowest ammonium concentrations during the incubation (0.5860.03 mmol L 21 ). The rate of photoammonification (Eq. (2)) for samples exposed to PAR+UVR reached 0.06 mmol L 21 h 21 . Bacterioplankton abundances during the incubation (Fig. 7B) only showed an increase in the treatment exposed to PAR radiation (151610610 5 cell mL 21 vs 13965610 5 cell mL 21 ) while values decreased in samples exposed to PAR+UVR as well as the dark control (12660 and 114622610 5 cell mL 21 , respectively). In spite of the observed differences, results between treatments were not significantly different between each other (one way ANOVA, P = 0.158).
Discussion
Our incident radiation data represents a seasonal survey of the study area that complements previous measurements [30]. The results show that irradiance levels increased 2.6, 2.6 and 4.2 times for PAR, UV-A and UV-B respectively in summer compared to winter, following a coherent seasonal trend. The depth of penetration of solar radiation (as PAR, UV-A and UV-B) between May 2011 and February 2012 varied in the study area, with a decrease in values between autumn 2011 and summer 2012. Maximum penetration in the water column was observed in autumn (April and May 2011), which coincides with the end of active upwelling conditions in the study area [35]. During this time, turbidity decreases in the water column allowing deeper penetration of solar radiation. Also important is the influence of freshwater inputs (as precipitation and river discharge) in the first meters of the water column [34] which modifies its optical properties compared to spring and summer. Nevertheless, UVR (both UV-A and UV-B) was measurable in the coastal area off central Chile during the entire study period (Fig. 2), suggesting a potential year-round occurrence of photochemical transformation of DOM in the first meters of the water column. Values of depth penetration (Z1%) reached 29.7 m for PAR, 7.4 m for UV-A and 5.1 m for UV-B. Although PAR Z1% seems deeper than expected for a coastal upwelling system [29], it is coherent with time data gathered historically at station 18 (unpublished data COPAS Time Series program). Although a few available data sets exist for the study area and are complementary with our results, they may not be entirely comparable because of the well-known differences between the instruments used. Our average UV-A values for winter 2011 are lower than data previously reported for 2003-2004 [30]. Our average UV-B values for winter and summer were also 3-4 times lower than a previous study [30]. A latitudinal comparison shows that UV-A (320-400 nm) values reported for lower and higher latitudes in Chile (Santiago, 33u339 and Punta Arenas, 53u089) are close to our values but we reports lower values of UV-B (280-320 nm) for the same latitudes [36]. The estimated penetration of solar radiation during this study is also lower than previous measurements in open ocean waters at the same latitude (36uS; 74uW) [7] while they are in the same range of estimations as Z10% reported for Seno Reloncaví and Valdivia in southern Chile [37]. However, since our Z1% estimations include the assumption of a homogeneous water column [3] and values of integrated solar radiation are affected by the intensity of incident solar radiation and the optical properties of the water, comparing our results with existing data must be done carefully. As stated previously, the variety of instruments used for underwater measurement of UVR precludes further comparisons [29].
High integrated values obtained in this study (up to 581, 25 and 1 W m 21 of PAR, UV-A and UV-B respectively, Fig. 2E) corresponded to the deepest values of Z1% (in autumn) while in summer, when the mixed layer heat balance is dominated by solar radiation [34], high integrated values responded to higher intensity of incident radiation but not deeper Z1%. This can be explained by the increased turbidity of the water column during the productive season which combines biological particles (phytoplankton and bacterioplankton) and high sedimentation rates previously observed in the study area [38].
We also report a persistent exposure during the year of the first meters of the water column to UV-A and UV-B. The effects of incident solar radiation have consequences for the photoproduction of bioavailable compounds for bacterioplankton [9] and can also have negative effects such as DNA damage [4], inhibition of photosynthesis [39,40] and decrease of bacterioplankton production [5]. However, the persistent exposure of the euphotic layer to UVR has also implications for biogeochemical processes which may result in increased availability of ammonium (derived from photodegradation of DOM) as well as enhanced heterotrophic N utilization and primary production. Further direct measurements of the optical properties of the water column are therefore needed to complement these results, including the characterization of chromophoric dissolved organic matter (CDOM) and its distribution, which is a major contributor to the attenuation of solar radiation in the ocean [3].
We evaluated the photoproduction of ammonium using DOM obtained from diatom cultures and natural samples (Time series coastal st 18). In this process, the exposure to sunlight (primarily UV radiation, [41]) causes the breakdown DOM and the consequent release of NH 4 + [15,19,42,43]. Photoproduction of ammonium was observed in all cases (Table 3), in agreement with previous studies (Table 4). Diatom-derived DOM exposed to PAR+UVR and UVR only resulted in ammonium production in two different types of cultures: C. muelleri and T. minuscule (Table 3). Because cultures were filtered through 0.7 mm, we assume that some bacterial activity (as ammonium consumption) may occur. This is particularly relevant for explaining the variability in T0 ammonium concentrations observed although the main factor influencing T0 variability may come from the differences in cell densities used in the cultures (Table 1). Nevertheless, ammonium was produced in samples exposed to PAR+UVR as well as in samples exposed to UVR only (the latter showing higher rates than values for PAR+ UVR exposure). Rates of photoproduction of ammonium for C. muelleri was estimated at 0.38, 0.36 and 0.51 mmol L 21 h 21 while exposed to PAR+UVR. Samples exposed to UVR only generated higher ammonium levels (0.61 and 1.49 mmol L 21 h 21 ). Exudates of T. minuscule exposed to PAR+UVR resulted in the generation of ammonium at a rate of 0.13 mmol L 21 h 21 . Rates obtained for samples exposed to UVR only were higher and reached 0.56 and 0.71 mmol L 21 h 21 . The rates of ammonium production obtained from DOM exudates from diatom cultures showed variability associated not only to the origin of DOM but also to its quality, a variable approached in this case by the use of cultures at different cell densities. In general, higher photoammonification rates were obtained with higher cell densities, which support the occurrence of photoammonification in high productivity regions such as the study area.
Photoproduction of ammonium was indeed observed during exposure of marine DOM to solar radiation (Table 3). According to culture-DOM results, ammonium photoproduction mainly occurred during the most productive seasons, spring (0.2 mmol L 21 h 21 ) and summer (0.06 mmol L 21 h 21 ) and mainly under PAR+UVR exposure. Interestingly, no net production of ammonium was observed during exposure to UVR only or PAR only. In fact, exposure to PAR resulted in increased ammonium consumption compared to dark and initial conditions. Accordingly, bacterioplankton abundance increased in samples exposed to PAR only compared to PAR+UVR, therefore enhancing ammonium consumption. On the other hand, exposure to UVR only resulted in ammonium consumption and decreasing bacterial abundance while increasing nitrite and nitrate concentrations were measured. This suggests active ammonium and nitrite oxidation in spite of UV exposure. This fact could explain the lower rates of ammonium photoproduction obtained for natural marine samples compared to cultured DOM. Consequently, the absence of ammonium production in samples exposed to PAR and the occurrence of ammonium production under PAR+UVR exposure suggest that active photoammonification can take place under full sunlight while PAR exposure enhances its biological consumption. This has been observed for microplankton, which can use UV-A as a source of energy as well as PAR for carbon fixation [44] while UV-B has an inhibitory effect. Furthermore, light stress (defined as high exposure to UV-B) seems to have enhanced inhibitory effects under nutrient limitation (particularly nitrate [45]), which is seldom observed in surface waters of our study area.
Our rates of ammonium photoproduction using marine DOM are smaller [12,15] or in the range [14,19,43] of results previously reported for marine waters (Table 4). They are nevertheless higher than rates obtained for oligotrophic and coastal lagoon waters [14,19]. This is coherent with previous observations of lower efficiency in photoammonification for coastal compared to off shore waters. Efficiency of photoproduction of ammonium is also dependent of the molar C:N ratio of DOM, which can explain the higher rates obtained with diatom exudates compared to naturally occurring DOM [46]. Also, at high ratios of bacterial activity and DOC, exposure to UVR does not necessarily result in increased availability of organic matter [8].
It is known that photochemical transformation of DOM can support heterotrophic and autotrophic plankton [41] and also increase bacterial production and abundance [9]. In order to determine the potential contribution of ammonium photoproduction in our experiments to local primary production we used phytoplankton uptake data reported for st.18 [26]. We estimated that the daily ammonium photoproduction from C. muelleri and T. minuscule exudates could support between 177 and 456% of the phytoplankton NH 4 + demand in spring and summer, respectively. Photoproduction of ammonium from natural DOM on the other hand has the potential for supporting between 50 and 178% of spring-summer phytoplankton demand [26]. Importantly, they could also sustain 100% of chemoautotrophic bacterial ammonium oxidation (the first step of the nitrification process) which can reach rates close to 50 nmol L 21 d 21 in surface waters of the study area [26].
These values are higher than the potential contribution of photochemically produced N to phytoplankton new production in the Baltic Sea where N deprived communities directly react to bioavailable N [41]. However, photoammonification and biological ammonium regeneration cannot be dissociated, the latter acting under full sunlight exposure and co-occurring with the former. Photoammonification has been suggested to be strong in shelf waters and marginal seas [14]. It has also been shown to decrease net community metabolism off the Eastern South Pacific [7]. Our results demonstrate that ammonium photoproduction under PAR+UVR occurs off central Chile in combination with its biological regeneration and consumption. Because net ammonium photoproduction is not usually accounted for in regional nitrogen budgets, our results have important implications for the understanding of mechanisms sustaining primary production in this coastal upwelling system.
Conclusions
We report a year-round influence of UVR in the first 2 to 7 meters of the water column in the coastal zone off central Chile. Net ammonium photoproduction via degradation of representative diatom exudates and natural DOM samples represents a potential contribution to the local nitrogen budget at short times scales (2 or 5 h). Photoammonification in culture-derived DOM was variable but showed higher rates in samples derived from higher cell density cultures. Naturally occurring DOM exposed to PAR+UVR resulted in net photoammonification at rates ranging between 0.06 and 0.2 mmol L 21 h 21 . No photoproduction of ammonium was observed after exposure to PAR, but increased consumption and bacterial abundance was observed.
Based in existing phytoplankton N uptake data for the study area [26], we estimate that net ammonium photoproduction could support between 50 and 178% of spring-summer phytoplankton NH 4 + demand. | v3-fos-license |
2020-01-23T09:22:41.457Z | 2019-01-01T00:00:00.000 | 210921749 | {
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} | pes2o/s2orc | Topological Descriptor of 2-Dimensional Silicon Carbons and Their Applications
Abstract The Chemical graph theory is extensively used in finding the atomic supplementary properties of different chemical stuructures. Many results of graph theory are commonly used in molecular structures and in general in Chemisty. In a molcular graph vertices are atoms while chemical bonds are given by edges. This article is about computing the exact values for some degree based toplogical descriptors of two molecular structures. Namely we work on the silicon-carbon Si2C3- III and SiC3-III for dimension two. We also discuss some applications of these results towards Chemistry.
Introduction
A combination of graph theory and Chemistry is an interesting branch of Mathematics called chemical graph theory. The molecules from Chemistry model in Mathematical ways in the form of molecular graph. A graph is a union of two sets namely vertices and edges. In a molecular graph vertices are atoms while chemical bonds are given by edges. Different techniques from graph theory use to apply on these molecular structures to get their different topological and structure properties. For example, the boiling point of chemical compound, which is a physical entity, can be estimated using degree and distance between the vertices of the chemical compound. Thus, we can say while modelling a chemical problem in terms of mathematical form, topology of its molecular structure plays an important role to give useful properties of corresponding chemical compound [1]. In 1988, it was accounted for that few hundred specialists worked in delivering around 500 research articles every year exploring various properties of chemical structures including two-volume of meticulous contents by Gutman in [2]. More applications of this interested branch of science particularly discussing topological indices are available in [3,4,5,6,7,8,9].
One of the promptly research directions among the researchers is the study of chemical compounds in term of mathematical modelling [4,5]. A broad proportion of chemical compounds exists which have interesting mathematical structures and have wide range of applications in industrial, pharmaceutical, research and commercial chemistry. Arrangements of atoms among a chemical compound have definite structural rules which have useful hidden properties. Thus, to explore these properties by use of mathematical tools, in terms of combinatorics and topology, play a significant rule in applied research. It is worth mentioning here that the mathematical chemistry obtains considerable contributions from the field of chemical graph theory [6,7]. There are many invariants of chemical graph theory namely, indices or descriptors, which are used in other sciences notably in pharmaceutical and chemical way [8,9]. More precisely the study of distance based and degreebased indices takes active part in the development of related fields [10]. It helps to gather huge data in the form of numerical values associated to chemical structures and get their comparisons using modern computer systems [11]. During the last decade of nineteenth century many topological descriptors have been introduced to fulfil the requirements of the chemists [12,13].
A graph G=(V,E) can be represented as combination of two sets namely edge set E and vertex set V. Edges are denoted by lines in a graph G. Number of edges shared by a vertex p called its degree μ(p) while distance between two vertices p and q is the minimum of the cardinality of the sets of all edges between them and is denote by d(p,q). One can represent a graph by many ways including a value namely by different topological descriptors which could be distance based or degree based.
The Zagreb indices are defined as follows by Gutman and Trinajstić [14,15]: The Zagreb coindices are defined as follows by Doslic' [16]: The multiple Zagreb indices are defined as follows by Ghorbani and Azimi [18]: We recommend the reader to study [19][20][21][22] for more details. Firstly this article deals with the computation of above mentioned indices for the molecular structres Si 2 C 3 -III[n,m] and SiC 3 -III[n,m]. Secondly we present comparisons of the results in the form of tables and functional graphs.
Methods
To process our outcomes, we utilize the strategy for combinatorial registering, vertex segment technique, edge segment strategy, graph hypothetical instruments, investigative strategies, degree tallying strategy and whole of degrees of neighbor's technique. Likewise, we used computer algebra systems like Matlab and Maple for estimation and numerical sketching, respectively.
Two dimenstional Silicon Carbide Si 2 C -III[n,m]
The silicones are semiconductor in nature that utilizes in assembling of some different materials. It is organism used nearly in all the most recent electronic based gadgets. A be notable among these discovered structures are the 2 dimensional silicon carbon having single layer blends having particular stoichiometric which were wrapped [23]. It's based particle the molecule swarm streamlining given to be (PSO) system combined with deep practical postulate enhancement.
Some sheets of the graphene were effectively disconnected in 2004 [24]. This two-dimensional structure come out to be used in the additional conventional particularly in their mechanical and optical possessions. Moreover, these gadgets possess distinguish electric properties of graphene draw which attracts the researcher to work on this two dimensional structure.
Till this date study of these silicon sheets is quite open although a lot of work has already been done. There is another form of these structures namely two dimensional (Si-C) single layers which can be seen as distinctive objects among the pure 2 dimensional single-layer carbon -graphene and the pure 2 dimensional solitary layersilicone. Many attempts are accompanied for forestalling the silicon carbon (Si-C) sheet [25]. Figure 1 give the structure for the above-mentioned sheets.
To have idea about the corresponding molecular graph, we characterize associated values of the one dimensional cells in succession (chain). It is easily seen in Figure 2 that in what way the cells associate in bolt (chain) and how one line interfaces with another line. As a first part we work on the molecular structure given by Si 2 C 3 -III[n,m].
Results
For this structure the cardinality of vertex set is 10 mn. Also, the cardinality of the edge set is 15mn-2n-3. We make partitions of vertex set and edge set to compute the topological descriptors for this chemical structure. Let n, m≥1. The vertex is partitioned into three sets depending upon degrees of the vertices. Namely we denote by V i the
The first and second Zagreb coindices
Equations (3), (4) and Theorem 1,2 we get our results as:
Multiple Zagreb indices
Now equations (7), (8) are used to compute following indices as:
2D Silicon Carbide SiC 3 -III[n,m]
The two dimensional Silicon Carbide SiC 3 -III, the molecular graph, is draft in Figure 6. The one dimensional cells are shown in the straight line in the form of chain. The rows are given by n and columns are given by m. Figure 7 describes the structure exhibited in what way the cells interface straight (chain) and how one line associates with another column.
Results
For this structure the cardinality of vertex set is 8 mn. Also, the cardinality of the edge set is 12mn-3n-2m. We make partitions of vertex set and edge set to compute the topological descriptors for this chemical structure. Let n, m≥1. The vertex is partitioned into three sets depending upon degrees of the vertices. Namely we denote by V i the set containing vertices of degree i. For Si 2 C 3 -III[n,m], we have |V 1 |=3, |V 2 |=6n+4m-6 and |V 3 |=8mn-6n-4m-1. The partition of edges is shown in the following Table.
Zagreb Coincides
Equations (3), (4) and Theorem 1, 2 leads us to compute the following results as below:
Results Comparisons and Discussion
A brief comparison of the indices has shown growth in the values of the calculated topological descriptors. This is clear from the Table 3 and Table 4 Table 5 and Table 6 that the numerical values of these indices are in increasing order as values of n and m are increasing. The corresponding behaviour of the graphs of these indices are shown in Figures 8-10 for some estimated values of n and m.
The topological descriptors computed in this article are used to compute the energy of electron particles π [15]. Thus, above mentioned comparison shown that the energy of electron particles increases as we increase the value of m and n for both types of Silicon sheets.
Applications of Zagreb Indices
The Zagreb coindex demonstrations a virtuous correlation of the materialization in heat of heptane's and octane.
Thus, our calculation done for the Zagreb coindices has shown an imperative statute for the materialization heat absorbed by heptanes and octane as there standards are in growing behaviour [26,27]. The multiple Zagreb indices are valuable for investigations of the ingredient and pharmacological possessions of medication in nuclear configurations. Therefore, on account of Si 2 C 3 -III[n,m], and SiC 3 -III[n,m], its expanding esteems are valuable in the fast activity during synthetic reaction for drugs [28].
In [29], the authors utilized these topological descriptors to demonstrate the mitigating movement of different acids particularly in N-arylanthranilic. In [30], the authors established the fact that these descriptors are important in demonstrating the division guaranteed and leeway of cephalosporin's inhumanness [31].
Applications of Silicone carbides
In Physical perspective, unadulterated silicon carbide is gotten as dry gems, along with thickness of around 3 g/ mL and a peak dissolving purpose of 2730C 0 . Moreover, generally this is found as a somewhat blue dark, radiant crystalline strong, because of modest quantities of iron or different polluting influences from the modern generation. In synthetic perspective, silicon carbide is an entirely steady furthermore, synthetically inactive multifaceted. This is indeed incredibly tough material, usually having 9 hardness rating, which is near to the precious stone. This is additionally portrayed by there extraordinary warm conductivity, peak temperature equality, below warm extension, protection from substance response, and capacity to work as a semiconductor. For more details, see books [32,33].
In addition, silicon carbide is broadly utilized as a rough. It is utilized to make different materials, for example, polishes, granulating wheels, spiteful apparatuses, hard earthenware production, vehicle parts, recalcitrant linings, high temperature blocks, warming components, wear-safe parts for siphons and even gems. It is additionally a significant material in the hardware business and utilized for making light LEDs and semiconductor electronic devices. Moreover, Silicon carbides residue and filaments delivered during the handling found to be primary dangers of this quantifiable. The silicon carbides residue may also bother eyes, coating, and superior respiratory framework and lead to lung cancer and fibrosis. For more details, see book [34].
Concluding Remarks
This article is about the computation of different topological descriptors of the chemical structures Si 2 C 3 -III[n,m], and SiC 3 -III[n,m]. We have computed the exact values of Zagreb type indices. The results are very useful and helpful for both chemical and pharmaceutical point of view. It gives interesting comparisons in terms of tables and three dimensional graphs. | v3-fos-license |
2020-06-11T09:05:27.021Z | 2020-06-01T00:00:00.000 | 219563895 | {
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} | pes2o/s2orc | Dietary Supplementation of Postbiotics Mitigates Adverse Impacts of Heat Stress on Antioxidant Enzyme Activity, Total Antioxidant, Lipid Peroxidation, Physiological Stress Indicators, Lipid Profile and Meat Quality in Broilers
Simple Summary To mitigate the adverse impacts of stressful environmental conditions on poultry and to promote the animal’s health and growth performance, antibiotics at sub-therapeutic doses have been added to poultry diets as growth promoters. Nevertheless, the improper and overuse of antibiotics as feed additives have played a major role in the emergence of antibiotic-resistant bacteria and increased levels of antibiotic residues in animal products, which have disastrous effects on the health of both animals and humans. Postbiotics, used as dietary additives for livestock, could be potential alternatives to antibiotics. Postbiotics produced from the probiotic Lactobacillus plantarum have been the subject of several recent kinds of research. However, the researchers have very rarely considered the effect of postbiotics on the broilers under heat stress. Abstract The purpose of this work was to evaluate the impacts of feeding different postbiotics on oxidative stress markers, physiological stress indicators, lipid profile and meat quality in heat-stressed broilers. A total of 252 male Cobb 500 (22-day-old) were fed with 1 of 6 diets: A basal diet without any supplementation as negative control (NC); basal diet + 0.02% oxytetracycline served as positive control (PC); basal diet + 0.02% ascorbic acid (AA); or the basal diet diet + 0.3% of RI11, RS5 or UL4 postbiotics. Postbiotics supplementation, especially RI11 increased plasma activity of total-antioxidant capacity (T-AOC), catalase (CAT) and glutathione (GSH), and decreased alpha-1-acid-glycoprotein (α1-AGP) and ceruloplasmin (CPN) compared to NC and PC groups. Meat malondialdehyde (MDA) was lower in the postbiotic groups than the NC, PC and AA groups. Plasma corticosterone, heat shock protein70 (HSP70) and high density lipoprotein (HDL) were not affected by dietary treatments. Postbiotics decreased plasma cholesterol concentration compared to other groups, and plasma triglyceride and very low density lipoprotein (VLDL) compared to the NC group. Postbiotics increased breast meat pH, and decreased shear force and lightness (L*) compared to NC and PC groups. The drip loss, cooking loss and yellowness (b*) were lower in postbiotics groups compared to other groups. In conclusion, postbiotics particularly RI11 could be used as an alternative to antibiotics and natural sources of antioxidants for heat-stressed broilers.
Introduction
Environmental stressors such as disease and heat stress are the major problems faced by global poultry production, having negative influences on animal physiology, behaviour, health and productive performance, causing tremendous economic losses [1][2][3][4][5]. They can adversely affect the biological macromolecules such as proteins, lipids, carbohydrates and DNA by the generation and accumulation of reactive oxygen species (ROS) and free radicals in the cells while performing their normal metabolic functions [6][7][8], resulting in cell damage and the appearance of pathological symptoms [9,10]. To mitigate the adverse impacts of stressful environmental conditions on poultry and to promote the animal's health and growth performance, antibiotics at sub-therapeutic doses have been added to poultry diets as growth promoters [11][12][13]. However, the improper and overuse of antibiotics as feed additives have played a major role in the emergence of antibiotic-resistant bacteria and increased the levels of antibiotic residues in animal products, which have disastrous effects on the health of both animals and humans [14,15]. Thus, the use of antibiotics in farming livestock has been prohibited in the EU [16,17]. Ascorbic acid is a natural antioxidant and health-promoting agent that has great potential to substitute antibiotics as growth promoters in fighting bacterial infections [18,19], and have been thought to be beneficial for heat-stressed broiler chickens. Dietary supplementation of ascorbic acid has the advantage of compensating for incompetent biosynthesis of ascorbic acid and has the potential to ameliorate the harmful effects of hot climate in broiler chickens [20]. Data from several studies suggest that ascorbic acid supplementation may compensate for the reduction in growth rate and feed intake [21,22], improve overall growth performance, antioxidant status and meat quality, and reduce serum concentrations of corticosterone, acute phase proteins, cholesterol and lipid oxidation [23,24].
Using postbiotics as dietary additives for livestock and potential alternatives to antibiotics, postbiotics produced from the probiotic Lactobacillus plantarum have been the subject of several recent kinds of research. The mechanism of action of postbiotics is not different from that of probiotics, owing to the fact that the same secondary metabolites from probiotics are present in the postbiotics, but not the living cells [25]. Postbiotics contain several antimicrobial components including bacteriocins and organic acids, which can minimise the pH of the gut and prevent the proliferation of pathogens in both the feed and animal gut [26]. Recent evidence suggests that the postbiotics produced by L. plantarum strains have an inhibitory effect on several gut pathogens, such as vancomycin-resistant Enterococci, Listeria monocytogenes, Salmonella typhimurium and Escherichia coli [27][28][29][30]. It has recently been observed that the dietary supplementation of postbiotics promoted the health and growth performance in broilers [31][32][33], layers [34,35] and piglets [36,37]. More recently, postbiotics have been revealed to enhance the growth performance, rumen fermentation, immune status and gastrointestinal health in small ruminants [38,39]. Under normal environmental temperature, dietary supplementation of postbiotics improved health and growth performance of broiler chickens by promoting their immune status, growth genes expression and gut health, as their supplementation significantly improved the intestinal villus, decreased the population of Enterobacteriaceae and faecal pH, and increased the population of lactic acid bacteria [25,[31][32][33]40]. Moreover, improvements in broiler meat quality and reduction in plasma cholesterol were observed with dietary supplementation of postbiotics in broilers [35,[40][41][42]. Our recent findings from a companion study [43] revealed that dietary supplementation of postbiotics produced from L. plantarum increased body weight, body weight gain, feed conversion ratio (FCR), intestinal villus height, immune response, insulin-like growth factor 1 (IGF-1) and growth hormone receptor (GHR) mRNA expression, caecum non-pathogenic bacteria population, and reduced Enterobacteriaceae and E. coli population in heat-stressed broilers.
Aside from developing a healthy gut and promoting growth performance, a preliminary study from this laboratory revealed that the postbiotics produced by L. plantarum have high antioxidant activities [44]. Similarly, bacterial cultures of L. plantarum were reported to exhibit high antioxidative activities [45,46]. Whilst considerable research has investigated the beneficial impacts of postbiotics on broiler chickens under normal temperature, there is still a scarcity of information on their impacts on heat-stressed broilers. Therefore, the outlook of this work was to examine the impacts of feeding three different postbiotics on antioxidant enzyme activity, total antioxidant, lipid peroxidation, heat shock protein 70, acute phase proteins, lipid profile and meat quality in heat-stressed broiler chickens.
Postbiotics Production
The three different Lactobacillus plantarum strains, RS5, RI11 and UL4 were procured from the Industrial Biotechnology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia. The DNA was sequenced by Moghadam et al. [47] who differentiated between the L. plantarum strains, while their ability for producing different amino acids was recently determined by Lim et al. [48]. The three different cultures were preserved by the revival of cultures following Foo et al.'s procedure [49]. The cultures were kept at −20 • C in De Man, Rogosa and Sharpe MRS medium (Merck, Darmstadt, Germany) with 20% (v/v) glycerol.
A volume of 100 µL from each stock culture was activated in 10 mL MRS broth, incubated at 30 • C for 48 h and sub-cultured in the same media for another 24 h. The activated cultures were spread onto a plate and incubated at 30 • C for 48 h. An individual colony was picked from the plate, inoculated twice into MRS broth (10 mL) and incubated at 30 • C for 48 h and 24 h. Active cells of L. plantarum (RI11, UL4 and RS5) were first washed using a 0.85% (w/v) NaCl (Merck, Darmstadt, Germany) sterile solution, then adjusted to 10 9 CFU/mL and used as an inoculum. For preparing the working cultures of the three L. plantarum strains (RS5, UL4 and RI11), 10% (v/w) 10 9 CFU/mL active bacterial cells were inoculated into MRS media, incubated for 10 h at 30 • C, and centrifuged at 10,000× g for 15 min at 4 • C. Cell-free supernatant (CFS) was filtered using a 0.22 µm cellulose acetate membrane (Sartorius Minisart, Gottingen, Germany) following the procedure outlined in detail by Loh et al. [50]. The harvested CFS (postbiotic) was kept at −20 • C until further analysis.
Ethical Note, Birds, Experimental Design and Housing
The feeding trial was performed at the research facilities of the Institute of Tropical Agriculture and Food Security (ITAFoS), University Putra Malaysia. The experiment was conducted in conferment with the approved guidelines by the Animal Ethics Committee of the University Putra Malaysia (protocol no. UPM/ACUC/AUP-R085/2018), which ascertains that the use and care of research animals are ethical and humane. Two hundred and fifty-two Cobb 500 male chicks (one-day-old) were supplied by a local hatchery. The chicks were housed in wire-floor cages placed in two identical rooms. The rooms were environmentally controlled with each of the two measuring 9.1 × randomised 3.8 × 2.3 m, length × width × height, whereas measurement of each cage was 120 (length) × 120 (width) × 45 (height) cm. The birds were reared following the management recommendations of Cobb 500 from 1 to 21 days of age (starter period). The chickens in the two rooms were maintained at the recommended temperature of 32 ± 1 • C on the first day, and gradually reduced to around 24 ± 1 • C by 21 days of age. During the finisher period (day 22 to day 42), the birds were divided into 6 treatment groups, 7 replicates per group with 6 chicks in each replicate. The birds were offered 1 of 6 diets: (1) A basal diet without any supplementation as negative control (NC); (2) basal diet + 0.02% (w/w) oxytetracycline as positive control (PC); (3) basal diet + 0.02% (w/w) ascorbic acid as antioxidant control (AA); or basal diet + 0.3% (v/w) of (4) RI11, (5) RS5 or (6) UL4 postbiotics. The basal diets were formulated using FeedLIVE software Version 1.52 (Live Informatics Company Ltd., Nonthaburi, Thailand) [43] following the nutrient specifications of the Cobb 500 Nutrition Guide. From day 22 to day 42, broilers were subjected to a high temperature for 3 h per day from 11:00 am to 2:00 pm at 36 ± 1 • C. It took approximately 45 min for the temperature to escalate from 24 to 36 • C. Nonetheless, it took 1 h and 30 min for the temperature to decline from 36 to 24 • C. The management and environmental conditions of this current experiment are described in our recently published companion study [43].
Samples Collection
At 42 days of age, around 2 h and 45 min after the daily heat stress, 2 chickens from each cage (14 chickens per treatment group) were selected at random and slaughtered following the Halal practice, as recommended by the Malaysian Standard [51]. Blood was collected at exsanguination into BD Vacutainer ® EDTA blood tubes (New Jersey, BD, USA) and kept on ice. Upon centrifugation at 3500× g for 15 min at 4 • C, harvested plasma samples were (1.5 mL microcentrifuge tubes) stored at −80 • C for later determination of total antioxidant capacity (T-AOC), glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), corticosterone (CORT), ceruloplasmin (CPN), alpha 1-acid glycoprotein (α1-AGP), heat shock protein 70 (HSP70) and lipid profiles. Whole breast muscle was collected for the meat quality determination. The thigh muscle was collected for lipid peroxidation measurement.
Total Antioxidant Capacity
Total antioxidant capacity (T-AOC) was measured by a colourimetric method (ABTS) using a commercial total antioxidant capacity assay kit (Elabscience, E-BC-K219, Houston, TX, USA) according to the manufacturer's protocol. The principle of this method, to determine the T-AOC from plasma, is that ABTS is oxidised to green ABTS + by appropriate oxidant, which can be inhibited in the presence of antioxidants. Briefly, 10 µL of plasma sample was loaded into a microplate well and mixed with 20 µL of application solution. Then, 170 µL of ABTS working solution was added into each well, and incubated at room temperature for 6 min. Finally, the absorbance of the colour was measured at 405 nm by a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Blank contains distilled water plus application and working solutions were abstracted from samples and standard absorbance. The trolox solution was diluted with distilled water to prepare serial concentration (0.1, 0.2, 0.4, 0.8, and 1.0 mM) and the standard curve was plotted to determine T-AOC concentration in plasma.
Superoxide Dismutase Activity
Superoxide dismutase (SOD) assays were carried out using EnzyChrom™ Superoxide Dismutase Assay Kit (ESOD-100, BioAssay Systems, Hayward, CA, USA) based on the protocol provided by the manufacturer. The detection range of the kit was 0.05-3 U/mL SOD. The test depended on the addition of xanthine oxidase to the sample as a source of superoxide, and this superoxide reacted with a specific dye to form a coloured product. Based on the activity of SOD in the sample, which acted as a superoxide scavenger, the superoxide was reduced, and then the intensity of colour was decreased. The activity of SOD was determined by measuring the colour intensity at 440 nm using a Multiskan GO microplate reader (Thermo Scientific, Waltham, MA, USA). The concentration of SOD in the sample was quantified using standard curve of known concentration of SOD.
Catalase Activity
Catalase (CAT) activity was measured from plasma using the EnzyChrom TM catalase assay kit (ECAT-100, BioAssay Systems, Hayward, CA, USA), according to the manufacturer's instructions. The detection range of the kit was 0.2-5 U/L CAT. The test depends on the degradation of H 2 O 2 using redox dye. After the preparation of the assay, 10 µL of the sample, positive control and assay buffer as blank plus 90 µL of substrate buffer (50 µM) were loaded into the micro-plate well, then the plate was shaken and incubated at room temperature for 30 min. During the incubation time, the standard curve was prepared by mixing 40 µL of the 4.8 mM H 2 O 2 reagent with 440 µL of distilled water in the serial concentration, then 10 µL of the standard solution with 90 µL of assay buffer were placed into standard wells. After incubation, 100 µL of detection reagent was combined in each well and incubated for 10 min at room temperature. Finally, the optical density of CAT was read at 570 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). The standard curve was used to calculate the CAT activity in the plasma samples
Glutathione Peroxidase Activity
Glutathione peroxidase (GPx) activity was analysed in plasma samples using the EnzyChrom TM Glutathione Peroxidase Assay Kit (EGPx-100, BioAssay Systems, Hayward, CA, USA), which directly measures the consumption of NADPH in the enzyme-coupled reactions. The assay was carried out as recommended in the manufacturer's protocol. The detection range of the kit was 40-800 U/L GPx. Approximately, 10 µL of the sample plus 90 µL of working reagent (80 µL assay buffer, 5 µL glutathione, 3 µL NADPH (35 mM), and 2 µL gr enzyme) were loaded into the microplate well and the plate was tapped to mix. Next, 100 µL of substrate solution was added to each sample and control well. The optical density of the samples and standards were measured immediately at time zero (OD0), and again at 4 min (OD4). The absorbance of the GPx activity was recorded at 340 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). The NADPH standards were used to plot the standard curve. The standard curve was used to calculate the GPx activity in the plasma samples.
Glutathione Activity
Glutathione (GSH) activity was measured in plasma using QuantiChrom TM Glutathione Assay Kit (DIGT-250, BioAssay Systems, Hayward, CA, USA) following the manufacturer's protocol. The principle of the assay depended on the reaction of 5,5'-dithiobis-2-nitrobenzoic acid with reduced glutathione to form a yellow product. Briefly, 120 µL of 20-fold diluted sample was mixed with 120 µL of reagent A into 1.5 mL tube, centrifuged at 14,000 rpm for 5 min and 200 µL of supernatant was transferred into the microplate well. Then, 100 µL of reagent B was added to each well of samples, the plate was tapped for mixing, and incubated for 25 min at room temperature. Next, 400 µL of the calibrator was mixed in serial dilution with distilled water into separate wells as the standard and 300 µL of distilled water was pipetted into a separate well as a blank. After incubation, the absorbance was read at 412 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). The GSH concentration in the plasma was calculated using the standard curve of glutathione.
Plasma Lipid Profile
Plasma total cholesterol (TCHOL), high density lipoprotein (HDL) and triglyceride (TG) were analysed using an automatic analyser 902 (Hitachi, Munich, Germany), while low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) levels were estimated with the protocol described by DeLong et al. [52]. The level of VLDL was calculated by dividing plasma TG by five. The LDL level was obtained using the following equation:
Lipid Peroxidation
Thiobarbituric acid reactive substances (TBARS) in the thigh meat were measured with the method described by Lynch and Frei [53] and modified by Mercier et al. [54]. About 1 g of meat sample was homogenised in 4 mL 0.15 M KCl + 0.1 mM BHT for 1 min at 6000 rpm with Ultraturrax homogenizer. Two hundred microliters of the homogenised sample was mixed with a solution (TBARS solution), heated in a water bath set at 95 • C for 1 h, for a pink colour to develop. The formation of a pink coloured sample mixture is the result of the reaction of TBARS end products (malondialdehyde) with thiobarbituric acid (TBA). The following step was the cooling of the sample under tap water for 5 min. Three millilitres of n-butyl alcohol was then added to the extracts and homogenised. The next step was the centrifugation of the mixture for 10 min at 5000 rpm. The absorbance of the supernatant was read with a spectrophotometer (Secomam, Domont, France) at 532 nm wavelengths against n-butyl alcohol as a blank. TBARS concentration of samples was determined from a standard curve of 1, 1,3,3-tetraethoxypropane and calculated as µg malondialdehyde (MDA)/g meat.
Plasma Corticosterone
The corticosterone (CORT) plasma concentration was measured using a CORT ELISA kit specific to chickens (QAYEE-BIO, Shanghai, China) in accordance with the instructions of the manufacturer. Fifty microliters of appropriately diluted samples and standard were loaded in duplicate into predetermined microplate wells, mixed with 50 µL of horseradish peroxidase solution, gently shaken and incubated at 37 • C for 60 min. Then, the contents of the wells were discarded and washed using washing solution. The last step was repeated five times. Then, 50 µL of chromogen solution A and 50 µL of chromogen solution B (of each) were pipetted into each well and incubated for 10 min at 37 • C while protected from light. After the incubation, 50 µL of a stop solution was pipetted into each well and the absorbance was immediately measured using a BioTek™ ELx800™ microplate reader (BioTek, Winooski, VT, USA) at 450 nm. A standard diluent (without horseradish peroxidase (HRP) or sample) was used as a blank, which was later subtracted from the absorbance of standards and samples. CORT concentration in plasma was calculated using an equation generated from the slope of the standard curve.
Heat Shock Protein 70 (HSP70)
The heat shock protein 70 (HSP70) plasma concentration was measured using chicken HSP70 ELISA kits (QAYEE-BIO, Shanghai, China) based on the manufacturer's instructions. Except for the differences in assay standard, the principle, materials and steps used in this assay were similar to those described for plasma corticosterone determination (Section 2.7). HSP70 concentration in plasma was calculated using an equation generated from the slope of the standard curve.
Plasma Acute Phase Proteins
The α1-AGP plasma concentration was measured using a commercial ELISA kit specific to chicken (QAYEE-BIO, Shanghai, China) following the manufacturer's protocol. Except for the differences in assay standard, the principle, materials and steps used in this assay were similar to those described for plasma corticosterone determination (Section 2.7). α1-AGP concentration in plasma was calculated using an equation generated from the slope of the standard curve.
Ceruloplasmin (CPN)
The ceruloplasmin (CPN) concentration was determined by the method of Martinez-Subiela et al. [55]. The method used in this work was based on the rate of coloured product development from CPN and the substrate 1,4-phenylenediamine-dihydrochloride. Approximately, 8.15 g of sodium acetate trihydrate was dissolved in 100 mL distilled water and the pH of the solution was adjusted to 6.2 with glacial acetic acid. Then, 0.246 g of 1,4-phenylenediamine-dihydrochloride (Sigma Chemical, P1519) was added to the prepared buffer and kept in the dark for a minimum of 45 min. A 100 µL of the working solution and 10 µL of samples or standards were loaded into appropriate microplate wells, gently shaken and kept in the dark for 20 minutes. The ceruloplasmin oxidase activity was measured at 550 nm using a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). A standard curve was carried out with serial dilution of pig plasma of known CPN concentration, calibrated against a combination of purified CPN (Sigma-Aldrich, St. Louis, MO, USA) and saline buffer to obtain different concentrations of 12.75 (20 µL pig plasma + 60 µL saline buffer), 6.375, 3.187, 1.593, 0.796, 0.398, 0.199, and 0.099 mg/mL CPN. A blank, containing phenylenediamine dihydrochloride solution and distilled water absorbance, was identified and subtracted from the samples and standard absorbance.
Drip Loss
Drip loss of the meat samples was evaluated using the method described by Honikel [56]. After slaughtering, approximately 25-35 g of fresh meat samples from pectoralis major muscle was collected, individually weighed and recorded as the initial weight (W1). Polyethene plastics bags used to pack each sample were sealed and the vacuum packages were stored at 4 • C in the chiller. Then, at 7 days post-storage, the final weight (W2) was measured immediately after removing the samples from the bags and blotted dry. The calculation of the percentage drip loss was done by differences in the final and initial weight of the sample. The sample weight was divided by the initial sample weight after 7 days of storage using the following equation: W1 = initial sample weight on day 0; W2 = final sample weight after 7 days of storage.
Cooking Loss
Cooking losses of the meat samples were obtained according to Honikel [56]. Fresh meat samples from pectoralis major muscle were weighed individually and recorded as the initial weight (W1). Samples were then cooked in a water bath at 80 • C for 20 min in plastic bags. Following that, the samples were cooled at room temperature and blotted gently and reweighed as a final weight (W2). Cooking loss percentage was quantified as the initial and the final weight difference using the following equation: where W1 = weight before cooking; W2 = weight after cooking.
Shear Force (Tenderness)
The shear force of the meat sample was determined with the protocol according to Sazili et al. [57]. Upon the determination of cooking loss, the same meat samples proceeded to a shear force determination using a TA.HD plus ® texture analyser (Stable Micro Systems, Surrey, UK) having a Volodkevitch bite jaw equipment. Meat samples length were cut in parallel to the direction of the muscle fibres from each sample in triplicate blocks, each of them measuring 1 cm (height) × 1 cm (width) × 2 cm (length). Each block was sheared with the Volodkevitch bite jaw on the middle of the block, perpendicular to the direction of the fibres. Tenderness of meat is inversely comparable to the shear force values. Shear force values were expressed as the average peak positive force value of triplicate blocks from each individual meat sample, and the results were expressed in grams.
Colour
A ColorFlex EZ spectrophotometer (Hunter Associates Laboratory, Inc., Reston, VA, USA) was used for meat colour determination following the International Commission on Illumination (CIE) Lab-values. Before using it, the colourimeter was calibrated against black and white tiles. The frozen meat samples were thawed overnight into a 4 • C chiller. The thawed meat samples were subjected to blooming for 30 min and transferred into the ColorFlex sample cup with the bloomed meat surface facing the base of the cup. Meat colour was measured in triplicate (the cup was rotated clockwise to 90 • in the second and third reading) and to obtain the average values of lightness (L*), redness (a*) and yellowness (b*) readings [58].
Meat pH
The breast muscle samples stored at −80 • C were removed and pulverised using a mortar and pestle with presence of liquid nitrogen. Half a gram of each sample was homogenised (Wiggen Hauser ® D-500, Berlin, Germany) for 30 seconds in 10 mL ice cold 5 mM sodium iodoacetate (Merck Schuchardt OHG, Hohenbrunn, Germany), and 150 mM KCl solution for 20 seconds to stop glycolysis process (specifically glyceraldehyde 3-phosphate dehydrogenase) and its subsequent lactic acid production [58]. A pre-calibrated pH meter (Mettler-Toledo AG, Zürich, Switzerland) was used for the measurement of pH and calibration was done prior to usage using pH 4.0 and 7.0 buffers.
Statistical Analysis
All statistical analyses were analysed using the 9.4 Version of Statistical Analysis System (SAS) software (SAS Inc., Cary, NC, USA). All data were analysed as 1-way ANOVA using the general linear model procedure. The analysis involved diet as the main effect in a completely randomised design. Where appropriate, means were separated by Duncan's multiple range test. The statistical significance was considered at p < 0.05.
Antioxidant Enzyme Activities
The SOD and GPx activities did not differ (p > 0.05) among treatment groups ( Table 1). The CAT activity was significantly (p < 0.05) enhanced in the RI11 group compared to NC and PC groups. The GSH activity was also significantly (p < 0.05) higher in UL4 and RI11 groups compared to NC and PC groups. Birds fed with the RI11 diet had significantly (p < 0.05) higher T-AOC activity than their counterparts fed with PC, NC and UL4 diets. The broilers fed with RS5 diet had higher (p < 0.05) T-AOC than broilers fed with NC diet.
Acute Phase Proteins (APPs) and Heat Shock Protein 70 (HSP70)
The impacts of feeding different postbiotics on APPs (α1-AGP and CPN) and HSP70 in broiler chickens under heat stress conditions are summarised in Figure 1. CPN concentration was significantly (p < 0.05) lower in birds fed RI11 diet as compared to the birds fed NC, PC and AA diets. However, difference in CPN was neither observed between the NC and PC groups nor between AA, UL4 and RS5 groups. The RI11 group recorded the lowest α1-AGP concentration compared to other groups. The UL4 group also had significantly lower α1-AGP plasma concentration compared to the NC group. HSP70 concentration was not affected by dietary treatment (p > 0.05).
Plasma Corticosterone
The plasma corticosterone level was not affected (p > 0.05) by dietary treatment (Figure 2).
Plasma Lipid Profile
Feeding broilers with RI11 diet resulted in lower (p < 0.05) total cholesterol as compared with broilers fed NC, PC or AA diets, but not different (p > 0.05) when compared with other postbiotic groups ( Table 2). No differences (p > 0.05) were noted for the cholesterol level between PC, UL4, RS5 and AA groups or between NC and PC treatment groups. Triglyceride and VLDL were lower (p < 0.05) in postbiotic groups compared with NC, but not different (p > 0.05) from PC and AA treatment groups. The triglyceride and VLDL concentrations were not different (p > 0.05) in broilers fed AA, PC and NC diets. The LDL level was lower (p < 0.05) in birds fed with the postbiotics and AA diets compared with birds that received the PC and NC diets. No differences (p > 0.05) were observed between NC and PC groups for LDL concentration. Dietary treatment had no effect (p > 0.05) on HDL concentration. Table 3 shows the effects of different postbiotics supplementation on pH, drip loss, cooking loss, shear force and colour of pectorals major muscle from heat-stressed broiler chickens. The pH was significantly higher in the RI11 group as compared with RS5, NC and PC treatment groups, whereas the pH in RS5 group was not different (p > 0.05) compared with those of the UL4 and AA groups. The latter treatment groups were higher (p < 0.05) in breast meat pH than NC and PC groups. RI11 and UL4 groups had lower (p < 0.05) meat drip loss as compared with the meat from the NC treatment group. The drip loss in the meat from heat-stressed broiler chickens was not different between PC, AA, RI11, UL4 and RS5 treatment groups. The cooking losses from RI11 and RS5 postbiotics groups were significantly lower compared to the AA, PC and NC treatment groups. However, there were no significant differences between the latter groups in the cooking loss. Shearing force of pectoralis major muscle of heat-stressed broiler chickens was higher (p < 0.05) in the NC group compared to AA, RI11, RS5 and UL4 treatment groups. No differences (p > 0.05) were recorded for the share force among PC, AA, RI11, UL4 and RS5 treatment groups or between the NC and PC treatment groups. Birds fed RI11, UL4 and AA diets had lower (p < 0.05) lightness values compared to PC and NC treatment groups. No significant differences were observed for lightness among birds fed on AA, RI11 and UL4 diets or between birds fed RS5, NC and PC diets. The meat yellowness was higher (p < 0.05) in the NC, PC and AA treatment groups compared to the RI11 postbiotics group. No differences were observed for redness among all the treatment groups.
Meat Quality (pH, Drip Loss, Cooking Loss, Shear Force and Colour) and Lipid Peroxidation (TBARS)
For the lipid peroxidation), there were significant reductions (p < 0.05) in thigh MDA content of RI11 and UL4 treatment groups compared to PC, NC and AA treatment groups. No differences were observed for MDA in between PC and AA treatment groups. The MDA content of the RS5 group was not different from PC and AA treatment groups.
Antioxidant Activities and Lipid Peroxidation
Heats stress increases core body temperature, which triggers an increment in the production of free radicals leading to oxidative damage [59,60]. Oxidative stress leads to the production of varieties of ROS, including hydroxyl free radical and superoxide anions. Several studies showed that overflow of ROS could damage the biological macromolecules such as proteins and nucleic acids, and produce huge amounts of MDA causing tissue damage, consequently leading to the development of diseases [61]. In birds, the main antioxidant enzymes are glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). These enzymes are a higher order of antioxidant defence acting to transform reactive species into non-radical and non-toxic products [62,63].
Postbiotic supplementation in the present study did not affect the SOD and GPx activities. However, the CAT and GSH activities were enhanced in the postbiotic groups, especially in RI11 and UL4 birds as compared with the negative control and other treatment groups. Likewise, two studies reported that broilers under heat challenge had increased activities of CAT, GPx, GSH and SOD [60,64]. Heat-stressed birds fed with postbiotics also had significantly higher T-AOC in the present study. The present study results were in line with the findings of Wang et al. [65] who found that broilers fed probiotic Lactobacillus johnsonii BS15 had significantly higher blood T-AOC, SOD and CAT activities compared to a negative control group. Hence, the dietary postbiotics showed the capacity to improve antioxidant activities (concentrations of CAT, GSH and T-AOC) in the plasma of heat-stressed broilers. Postbiotics are a natural source of antimicrobial and antioxidant that can safely alleviate the stress and improve the health of animals. This finding is consistent with Akbarian et al. [66], who observed dietary supplementation with another natural source of antioxidant (Xanthorrhiza and Origanum compactum) in heat-stressed broilers, which led to increased mRNA levels of SOD, GPx, and CAT, in the hepatic system. As postbiotics possess probiotic characteristics [34,39,40,43,67,68], probiotic studies can provide useful information to understand how postbiotics could improve the antioxidant capability and develop the oxidative resistance in the body under heat stress. Several studies reported that the supplementation of probiotics in poultry diets reduced the adverse effects of oxidative stress and enhanced the activity of antioxidant enzymes [61,69], which might reduce cell damage by inhibiting the production of ROS and finally improving the health of animals [70,71]. Our results were consistent with Shen et al. [72] who reported that blood antioxidant capacities were significantly intensified by the incorporation of probiotic L. plantarum in the diets and promoted growth performance in broilers.
This study is the first attempt to provide data on the effect of postbiotics on antioxidant activities in heat-stressed broilers. However, probiotics have been reported for their ROS removal capacity and promoting broiler health under normal [61] and high-temperature conditions [73].
Generally, broiler chickens are characterised by their high lipid contents which increase the exposure to lipid peroxidation, which could harm the body due to ROS production to maintain nutritional and physiological demands. Lipid peroxidation is a sequela of reduced antioxidant protection as the production of free radicals and ROS increases. The MDA level is often used as an endogenous reflection of lipid peroxidation [74].
Antioxidant enzymes such as CAT, GPx, GSH and SOD are vital in scavenging excess ROS at the cellular level, thereby affecting the stability of lipid peroxidation of broiler meat during storage [75]. In this study, there was a significant reduction in MDA content in broilers fed RI11 and UL4 under heat stress compared to negative control and other groups. The postbiotics herein showed probiotic antioxidant benefits and could promote the function of CAT and other antioxidant enzymes. This may be associated to the postbiotic ability to confer effective antioxidant protection in reducing lipid peroxidation during the growth phase of the birds, despite being under heat challenge.
Acute Phase Proteins, HSP70 and Corticosterone
APPs are blood proteins majorly produced in the hepatic system and their concentrations change under conditions such as inflammatory reactions, infections, and tissue injuries [4]. APPs also function to restore homeostasis and prevent microbial proliferation following stimulation by non-specific innate immune cells [76]. In this study, the CPN and α1-AGP concentrations were significantly reduced in the heat-stressed birds fed either with postbiotics (RI11, RS5, UL4) or ascorbic acid (AA) compared to the negative control. These findings indicate the beneficial role of the postbiotics (especially RI11) in improving immune response so as to annul the negative effect of heat stress on the broiler chickens. The capacity of ascorbic acid to produce such a beneficial effect was also illustrated, though the concentrations of the APPs were still higher compared with the RI11 fed birds.
Various authors have reported different response kinetics of APPs in avian species. APPs such as AGP were shown to be faster reacting than CPN upon exogenous administration of corticosterone [77]. In contrast, corticosterone was markedly elevated after 2 h of feed deprivation compared to AGP and CPN which showed similar response only after 30 h [4]. Such discrepancies suggest species differences in either stress exposure or capacity for APPs synthesis.
In this study, despite the lower corticosterone level in broilers fed with postbiotics as compared to the NC, the effect was not significant. Changes in corticosterone levels could be influenced by the duration of heat exposure, and the benefits of postbiotic supplementation might be masked during chronic heat stress. As reported by Sohail et al. [78], broilers supplemented with prebiotic and probiotic mixture recorded lower serum corticosterone concentrations on day 21 following heat exposure compared with the negative control group.
Another important plasma protein is HSP70, as their levels increase during cellular insult, which is useful in predicting the degree of thermal stress in broilers [77]. HSP70 is a type of heat shock protein that is highly conserved and promptly synthesised in response to stressors such as feed restriction, crating, transportation, unpleasant human contact, and elevated temperature [77,[79][80][81].
In this study, the HSP70 level was not significantly different between the heat-stressed broilers supplemented with postbiotics, antibiotics, ascorbic acid, and the negative control. HSP70 acts as a chaperone through interaction between proteins to defend synthesised proteins against additional injury [82]. Hence, HSP70 induction guards against stresses such as hyperthermia, ischemia, and inflammation. For instance, HSP70 levels increased significantly in broilers exposed to heat stress [4,80]. Broilers restricted from feeding had significantly lower HSP70 and S. enteritidis colonization compared with the control group after heat exposure [83]. Ascorbic acid also induced increased body temperature in heat-stressed chickens, which correlated with the HSP70 response [1]. Another study reported that the concentration of HSP70 decreased after exposure the birds to long-term heat stress and this could be that the birds developed tolerance to heat after long heat stress, or due to material insufficiency after long-term stressing [84,85]. These results are inconsistent with the present study, as we observed no difference in HSP70 response following postbiotic and ascorbic acid supplementation. However, the expression of HSP70 could be influenced by various factors including the type of target organ and age of the birds [86]. Yan et al. [87] showed that HSP70 induction is organ dependent following the induction of the protein in heat-stressed chickens. Differences in the management system, breeds, and the degree of thermal stress could explain the inconsistent result between the present study and other related studies.
Plasma Lipid Profile
The birds supplemented with various types of postbiotics had lower total cholesterol and triglyceride levels. Nevertheless, the effect of total cholesterol in the former groups was not different compared with AA fed birds. The present result is in agreement with that of [25] who found decreased cholesterol levels following supplementation with various combinations of postbiotics. Kareem [88] reported that supplementation of postbiotics and inulin in broiler diets decreased serum cholesterol, triglyceride and LDL compared with the antibiotic and negative control group. Similar results were found in the reduction of cholesterol profile in plasma of laying hens [34], post-weaning rats [50] and piglets [89] fed postbiotics produced from L. plantarum.
Our result corroborated that of [35], where plasma cholesterol concentration was reduced in egg yolk following dietary feeding with postbiotics. L. plantarum metabolites have been reported to have a cholesterol-lowering effect in rats [49]. This could be related to the increase in the population of LAB, as found in this study. Through conjugation, the primary bile acids are transformed into secondary products by the intrinsic ability of LAB. Therefore, the volume of bile acid in the intestine is reduced. Another likely pathway for the effect observed in this study is the enhancement of both faecal excretion and turnover of bile acids [49].
Cholesterol is a precursor for bile acid synthesis, which explains the reason for the higher rate of cholesterol break down [90]. Postbiotics used in this study could increase the population of lactic acid bacteria, production of enzymes disintegrating bile salts and de-conjugating them in the gut, as well as reduction of the gut pH that can be efficient in decreasing the cholesterol of blood by reducing non-conjugate bile acids solvability at low pH, leading to less absorption from the intestine and more excretion in the faeces [88]. Other authors have demonstrated that cholesterol levels can be reduced when cholesterol is incorporated into the bacterial cellular membrane [91]. In the present study, postbiotics, especially RI11 supplemented in broiler diets under heat stress, showed the same trend of previous studies in terms of reducing plasma cholesterol profile levels.
Meat Quality
Aside from the health implications, the quality of the meat obtained from the chicken muscle is crucial in the economics of meat processing industries. The water holding capacity (WHC) of meat is indicated by the drip and cooking losses, while the latter determines profit in meat sales [92].
In this study, the higher drip losses in the meat of heat-stressed broilers fed only a basal diet are comparable to earlier works reporting the adverse effect of exposure of broilers to heat stress on meat quality. Both acute and chronic heat stress exposure could negatively affect meat quality [93,94]. The former occurs by alteration in aerobic metabolism, glycolysis, and intramuscular deposition of fat, thus leading to pale meat colour, reduced WHC, and higher shear force [95,96]. Acute heat stress induces changes in blood acid-base status and integrity of muscle membrane [97]. Findings from the present study showed broilers fed with postbiotics had significantly lower drip loss and cooking loss than the NC, OTC and AA fed groups. This is consistent with other studies conducted using postbiotics and probiotics. Although the experiments were conducted under normal ambient temperature, broilers fed combinations of postbiotics and inulin had lower drip loss as compared with control groups [31]. Additionally, dietary probiotics resulted in significant reductions in drip loss in the breast muscle of chickens [98].
Ali [99] also observed no significant change in shear force in breast muscle of broilers fed with or without probiotics. Shearing force in the present study was higher (p < 0.05) in the antibiotic and NC birds as compared with those fed various postbiotics. This might be due to the capacity of the postbiotics to annul the effect of thermal stress in the broilers as it relates to antioxidant enzyme activities. Similar findings were reported in one study based on the lower shear force in the birds fed probiotics [98]. Another study reported that the meat pH, colour, drip loss, cooking loss and tenderness were improved by feeding probiotics which induced the differential expression of carbohydrate metabolism, cytoskeleton, chaperone and transportation proteins. These proteins participate in carbohydrate and energy metabolism as well as tight junction pathways, suggesting roles in the organization of meat quality improvement [100]. The same previous study documented that the improvement in the meat quality traits in the broilers fed probiotic Enterococcus faecium may be attributed to the up-regulation of the protein's related substrate metabolism, antioxidant and immune systems which lead to alterations of the pentose phosphate and citric acid pathways and increase the metabolism of amino acids and improve antioxidant and immune capacity leading to improved muscle biochemical indexes (pH, colour, WHC and texture). The postbiotic inclusion in the broiler diets under heat stress is expected to deliver the same action of probiotics on the meat quality, as mentioned.
Another indicator of meat quality is its pH [101]. The pH value of 6.0 is considered good for broiler breast meat [101]. The pH of the breast meat of all the treatment groups ranged from 5.7-6.0, thus signifying good quality. The pH of the birds fed with postbiotics (RI11, RS5, UL4) was higher (p < 0.05) compared with the PC and NC groups. This is another indication of the role of the postbiotics administered to the heat-stressed birds in maintaining the meat quality. Our observation is different in comparison to the study where dietary supplementation with postbiotics and inulin resulted in significantly lower pH in broiler meat [31]. The fact that the birds in this study were under heat challenge could explain the disparity between our results and the previous study. Nevertheless, since the final pH of the breast meat was within the normal range, the difference offers no significant impact.
Meat colour as an indicator of broiler meat quality was also examined in the present study. Broilers fed postbiotics (UL4, RI11 and RS5) and ascorbic acid (AA) had significantly lower lightness values compared to the negative control and antibiotic group, while birds fed RI11 had the lowest lightness level. Lightness is used as a scale of breast muscle colour [102]. Myoglobin in the meat is responsible for absorbing green light, leading to the more yellowish and less reddish appearance of meat [103]. Therefore, the lower lightness in the treated broiler chickens could be attributed to the higher pH of the muscle [104]. Such colour changes were reported in previous studies following probiotics feeding [99,104,105]. Additionally, the redness level was not significantly different between the treatment groups in the present study, which is consistent with the results of [106]. However, [31] reported changes in redness level in breast meat after combined feeding of postbiotics and inulin.
Based on the review by [97], we suggest there are three main pathways through which postbiotics exert their action in annulling the adverse effects of heat stress on meat quality in broiler chickens. First, is the prevention of a rapid drop in pH which could be detrimental to meat quality. Low pH has been associated with low redness, high drip and cooking losses in breast meat of chickens [107][108][109]. Second, the enhancement of antioxidant activities of GPx, SOD, and catalase, is vital to alleviate the effect of heat stress on meat quality through oxidative damages. The third likely mechanism is lowering the corticoid hormone levels (secretion of corticosterone). Sato et al. [110] reported that corticosterone accelerates ROS production, this could result in pale and high drip loss of broiler meat.
Conclusions
The findings of the current study presented that feeding postbiotics, especially RI11, enhanced the antioxidant activities, meat quality (pH, WHC, colour and tenderness) and reduced acute phase proteins (AGP and CPN), plasma cholesterol and lipid peroxidation in broiler chickens exposed to heat stress conditions. Hence, postbiotics can be used as antioxidant agents in production of poultry under hot weather conditions. | v3-fos-license |
2014-10-01T00:00:00.000Z | 2008-07-19T00:00:00.000 | 26542140 | {
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} | pes2o/s2orc | Ethyl 4-(tert-butylamino)-3-nitrobenzoate
In the title compound, C13H18N2O4, intramolecular N—H⋯O, N—H⋯N and C—H⋯O (× 3) hydrogen bonds generate S(6) and S(5) ring motifs. There are two crystallographically independent molecules (A and B) in the asymmetric unit. The nitro group is coplanar with the benzene ring, with O—N—C—C torsion angles of −0.33 (13) and 0.93 (14)° in molecules A and B, respectively. In the crystal structure, neighbouring molecules are linked together by intermolecular C—H⋯O hydrogen bonds. In addition, the crystal structure is stabilized by π–π interactions with centroid–centroid distances ranging from 3.7853 (6) to 3.8625 (6) Å.
In the title compound, C 13 H 18 N 2 O 4 , intramolecular N-HÁ Á ÁO, N-HÁ Á ÁN and C-HÁ Á ÁO (Â 3) hydrogen bonds generate S(6) and S(5) ring motifs. There are two crystallographically independent molecules (A and B) in the asymmetric unit. The nitro group is coplanar with the benzene ring, with O-N-C-C torsion angles of À0.33 (13) and 0.93 (14) in molecules A and B, respectively. In the crystal structure, neighbouring molecules are linked together by intermolecular C-HÁ Á ÁO hydrogen bonds. In addition, the crystal structure is stabilized byinteractions with centroid-centroid distances ranging from 3.7853 (6) to 3.8625 (6) Å .
Comment
As a part of our ongoing studies on new nitro benzoic acid derivatives, we have synthesized the title compound (I) employing a modified protocol of previous procedure (Göker et al., 1998). The nitro benzoic acid intermediates are a convenient starting material for the synthesis of heterocycles targeting important biological processes, e.g. antifungal (Fluconazole) (Anderson, 2005) and proton pump inhibitor (Omeprazole) (Kakei et al., 1993). The crystal structure of the tert-butylamino functionalized nitro benzoic acid (I) has been determined, and herein, we present a full report on its crystal structure.
In the title compound (I) (Fig. 1), intramolecular N-H···O (x 2), N-H···N (x 2), and C-H···O (x 3) hydrogen bonds (Table 2) generate S(6) and S(5) ring motifs, respectively (Bernstein et al., 1995). There are two crystallographically independent molecules, A and B in the asymmetric unit of the title compound. The nitro group is coplanar with the benzene ring with torsion angle of -0.33 (13) and 0.93 (14)° in the molecule A and B, respectively. In the crystal structure neighbouring molecules are linked together by intermolecular C-H···O hydrogen bonds (Table 1). In the crystal packing (Table 2 & Fig. 2), molecules are stacked down the b axis, being consolidated by π-π interactions with centroid to centroid distances ranging from 3.7853 (6) -3.8625 (6) Å.
Experimental
Ethyl 4-fluoro-3-nitrobenzoate (200 mg, 0.93 mmol) was dissolved in dry dichloromethane (10 ml). N, N-diisopropylethylamine (DIPEA) (0.20 ml, 1.12 mmol) was added to the stirred mixture. Then, tert-butylamine (0.11 ml, 1.03 mmol) was added dropwise using syringe and stirred at room temperature under N 2 overnight. After completion of the reaction, the mixture was washed with 10% NaCO 3 (10 ml). The aqueous layer was washed with dichloromethane (3 x 15 ml). The organic layers were collected and dried over MgSO 4 (anhydrous). The solvent was removed under reduced pressure to yield the crude product. Recrystallisation with hot hexane revealed the title compound (I) as bright yellow crystals.
Refinement
The H-atoms attached to N2A and N2B were located from the difference Fourier map and refined as riding with the parent atom with an isotropic thermal parameter 1.2 times that of the parent atom. The rest of the hydrogen atoms were positioned geometrically [C-H = 0.95-98 Å] and refined using a riding model. A rotating-group model was used for the methyl groups. The highest peak is located 0.63 Å from C6B and the deepest hole is located 0.59 Å from N1A. Ethyl 4-(tert-butylamino)-3-nitrobenzoate | v3-fos-license |
2017-08-03T01:36:17.847Z | 2011-12-12T00:00:00.000 | 9596191 | {
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} | pes2o/s2orc | Decrease in the production of beta-amyloid by berberine inhibition of the expression of beta-secretase in HEK293 cells
Background Berberine (BER), the major alkaloidal component of Rhizoma coptidis, has multiple pharmacological effects including inhibition of acetylcholinesterase, reduction of cholesterol and glucose levels, anti-inflammatory, neuroprotective and neurotrophic effects. It has also been demonstrated that BER can reduce the production of beta-amyloid40/42, which plays a critical and primary role in the pathogenesis of Alzheimer's disease. However, the mechanism by which it accomplishes this remains unclear. Results Here, we report that BER could not only significantly decrease the production of beta-amyloid40/42 and the expression of beta-secretase (BACE), but was also able to activate the extracellular signal-regulated kinase1/2 (ERK1/2) pathway in a dose- and time-dependent manner in HEK293 cells stably transfected with APP695 containing the Swedish mutation. We also find that U0126, an antagonist of the ERK1/2 pathway, could abolish (1) the activation activity of BER on the ERK1/2 pathway and (2) the inhibition activity of BER on the production of beta-amyloid40/42 and the expression of BACE. Conclusion Our data indicate that BER decreases the production of beta-amyloid40/42 by inhibiting the expression of BACE via activation of the ERK1/2 pathway.
BER can pass through the blood-brain barrier and reach the brain parenchyma in a dose-and time-dependent manner [24], and has multiple neuropharmacological properties including neuroprotective and neurotrophic effects. It also stimulates anti-neuronal apoptosis, improves cerebral microcirculation, reduces depression, and inhibits acetylcholinesterase [25][26][27]. Notably, one study [28] has reported that BER can decrease the production of Aβ 40/42 , but the mechanism remains unclear. Further investigation of how BER inhibits the expression of BACE may have significant impact on the treatment of AD. In this study, we therefore focused on the mechanism of BER on BACE and Aβ 40/42 inhibition, using HEK293 cells stably transfected with APP695 containing the Swedish mutation.
Results
Effects of BER and U0126 on the proliferation and cytotoxicity of HEK293 cells The MTT assay was used to detect the treatments on the proliferation of HEK293 cells. Relative to the vehicle group, no significant declines were observed in the cells receiving treatments (P > 0.05) ( Figure 1A, B and 1C). The LDH release of cultured medium was used to assay the treatments for the cytotoxicity of HEK293 cells. Compared with vehicle treatment, BER and U0126 showed no significant effects on the release of LDH in the culture medium (P > 0.05) ( Figure 1D, E and 1F), but 3% H 2 O 2 significantly increased the release of LDH in the culture medium (P < 0.01).
Effects of BER and U0126 on the expression of BACE
We assayed the expression of BACE in HEK293 cells by WB. BER (1 μM, 5 μM, 10 μM, and 20 μM) was found to significantly reduce the expression of BACE for 48 hours of incubation ( Figure 3A). BER (5 μM) was found to significantly reduce the expression of BACE for 8 hours, 24 hours, 48 hours, and 72 hours of incubation ( Figure 3B). However, U0126 (0.5 μM) was found to significantly increase the expression of BACE and alleviate the inhibition of BER (5 μM) on the expression of BACE (Figure 3C).
Discussion
In this study, we observed that BER significantly decreased the production of Aβ 40/42 and the expression of BACE via activation of the ERK1/2 pathway in a dose-and time-dependent manner. We also found that U0126, an antagonist of ERK1/2 pathway, abolished the effects of BER on both Aβ 40/42 and BACE. BER had previously been demonstrated to be able to reduce cancerous conditions by inhibiting the proliferation of tumor cells [29,30], but we did not find that BER could inhibit the proliferation and show cytotoxicity toward HEK 293 cells by MTT and LDH assays. From this, it can be concluded that the inhibition of BER on the production of Aβ 40/42 is not associated with the anti-proliferative or cytotoxic qualities of BER.
The enzyme BACE is crucial to the production of Aβ 40/42 and the expression of BACE increases in the brains of AD patients [5]. For this reason, BACE has been considered as a therapeutic target for AD treatments. On the other hand, the expression and activity of BACE is regulated by the ERK1/2 pathway in a doseand time-dependent manner [31], and BER increases the expression of LDLR and glucose uptake by activating the ERK1/2 pathway [10,15]. So berberine-induced reduction of BACE1 protein levels is related to ERK1 activation. Furthermore, though BER has been shown unable to inhibit the activity of BACE in vitro [25], the ERK1/2 pathway negatively modulates BACE1 activity in vivo [31]. Thus, we think that BER might also decrease the production of Aβ 40/42 by inhibit BACE1 activity via activating ERK1/2 pathway, and it need to be studied in the next study.
At the same time, BER may decrease the production of Aβ 40/42 by affecting the activity of α-secretase and γsecretase. It has been reported that ERK1/2 is an endogenous negative regulator of γ-secretase activity, and NSAIDs can inhibit γ-secretase activity by inhibiting the Rho-ROCK pathway [6,32,33]. BER inhibits tumor cell migration by inhibiting the Rho-ROCK pathway in HONE1 cells [34], so it is possible that BER inhibits the activity of γ-secretase by activating the ERK1/2 pathway and inhibiting the Rho-ROCK pathway. Moreover, BER, an acetylcholinesterase inhibitor, may be able to upregulate α-secretase activity by promoting the translocation of α-secretase to the cell surface [35]. All these possibilities require further study.
Conclusion
In this study, we demonstrated that BER can decrease the production of Aβ 40/42 by inhibiting the expression of BACE via activation of the ERK1/2 pathway. In previous studies, we demonstrated that BER improved impaired spatial memory and increased both the activation of microglia and the expression of insulin degrading enzyme (IDE) in the rat model of AD [36][37][38]. Other researchers have demonstrated other pharmacological effects of BER in HEK293 cells, e.g., inhibiting Aβ 42 aggregation and attenuating the Tau hyperphosphorylation induced by calyculin A [39,40]. Together, we consider BER to be a very promising drug for use in AD patients.
Methods
Cell culture and treatments
MTT analysis
After the cells were treated in the manner described above, 10 μl of 1 mg/ml MTT stock (Sigma, St. Louis, MO, U.S.) were added to each well and the incubation continued for another 4 hours. One hundred microliters of a solution containing 20% SDS and 50% dimethylformamide (pH 4.8) were then added to each well. After overnight incubation, absorption values at a wavelength of 570 nm were determined by spectrophotometer.
Cellular toxicity analysis
HEK293 cells were plated at a density of approximately 1 × 10 4 cells per well on 24-well plates. After 24 hours of incubation, the conditioned media were replaced with new media containing BER, U0126, and BER with U0126 at the final concentrations and the final times indicated. Lactate dehydrogenase (LDH) activity was determined to evaluate the cell toxicity of BER, U0126, and BER with U0126 by using cytotoxicity detection kits (Njjcbio Institute, China) according to the manufacturer's instructions. Hydrogen peroxide (3%) was used as a positive control and added to the conditioned media during the last hour of incubation. The baseline was determined in control wells containing no cells and the values obtained there were subtracted from those obtained from experimental wells.
Sandwich ELISA
HEK293 cells were plated at a density of approximately 4 × 10 4 cells per well on 6-well plates. After 24 hours of incubation, the conditioned media were replaced by new media containing BER, U0126, and BER with U0126 at the final concentrations and final times indicated. The cultured media were harvested and extra cellular Aβ levels were determined by using the Human Aβ 40/42 Assay Kit (Cusabiao Biotech Co., Ltd., U.S.) according to the manufacturer's instructions.
Statistical analysis
All of the data were expressed as mean ± SD and the analysis was carried out using the one way analysis of variance (ANOVA). Values of P < 0.05 were considered statistically significant. | v3-fos-license |
2020-01-05T00:03:03.765Z | 2017-01-01T00:00:00.000 | 209711419 | {
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} | pes2o/s2orc | Differential Effects of Excess Potassium and Sodium on Plant Growth and Betaine Accumulation in Sugar Beet
The effects of long-term NaCl and KCl treatment on plant growth and betaine accumulation were investigated in sugar beet. Leaf fresh weight of the plants treated with 300 mM KCl and NaCl for 15 days were declined when compared with the control plants. Photosynthetic activity, chlorophyll content and magnesium content were decreased in the plants treated with 300 mM KCl, whereas these values were essentially maintained in the control and 300 mM NaCl treated plants. K+ content in 300 mM KCl treated plants were significantly higher than the Na+ content in the 300 mM NaCl treated plants. These effects were more severe in developing leaves than the mature leaves. Betaine and choline monooxygenase (CMO) accumulation levels were increased in plants exposed to 300 mM KCl and NaCl treatment, with a higher increase in NaCl treated plants. The betaine/Na+ ratio increased during the 300 mM NaCl treatment but remained constant during the KCl treatment. Results indicate the presence of a better adaptive system to high NaCl than KCl in developing leaves of sugar beet.
Introduction
Salt stress is a major factor that decreases crops yield, especially in semi-arid and arid areas (Munns and Tester 2008). World over, attempts are being made to explore and grow salttolerant species with potential economic and ecological value in salinized soils (Rozema andFlowers 2008, Flowers andColmer 2015). Sugar beet is an economically important, salt-tolerant and widely distributed plant, which can grow under 300 mMNaCl treatment (Yamada et al. 2009). It accumulates glycine betaine as an osmoprotectant under NaCl stress and is an excellent model plant to study the role of betaine in regulating salt stress. In plants, betaine is synthesized via two steps of choline oxidation: choline→betainealdehyde→betaine (Rathinasabapathi et al.1997, Takabe et al. 2006. These steps are catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively, wherein the first step is rate-limiting.
Potassium (K) is ubiquitous in all higher plants and plays a vital role in a wide range of biochemical and biophysical processes. Typical K + concentration in growth medium is in the range of 1-2 mM (Cakmak 2005). However, most plants accumulate K + too much higher concentrations than required for normal metabolism (Leigh and Wyn Jones 1984), which is termed as luxury uptake (Winkler and Zotz 2010). In plants, the total K + is located in the cell vacuole, while a small proportion is also localized in the cytosol (Leigh and Wyn Jones 1984). In the past, extensive studies have demonstrated physiological and biochemical implications of K + deficiency in plants (Cakmak 2005, Ashley et al. 2006). Notwithstanding, a few studies have reported the effect of high K + levels in plant system (Ramos et al. 2004, Yao et al. 2010. It has been shown that 300 mM KCl for Chenopodium album (Yao et al. 2010) and 350-500 mM KCl for Atriplex nummularia (Ramos et al. 2004) were more toxic than the treatments with the same concentrations of NaCl. In agricultural land, 300 mM KCl might be too high. But, saline water, containing >400,000 ppm total salts, has been reported (Egan and Ungar 1998). In addition, over-fertilization of soils used for agricultural and horticultural purposes is a growing environmental concern. Over-use of compost, manure or other organic materials can cause adverse effects on plant growth and the environmental contamination in drinking water. Considering the remediation of soil by halophyte plants and sea water farming, high KCl treatment would be interesting. In addition, it is uncertain whether high K + can induce the accumulation of organic osmoprotectant.
With above background, we conducted a series of experiments to investigate the differential effects of high K + and Na + on the plant growth and betaine biosynthesis in sugar beet.
Seed germination, plant materials and salt treatments
Seeds of sugar beet (Beta vulgaris L., cv. NK-210mm-0) were germinated on paper towels moistened with distilled water under dark conditions at 25°C. After germination, seedlings were transplanted into plastic pots (100 mL) containing sterile vermiculite. Plants were grown with half strength Murashige and Skoog solutions (½ MS) in a growth chamber set at 16 h light (25°C, 100 µE m -2 s -1 )/8 h dark (20°C) cycle and 60% relative humidity (Yamada et al. 2009). When seedlings were 3 weeks old, these were transferred to the growth medium containing various concentrations of NaCl (0, 50, 100, 200, 300 and 400 mM) and KCl (0,10,50,100,200,300 and 400 mM). The plants were allowed to grow further for next 30 days. One hundred millilitres of ½ MS solution containing various concentrations of the salts were applied to the culture medium at 2 days interval. True leaves of first, second and third pairs, designated as mature (L1), developing (L2) and young (L3) leaves, were used for further biochemical estimations. Plant cultivation was carried out at least three biological replicates.
Determination of leaf succulence and water content
The degree of leaf succulence was calculated as the ratio of initial fresh weight to the dry weight (Yao et al. 2010). Four leaf discs (2 discs from tip region, 2 discs from basal region) per leaf were cut with a cork borer (ϕ=1 cm). Leaf discs were then oven-dried for at least 5 days at 55°C in a glass Petri dish. Dry weight was determined by an analytical weighing balance (Shimadzu Co, Japan).
Measurement of total chlorophyll content and photosynthetic activity
For the measurement of chlorophyll content, 50 mg leaves were homogenized in 1.8 mL of 100% acetone. After centrifuga-tion at 20,000 × g at 4°C rotor temperature for 15 min, the absorbance of supernatant was read at 646.6, 663.6 and 750 nm using a spectrophotometer (BioSpec-1600, Shimadzu) (Yamada et al. 2015). Photosystem II activity (PSII activity) was measured by a portable Mini PAM (Pulse Amplitude Modulation) fluorometer (PAM-2000, Walz, Germany) at 25°C using 30 min dark adapted leaves and data acquisition software (DA-2000, Walz) (Hoshida et al. 2000).
Measurement of ions and osmotic concentration
Fresh leaves (200 mg) were homogenized in eppendorf tubes 25°C, and centrifuged at 20,000 × g at 4°C rotor temperature for 15 min. Osmotic concentration in aliquots (10 µL) of the supernatant was analyzed by a vapor pressure osmometer (model 5520; Wescor, Logan, Utah, USA). Cellular ions were determined using Shimadzu Personal Ion Analyzer PIA-1000 (Shimadzu, Japan) as described previously (Waditee et al. 2007). For Na + and K + extraction, 100 mg fresh leaves were homogenized in 1 mL of sterile distilled water. For Mg 2+ extraction, fresh leaves were dried at 60°C for 1 week. The dried leaves were ground in a mortar and 20 mg of powdered material was extracted with 13 M HNO 3 for 1 h. After air-drying, it was re-extracted with sterile distilled water. Then, the contents were centrifuged at 20,000 × g at 4°C for 15 min and the supernatant was used for the measurement of Mg 2+ .
Analysis of betaine
Betaine was extracted as described by Waditee et al. (2005). The plant tissue (100 mg FW) was extracted in an extraction buffer (methanol:chloroform: water = 12:5:1) and centrifuged at 20,000 × g at 4°C for 15 min. The supernatant was re-extracted with a mixture containing 25% (v/v) chloroform and 37.5% (v/v) water and again centrifuged at 20,000 × g for 15 min. The supernatant, thus obtained, was dried and dissolved in 100 µL water. Betaine was measured with the time of flight mass spectroscopy (KOM-PACT MALDI TOF-MS, Shimadzu/Kratos) using d 11 -betaine as the internal standard.
CMO protein expression and statistical analysis
SDS-PAGE and immune-blot analysis were carried out according to the standard protocol as described by Waditee et al. (2005). Protein contents were determined by Bradford method (Waditee et al. 2007). All values are presented as mean±standard errors of three replicates.
Effects of KCl and NaCl on the growth of sugar beet
The inclusion of 10-50 mM NaCl or KCl in the growth medium enhanced the growth, but at higher concentrations (>300 mM), the growth was inhibited, compared with the control plants (Fig. 1). The enhanced growth of sugar beet at lower concentrations of NaCl has also been reported previously (Russell et al. 1998, Liu et al. 2008, Wu et al. 2013. The growth medium of the control essentially did not contain Na + , but contains about 1 mM K + due to a major salt, KNO 3 . After 30 days, it has been found that plants treated with 400 mM NaCl survived, whereas those treated with ≥300 mM KCl were almost dead, indicating that growth inhibition by high KCl concentrations was more severe than that of NaCl. Figure 1: Photographs of sugar beet after KCl and NaCl treatments. Sugar beet was allowed to grow for 3 weeks before treating with various concentrations of NaCl and KCl. The plants were observed and photographed for next 30 days. The bar represents 10 cm. In the following experiment, the concentration of KCl and NaCl was fixed to 300 mM and the time course of fresh weight and succulence degree of L1 (mature leaf) and L2 (developing leaf) of B. vulgaris were determined. Fresh weight of L1and L2 leaves was decreased when treated with 300 mM NaCl or KCl as compared to control ( Fig. 2A). The degree of succulence of L1 leaves increased with increasing the time period of treatment, irrespective of the mode of treatment (Fig. 2B). In contrast, the degree of succulence of L2 leaves treated with 300 mM KCl increased significantly after 10 and 15 days as compared to control and NaCl treated plants. In control plants, leaf area of L1 remained constant whereas leaf area of L2 increased with increasing time period (data not shown). Leaf area of the plants treated with 300 mM of KCl or NaCl was lower than that of the control plants, and the decrease was more severe in Figure 2: Time course of leaf fresh weight and succulence degree of KCl and NaCl treated B. vulgaris. After treating the 3 weeks old seedlings of sugar beet with 300 mM NaCl or 300 mM KCl, fresh weight and succulence degree of its mature leaf (L1) and developing leaf (L2) were measured at different time intervals. The data are presented as mean±SE (n=3). The letters above the graph represent significant difference (P≤0.05) between WT type (white bar), NaCl treated plants (gray bar) and KCl treated plants (dark bar).
High KCl decreased chlorophyll content and photosynthetic activity
The leaf color of L2 leaves treated with 300 mM NaCl was changed to yellowish green (data not shown), indicating a decrease in chlorophyll content. However, when measured, the total chlorophyll content in L1 and L2 leaves of NaCl treated plants were similar to the control leaves (Figs. 3A and 3B). In contrast, the 300 mM KCl treatment reduced the chlorophyll content up to 50% after 10 and 15 days in L2 leaves (Fig. 3C), The maximum quantum yield of PSII (F v /F m ) was decreased after 10 days treatment with 300 mM KCl, whereas it was unaffected by the NaCl treatment (Fig. 3C).
KCl treated plants than in NaCl treated plants (data not shown).
Changes in cation content in response to salt stress
The content of K + increased with increasing the time period of exposure in both L1 and L2 leaves (Fig. 4A). After 15 days, the amount of K + in L1 and L2 leaves treated with 300 mM KCl was 371.2 and 674.8 µmol/gFW, respectively, thereby indicating that K + prefers to accumulate in developing leaves. A similar trend of changes was observed in NaCl treated plants (Fig. 4B); however, the amount of Na + in L1 and L2 leaves was 170.8 and 258.5 µmol/ gFW, respectively, which was 50% lower than K + . The osmolarity of L1 and L2 leaves in NaCl and KCl treated plants increased with increasing time period, whereas that in the control plants was constant. At 15 days after treatment, the osmolarity of L1 and L2 in KCl treated plants was 3.9-and 4.3-folds higher than control plants, respectively, whereas in NaCl treated plants it was 2.3-and 2.3-fold higher than in control plants (data not shown).
After 45 days of treatment with 300 mM KCl, the precipitation of salt was observed which was confirmed to be KCl based on the ionic analysis of the precipitate (data not shown). In contrast, no salt precipitation was observed in B. vulgaris plants treated with 300 mM NaCl, and the plants grew continuously. Since the interactive effects between excess K + and Mg 2+ deficiency has been reported earlier (Pujos andMorard 1997, Farhat et al. 2013), we measured the Mg 2+ content in L1 and L2 leaves after 5 and 15 days of salt treatments (Fig. 5). Mg 2+ contents in L1 leaves did not show a significant change compared to the control plants. However, the
Excess of KCl and NaCl induce greater accumulation of betaine
Betaine content increased with increasing the incubation time of NaCl in L1, L2 and L3 leaves, and the accumulation was the highest in L3 followed by L2 and L1 (Fig. 6A). These results are in conformity with the earlier findings of Yamada et al. (2009). In this study, we found that betaine content was increased under high KCl (300 mM) conditions (Fig. 6A). Betaine content was increased with increasing the incubation time of KCl in L1, L2 and L3 leaves, and the accumulation was the highest in L3 followed by L2 and L1. These results were similar to that by the NaCl treatment, but the betaine accumulation levels were slightly lower than that of the 300 mM NaCl treated plants.
Upon western blotting, no CMO bands were detected in the control plants, whereas these were detected in the L1 and L2 leaves of KCl and NaCl treated plants (Fig. 6B). However, the band intensity of L1 leaves was higher than that of the L2 leaves, and the Figure 6: Changes in CMO and betaine content in leaves of B. vulgaris after KCl and NaCl treatments. A) Time course of betaine content in leaves after KCl or NaCl treatment. Three week old seedlings of sugar beet were grown in the presence of 300 mM KCl or 300 mM NaCl for 1, 3, 5, 10, and 15 days. Then, betaine was extracted from the leaves and measured as described in materials and methods. Within each figure panel, different letters above the bars indicate significantly different means at P≤0.05. Values are expressed as mean±SE (n=3). B) Immunoblotting of CMO. Three-week old seedlings treated with 300 mM KCl or 300 mM NaCl for 15 days were harvested. The proteins were extracted from leaves and anal-Mg 2+ contents in L2 leaves of NaCl and KCl treated plants was reduced compared to the control plants. This reduction was more significant in the KCl treated plants after 15 days of exposure.
band intensity of KCl treated plants was slightly less than that of NaCl treated plants. The ratio of betaine/Na + was increased during the treatment, whereas the ratio of betaine/K + remained nearly constant (Fig. 6C).
ysed SDS-PAGE. BvCMO was detected by immuno-blot analysis using the antibodies raised against spinach CMO. C) Changes of betaine/cation ratio. The betaine/K + and betaine/Na + ratios were calculated based on the data of (A) and (B) and their time course changes were represented.
Discussion
The present data clearly shows that 300 mM KCl concentration resulted in the significant accumulation of K + , i.e. 371 and 675 µmol/g FW in mature (L1) and developing (L2) leaves of B. vulgaris, respectively which corresponds to 2.2 to 2.6-fold higher accumulation than that of Na + . Previously, Ramos et al. (2004) reported 1.3-fold higher accumulation of K + than Na + in the leaves of Atriplex nummularia at 350 mM KCl. This indicates that sugar beet accumulates more K + and lesser Na + than A. nummularia. Using 10 mM of KCl or NaCl, Reimann and Breckle (1993) demonstrated that K + uptake in C. album and C. schraderianum is much higher than that of Na + . The accumulation levels of Na + in C. album and C. schraderianum were relatively low compared to halophilic chenopod Atriplex prostrata (Reimann and Breckle 1993). The present study suggests that the selectivity for Na + and K + uptake in sugar beet is similar to that of C. album and C. schraderianum than the halophilic relative Atriplex spp.
It was found that betaine synthesis was enhanced under high K + conditions (Figs. 6A and 6B). The betaine/K + ratio was almost constant (0.04) during the treatment (Fig. 6C) regardless of the significant increase of K + in the 300 mM KCl treated plants (Fig. 4A). In contrast, the betaine/Na + ratio increased with increasing the time period of 300 mM NaCl treatment, and was found to be >0.1 after 15 days of treatment (Fig. 6C). If we assume that vacuole occupy 90% of cell volume and the preferred location of Na + accumulation is vacuole, whereas betaine is localized in cytosol, then betaine/Na + ratio of 0.1 would satisfy the osmotic balance between vacuole and cytosol. By contrast, K + is localized in both vacuole and cytosol. Then, the accumulation of betaine in cytosol under the 300 mM KCl treatment would be lower than that by the 300 mM NaCl treatment. Lower betaine contents in the 300 mM KCl treated plants were coincided with the lower accumulation levels of CMO protein in the 300 mM KCl treated plants (Fig. 6B). Another possibility is the different sensitivity of CMO promoter to Na + and K + ions. The reason behind low betaine levels in 300 mM KCl treated plants is yet to be clarified.
Sugar beet survived even when it is subjected to high NaCl concentrations (up to 400 mM), but the 300 mM KCl treatment significantly inhibited plant growth (Fig. 1). Morphological changes, such as chlorosis (yellow-green leaf), were evidently found in plants grown under 300 mM KCl treatment, but no major changes were observed under 300 mM NaCl treatment (Fig. 3), leading to increase succulence (Fig. 2B). The leaf chlorophyll contents remained unaffected after exposing to 300 mM NaCl concentra-tion, but decreased significantly when exposed to 300 mM KCl concentration. These observations are paralleled by similar results reported for C. album by Yao et al. (2010). Induction of high levels of reactive oxygen species (ROS) by high KCl treatment was demonstrated in C. album (Yao et al. 2010).
Mg is an important divalent cation within living cells and has a high affinity for water and forms a stable hydrate (Guo et al. 2016). In the cell, Mg exists either in the form of ions or is bound to the substances like ATP and RNA. Mg 2+ content in the plants treated with 300 mM KCl concentrations was drastically reduced (Fig. 5), thereby indicating that high K + inhibits Mg 2+ uptake in plant. It can be assumed that K + and Mg 2+ may possibly compete for the channel system or transporter (Winkler and Zotz 2010).
Previously, it has been reported that Na is excluded by the root, and K is selectively taken up at high rates by the leaves of C. album (Reimann and Breckle 1993). The results of the present study are in conformity with those observed in C. album. Due to higher accumulation of K in developing leaves than in mature leaves (Fig. 4), the damage by high K was more severe in developing leaves. In the field, high salinity is mostly induced by high NaCl concentrations. However, other types of salts such as KCl and K 2 SO 4 also occur naturally in the soils from different regions of the world (Egan and Ungar 1998). Heavy application of K fertilizers may also result in increased salinity of the soils (Cakmak 2013). High K uptake by the plants may result in Mg deficiency in grazing animals, which may induce disorders in the ruminant (Cakmak 2013). These facts indicate the importance to study the effects of high K concentrations on plants. In this study, we showed that the biosynthesis and accumulation of betaine in sugar beet were induced by slightly higher concentrations of KCl, but it was not sufficient for sugar beet to survive under severe KCl conditions. | v3-fos-license |
2016-05-12T22:15:10.714Z | 2009-06-04T00:00:00.000 | 9675166 | {
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} | pes2o/s2orc | SOD3 Reduces Inflammatory Cell Migration by Regulating Adhesion Molecule and Cytokine Expression
Inflammatory cell migration characteristic of ischemic damages has a dual role providing the tissue with factors needed for tissue injury recovery simultaneously causing deleterious development depending on the quality and the quantity of infiltrated cells. Extracellular superoxide dismutase (SOD3) has been shown to have an anti-inflammatory role in ischemic injuries where it increases the recovery process by activating mitogen signal transduction and increasing cell proliferation. However, SOD3 derived effects on inflammatory cytokine and adhesion molecule expression, which would explain reduced inflammation in vascular lesions, has not been properly characterized. In the present work the effect of SOD3 on the inflammatory cell extravasation was studied in vivo in rat hind limb ischemia and mouse peritonitis models by identifying the migrated cells and analyzing SOD3-derived response on inflammatory cytokine and adhesion molecule expression. SOD3 overexpression significantly reduced TNFα, IL1α, IL6, MIP2, and MCP-1 cytokine and VCAM, ICAM, P-selectin, and E-selectin adhesion molecule expressions in injured tissues. Consequently the mononuclear cell, especially CD68+ monocyte and CD3+ T cell infiltration were significantly decreased whereas granulocyte migration was less affected. According to our data SOD3 has a selective anti-inflammatory role in ischemic damages preventing the migration of reactive oxygen producing monocyte/macrophages, which in excessive amounts could potentially further intensify the tissue injuries therefore suggesting potential for SOD3 in treatment of inflammatory disorders.
Introduction
The inflammatory process is initiated by endothelial cell (EC) activation comprising upregulation of chemokines and cell adhesion molecules, leukocyte activation and transmigration, and secretion of proinflammatory factors by leukocytes [1]. The inflammatory reaction is necessary for tissue recovery as it provides the correct cytokine signals and cell machinery to clear up the site for regeneration of the tissue [2]. However, uncontrolled inflammation has unfavorable effects on the course of tissue healing since the inflammatory cells are also capable of inducing tissue damage [2] and therefore many conditions involving inflammation, e.g. autoimmune diseases and tissue transplantations, are treated with immunosuppressants to reduce harmful leukocyte infiltration. Among the most potent drugs are glucocorticoids that downregulate the expression of numerous inflammatory chemokines, cytokines and adhesion molecules [3][4][5], which, however, are not entirely without adverse effects such as delayed myocardial tissue healing, osteoporosis, and blood vessel calcification [6][7][8][9].
The most prominent outcome in the initial phase of inflammation is the enhanced production of cytokines, such as TNF-a and IL-1, and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) [10,11], which further induce expression of a number of inflammatory cytokines [4]. Many of the stimulus-specific pathways converge in the production of superoxide (O 2 2 ) and hydrogen peroxide (H 2 O 2 ) as signal mediators, which in turn result in e.g. NFkB activation responsible for numerous stress-related functions [12][13][14]. Leukocytes are thus recruited by expression of various cell adhesion molecules, e.g. selectins, and intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1, respectively) [15,16]. They promote rolling and firm adhesion of leukocytes to endothelial wall, the necessary interactions preceding transmigration [17]. To aid leukocyte migration the vessel wall cells change their morphology by assuming cytoskeletal and cell-cell junction modifications in response to e.g. ligand binding to ICAM-1 and VCAM-1 [18][19][20], and when stimulated by O 2 2 or TNF-a [21,22]. Previously, it has been shown that extracellular superoxide dismutase (SOD3) can attenuate tissue damage and inflammation but so far its mechanism of action has not been completely defined [23][24][25][26][27]. Since excess inflammation prevents the tissue injury recovery we investigated in the present study the effect of SOD3 overexpression on cell migration. We used two in vivo acute inflammation models to determine how SOD3 affects leukocyte extravasation, and compared the results to efficacy of the glucocorticoid immunosuppressant dexamethasone. The mouse peritonitis and rat hind limb ischemia models have been characterized previously: they induce rapid infiltration of leukocytes to the peritoneal cavity and large femoral muscles, respectively [28][29][30]. We then analyzed the proportions of the infiltrated leukocyte subtypes, and determined the effects on several mediators of the inflammatory reaction.
Ischemia model
Fischer 344 rats (Harlan, Horst, Netherlands) and Balb/C mice (local colony) were maintained in specific pathogen-free conditions and had access to food and water ad libitum. All experimental procedures were approved by the Experimental Animal Committee of University of Turku.
Ischemic hind limb injury was induced to male Fischer 344 rats (5 to 6 weeks old, 86-115 g) by surgical closure of the distal femoral artery, lateral circumflex femoral artery, and the proximal femoral artery. The animals were anesthetized for the procedure by intra peritoneal administration of fentanyl fluanisone (Janssen Pharmaceutica, Beerse, Belgium) and midazolame (Roche, Basel, Switzerland). Gene transfer was done immediately after the ligation by intra muscular injection of 0.5610 9 pfu adenovirus SOD3 (AdSOD3) or LacZ (AdLacZ) in 50 ml PBS as described [27,31,32]. Uninjured muscle tissue was used as control.
Peritonitis model
Gene transfer was done to 8-10 week old female balb/c mice with intra peritoneal injection of 0.5610 9 pfu AdSOD3 or AdLacZ. Acute peritoneal inflammation was induced 72 hours later by i.p. injection of 1 ml PBS containing 5% proteose peptone (BD Difco, Sparks, MD, USA) and 10 ng of IL-1b (R&D Systems, Minneapolis, MN, USA). As a control treatment, animals were given 50 mg/kg of Dexamethasone (Oradexon, Organon, Oss, Holland) half an hour before proteose peptone injection. Cells from the peritoneal cavity were collected 18 hours after the induction of inflammation by washing with 10 ml of RPMI containing 5 U/ml heparin (Løvens Kemiske Fabrik, Ballerup, Denmark). Cells from peritoneal lavage were counted and cytocentrifuged at 1000 rpm for 5 minutes (Shandon cytospin 3, Shandon, Pittsburgh, PA, USA). Slides were stained with Reastain Diff-Quick (Reagena, Toivala, Finland) to analyze different leukocyte subtypes.
Immunohistochemistry
Rat muscle samples were frozen in liquid nitrogen and embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Ten micrometer sections were fixed in acetone and stained with rabbit anti-rat CD3 and mouse anti-rat CD68 (Serotec, Oxford, UK). The sections were counterstained with hematoxylin/eosin (Sigma, Saint Louis, MI, USA). The number of CD3 + and CD68 + cells were analyzed from whole sections with Zeiss Axiovert 200 M (Carl Zeiss, Oberkochen, Germany).
Reporter assay
HEK 293T cells were used for in vitro assay to provide a general cell model that we have previously used in our reporter, expression, and cell signalling studies allowing the comparison of the data with our previous results. HEK 293T cells were transfected with SOD3 expression vector together with pNFkB Luc reporter (Stratagene, Cedar Creek, TX, USA). Luciferase activity was quantified with Tecan Ultra XFluor4 Fluorescence Reader (Tekan, Mannedorf, Switzerlard).
Quantitative PCR
Total RNA was extracted from a pool of four animals using Trireagent (Sigma, Saint Louis, MI, USA). The first strand synthesis was done with Revert-Aid M-MuLV (Fermentas, Burlington, Canada), and the following quantitative PCR with SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Primers and cycling conditions are shown in the Table 1.
Statistical Analysis
All results are expressed as mean6SEM. A paired t-test was used to determine differences between groups.
SOD3 inhibits leukocyte migration in acute ischemia
Neutrophils, macrophages, and other inflammatory cells mediate a number of important cellular functions in injured tissue [33,34]. Phagocytotic macrophages clear cellular debris and secrete inflammatory cytokines such as MIP-2, a strong neutrophil attracting agent leading to further increase in inflammatory signaling [35]. However, excessive inflammatory reaction may also contribute to tissue damage by enhancing macrophage infiltration, which increases tissue free radical load leading to further tissue injury [36]. In addition, decreased neutrophil accumulation leads to reduced infarct size, reduced vascular permeability, and resistance in ischemia/reperfusion (I/R) injury [37,38]. Thus, it is suggested that the tissue recovery is dependent on the factors regulating the inflammatory cell migration into the injuries. In the present work we studied leukocyte migration in acute ischemia and peritonitis models and analyzed the contribution of SOD3 on inflammatory cytokine and adhesion molecule expression.
We determined the effect of SOD3 overexpression on the degree of inflammation by analyzing the size of inflamed tissue and the number of infiltrated macrophages and T cells in acute ischemic injury model. In a mouse model of femoral artery ligation, macrophage infiltration into ischemic muscle reaches peak values 3 days after the injury [39]. Histological analysis of the rat hind limb ischemia showed 3-fold reduction in the inflamed tissue area as determined by the presence of CD68 + macrophages (p,0.001) in SOD3 vs. LacZ control animals 3 days after vessel ligation ( Figure 1). The reduction became even more prominent in later time points reaching 12-fold difference 10 days after vessel ligation. Additionally, the number of infiltrated CD68 + macrophages was 3-5 fold higher in LacZ control animals as compared to SOD3 animals (p,0.05). Maximal macrophage accumulation to the injured tissue was seen at 7-day time point in LacZ animals indicating that the inflammatory reaction was still developing in control animals at the initial phase of the follow-up period while the inflammation was decreasing in SOD3 animals. Throughout the experiment the number of macrophages remained higher in control animals than initially observed in SOD3 animals, which by the 10-day time point showed values close to the background levels further underlining the beneficial effect of SOD3. As compared to neutrophils and macrophages, the role of CD3 + T-cells in recovery of ischemic tissue has remained uncertain. Despite relatively low level of infiltration, studies suggest an early role for T-cells in attracting neutrophils and macrophages to site of myocardial or peripheral ischemia/reperfusion injury [40,41].
SOD3-mediated inhibition of leukocyte accumulation in peritonitis model
To confirm the findings and to further analyze the SOD3-derived selective inhibition of cell migration we examined leukocyte migration in a mouse peritonitis model, which provides an efficient way to analyze leukocyte traffic in an acute inflammatory response. To induce peritoneal inflammation we used 5% solution of proteose peptone supplemented with IL-1b and counted the numbers of different leukocyte subtypes from the peritoneal lavage 18 hours after induction of inflammation. The analysis of SOD3 overexpression derived inhibition of cell migration showed 20% (p = ns), 67% (p,0.001), and 33% (p,0.05) reduction in migrating neutrophils, monocytes/macrophages, and lymphocytes, respectively ( Figure 3B). Moreover, the total number of infiltrated leukocytes was decreased by 30% in SOD3 animals as compared to LacZ controls ( Figure 3A) (p,0.01), which is mostly caused by the effect of attenuated macrophage migration. The data effectively confirmed our findings in rat hind limb ischemia showing vastly stronger inhibition of macrophage infiltration as compared to other leukocyte subtypes.
Dexamethasone, a corticosteroid that reduces swelling and inflammation is a potent anti-inflammatory drug used to treat many bacteria-free inflammatory conditions, including rheumatoid arthritis and anaphylactic shock. Glucocorticoids exert their anti-inflammatory effect e.g. through repression of NF-kB mediated cytokine expression, which takes place after cytoplasmic glucocorticoid receptor translocates into the nucleus [5,42]. To compare SOD3 to clinically approved medication we gave an intra-peritoneal injection of dexamethasone (Oradexon) to animals 30 minutes before induction of peritoneal inflammation. Leukocyte traffic to the inflamed peritoneum was reduced by 20% (p,0.05) after treatment with 50 mg/kg dexamethasone ( Figure 4A). Monocyte/macrophage migration was reduced by 60% (p,0.01), and that of lymphocytes by marked 50% indicating tendency, while no significant difference was seen in neutrophil accumulation in this setting ( Figure 4B). PBS mock treated animals exhibited lower neutrophil and monocyte accumulation as compared to animals subjected to LacZ gene transfer, which may be result of the adenovirus vector used in the study. Intriguingly, SOD3 treatment reduced peritoneal monocyte and lymphocyte numbers to similar level as dexamethasone treatment although neutrophil numbers remained higher than what was observed in either PBS or dexamethasone treated animals.
SOD3 inhibits NF-kB activation and suppresses the inflammatory cytokine and adhesion molecule expression
Proinflammatory stimuli activate vascular endothelium leading to up-regulation of cell adhesion molecules and chemokines, NF-kB has been shown to be both necessary and sufficient for endothelial up-regulation of ICAM, VCAM, and MCP-1 [43]. Furthermore, ectopic expression of IkBa effectively abrogates expression of VCAM, IL-1, and IL-6 [44]. In vitro luciferase assay revealed 50% (p,0.01) decrease in NF-kB activity due to SOD3 transfection, which could at least partially be explained by increased IkBa expression ( Figure 5A) suggesting that SOD3 promotes cytoplasmic localization of NF-kB rendering it incapable of binding DNA. NF-kB plays a central part in responses to inflammatory signaling by regulating the expression of cytokines suggesting that reduced NF-kB activity could lead to reduction in expression of inflammatory cytokines and chemokines. Therefore, we quantified cytokine and chemokine expression level in vivo from rat muscle three days after vessel ligation and SOD3 gene transfer. Quantitative RT-PCR showed significantly reduced expression of TNFa, IL1a, IL6, MIP2, and MCP1 ( Figure 5B) in SOD3 animals suggesting reduced expression of several important inflammatory mediators. Specifically, MCP1 is an important macrophage attractant [45,46], possibly explaining markedly reduced macrophage accumulation. Furthermore, since TNFa, IL1a, and IL6 are important regulators of endothelial adhesion molecule expression we analyzed expression of ICAM, VCAM, E-selectin, and P-selectin from the tissue ( Figure 5C). We found significant reduction in adhesion molecule expression, which further confirms the reduction in overall inflammation in the muscle of SOD3 recipient rats as compared to LacZ control animals.
Discussion
Tissue damage launches rapid recruitment of inflammatory leukocytes into injured tissue due to activation of endothelial cells. Inflammatory reaction promotes tissue healing by eliminating pathogens, clearing cellular debris, and promoting cell proliferation. However, excessive inflammatory reaction promotes injury e.g. through neutrophil-derived superoxide production [47]. In fact, reactive oxygen species function as inflammatory mediators by activating expression of cytokines such as TNFa, IL-1, and IL-6 [48] and therefore ROS may contribute to tissue injury by not only directly damaging the tissue but also by enhancing further leukocyte accumulation.
In the current work we showed that SOD3 is an important mediator of reduced CD68+ macrophage migration into the inflammatory area. Macrophages accumulate in high numbers to ischemic muscle forming the primary leukocyte population three days after injury [39]. CD68 staining showed significantly reduced inflammatory area and macrophage migration in SOD3 treated ischemic tissue as compared to LacZ control animals (Figure 1). The SOD3-mediated reduction in macrophage accumulation was evident in all of the studied time points. T-cells accumulate to ischemic muscle in vastly lower numbers as compared to macrophages. However, their presence is required for efficient neutrophil traffic, and they attract macrophages by secreting IL-16 [40]. In our studies, SOD3 did not prevent initial low level T-cell migration, but efficiently inhibited further increase at 10-day time point (Figure 2). Late effect on T-cell migration suggests an indirect mechanism for SOD3 mediated inhibition in T-cell traffic, which might be result of general decrease in inflammation. Inflammatory cytokines secreted by infiltrating macrophages attract other leukocytes to injured tissue [49]. Thereafter, inhibition of macrophage infiltration could lead to overall reduction in inflammatory reaction.
Since the SOD3-derived reduction in inflammation showed selective inhibition of macrophage migration, we were prompted to confirm the finding and to better characterize the cell specific effect. The mouse peritonitis model supported the SOD3-derived reduction in the number of infiltrating leukocytes ( Figure 3A), which was predominantly due to reduced macrophage numbers ( Figure 3B). These results confirm the anti-inflammatory property of SOD3 and show a stronger inhibition of monocyte migration as compared to other analyzed leukocyte subtypes. The data suggest that reduced superoxide tissue concentrations caused by SOD3 overexpression may explain the anti-inflammatory effect of the enzyme. It has been previously shown that superoxide treatment of rat cerebral endothelial cells increases monocyte adhesion and migration, which, however, was not replicated by H 2 O 2 treatment but was instead abrogated by superoxide scavengers suggesting superoxide as an inflammatory mediator [21]. We have shown in our previous works that SOD3 overexpression in vivo efficiently decreases the production of superoxide in cardiovascular injuries including our hind limb ischemia model [25][26][27].
Anti-inflammatory medications currently available for clinical use include glucorticoid drugs such as dexamethasone. Dexamethasone binds the glucocorticoid receptor, which subsequently translocates to the nucleus and represses inflammatory gene expression by inhibiting e.g. NF-kB activity [4,5,42]. To compare the efficacy of SOD3 mediated anti-inflammatory effect to existing medication we determined the effect of Dexamethasone in mouse peritonitis. As a dose of 50 mg/kg, dexamethasone reduced leukocyte traffic in comparable levels to SOD3 gene transfer ( Figure 4A). Dexamethasone-mediated effect was equally effective in monocyte/macrophage and lymphocyte lineages whereas no significant effect was seen in neutrophils ( Figure 4B). Neutrophil accumulation has been shown to be at its highest as early as 4 hours after induction of inflammation in zymosan induced peritonitis [50]. Therefore, lack of effect on neutrophil migration could be due to late time point analyzed. The data suggests that SOD3 overexpression and dexamethasone administration have similar anti-inflammatory effect in acute inflammation and therefore suggesting SOD3 as a potential candidate molecule for clinical treatments.
NF-kB plays a crucial role in mediating inflammation due to its role in activating expression of pro-inflammatory genes such as cytokines TNFa and IL1a, and adhesion molecules ICAM-1 and VCAM-1 [43,44]. Since NF-kB is a redox sensitive transcription factor being activated by oxidative stress, [12,13] we analyzed the effect of SOD3 on NF-kB activity in vitro and showed significantly decreased activity. ( Figure 5A). The data is in line with previous work in cardiovascular and liver transplantation models showing that increased NAPDH oxidase-derived superoxide production correlates with increased NF-kB activity, which is attenuated by SOD3 overexpression [51][52][53][54].
Since cytokines TNFa, IL1a, IL6, MIP2, and MCP1 are known to contain NF-kB binding sites in their gene promoters and are thus up-regulated by NF-kB activation [55][56][57][58][59][60][61], we analyzed their expression levels in rat muscle by quantitative PCR. All of the analyzed cytokines and chemokines were significantly downregulated in SOD3 animals as compared to LacZ control animals ( Figure 5B). TNFa, IL1a, and IL6 promote inflammatory cell migration by up-regulating E-selectin, P-selectin, ICAM, and VCAM. Furthermore, macrophage recruitment has been shown to be strongly dependent on MCP-1 secretion [46], while MIP2 is a strong attractant for neutrophils [35]. MCP-1 deficiency does not reduce the number of resident macrophages in peritoneal cavity, but prevents macrophage migration in response to acute thioglycollate induced peritonitis [46]. Lu et al. showed 3-fold reduction in macrophage migration in 2,4-dinitro-1-fluorobenzene induced skin hypersensitivity model while neutrophil numbers remained unchanged. Thereafter, marked down-regulation of MCP-1 seen in ischemic muscle could explain the observed strong macrophage inhibition. Finally, due to reduced inflammatory cytokine expression, we conducted further expression analyses and found reduced expression VCAM, ICAM, E-selectin, and Pselectin ( Figure 5C). Reduced expression of common adhesion molecules highlights the anti-migratory role of SOD3. It has been shown that macrophage transmigration is strongly dependent on a 4 b 1 integrin -ICAM-1 interaction. Pre-treatment of recipient mice before intra venous macrophage injection with monoclonal antibodies for ICAM-1 reduced macrophage migration to atherosclerotic plaques by 65% [62]. In addition, rolling and attachment of P388D1 mouse monocyte cell line was inhibited by P-selectin and VCAM antibodies in an ex vivo isolated perfused carotid artery model [63] demonstrating the importance of these adhesion molecules on macrophage transmigration.
In conclusion, our novel observation shows that SOD3 gene transfer into hind limb ischemia or peritonitis results in significantly reduced leukocyte migration due to decreased cytokine and adhesion molecule expression. Further on, the data suggest more pronounced anti-inflammatory effect on macrophages as compared to other leukocyte subtypes in the models used in the current work. The observed anti-inflammatory effect in SOD3 treated mice was comparable or even higher than that of Dexamethasone, which recently has been shown to have cardiovascular side effects [64,65]. Our previous in vivo SOD3 overexpression models have suggested non-toxicity and beneficial effect on tissue protection and injury recovery [24][25][26]31] suggesting that SOD3 overexpression by exogenous administration or through increased endogenous production in injured tissues could provide a promising medication against excess inflammatory cell migration. | v3-fos-license |
2016-05-01T08:53:11.475Z | 2015-11-10T00:00:00.000 | 4641910 | {
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} | pes2o/s2orc | Murine versus human apolipoprotein E4: differential facilitation of and co-localization in cerebral amyloid angiopathy and amyloid plaques in APP transgenic mouse models
Introduction Amyloid β (Aβ) accumulates in the extracellular space as diffuse and neuritic plaques in Alzheimer’s disease (AD). Aβ also deposits on the walls of arterioles as cerebral amyloid angiopathy (CAA) in most cases of AD and sometimes independently of AD. Apolipoprotein E (apoE) ɛ4 is associated with increases in both Aβ plaques and CAA in humans. Studies in mouse models that develop Aβ deposition have shown that murine apoE and human apoE4 have different abilities to facilitate plaque or CAA formation when studied independently. To better understand and compare the effects of murine apoE and human apoE4, we bred 5XFAD (line 7031) transgenic mice so that they expressed one copy of murine apoE and one copy of human apoE4 under the control of the normal murine apoE regulatory elements (5XFAD/apoEm/4). Results The 5XFAD/apoEm/4 mice contained levels of parenchymal CAA that were intermediate between 5XFAD/apoEm/m and 5XFAD/apoE4/4 mice. In 5XFAD/apoEm/4 mice, we found that Aβ parenchymal plaques co-localized with much more apoE than did parenchymal CAA, suggesting differential co-aggregation of apoE with Aβ in plaques versus CAA. More importantly, within the brain parenchyma of the 5XFAD/apoEm/4 mice, plaques contained more murine apoE, which on its own results in more pronounced and earlier plaque formation, while CAA contained more human apoE4 which on its own results in more pronounced CAA formation. We further confirmed the co-aggregation of mouse apoE with Aβ in plaques by showing a strong correlation between insoluble mouse apoE and insoluble Aβ in PS1APP-21/apoEm/4 mice which develop plaques without CAA. Conclusions These studies suggest that both murine apoE and human apoE4 facilitate differential opposing effects in influencing Aβ plaques versus CAA via different co-aggregation with these two amyloid lesions and set the stage for understanding these effects at a molecular level. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0250-y) contains supplementary material, which is available to authorized users.
Introduction
The accumulation of amyloid β (Aβ) into plaques is one of the pathological hallmarks of Alzheimer's disease (AD) [13]. The vast majority of patients diagnosed with AD also have cerebral amyloid angiopathy (CAA), deposition of Aβ on the cerebral vessels [16]. Some individuals develop CAA in the absence of AD [3]. CAA often associates with hemorrhagic lesions, ischemic lesions, encephalopathy, and dementia [39].
The strongest known genetic risk factor for late onset AD is the ε4 allele of apolipoprotein E (apoE), while the ε2 allele is protective [7,8,33]. Human apoE4 carriers have higher amyloid plaque load as well as greater amounts of CAA [12,20,29]. ApoE influences deposition of Aβ in plaques and CAA likely through some common mechanisms affecting Aβ clearance and aggregation. For example, apoE4 slows down Aβ clearance and leads to higher Aβ concentration [6]. This could further increase Aβ accumulation in both plaques and CAA. ApoE colocalizes with both amyloid plaques and CAA [21,35,37], and blocking apoE/Aβ binding with a non-fibrillogenic synthetic peptide Aβ 12-28p reduces plaque load as well as CAA [27,40]. In addition, amyloid precursor protein (APP) transgenic mice lacking apoE have a marked reduction of fibrillar Aβ deposition and no CAA, suggesting that apoE facilitates Aβ deposition in both lesions [2,11,15].
Out of 299 amino acids, human apoE4 shares only 70 % homology with mouse apoE. Using APP transgenic (Tg) mice that develop Aβ deposition, it has been shown that mouse apoE is overall more amyloidogenic than any of the human apoE isoforms [9]. In addition, mouse apoE appears to be more prone to lead to parenchymal plaque formation while human apoE4 is more prone to lead to CAA formation in mice that generate wild type human Aβ peptide [10,24]. This conflicting pattern suggests that apoE may affect plaques and CAA deposition via a different mechanism(s). This difference is not likely caused by differential overall Aβ clearance rates since a change in Aβ clearance and consequent concentration would probably have the same impact on both plaques and CAA. Another possibility is that this difference is mediated by differential co-aggregation of apoE with Aβ in parenchymal plaques or CAA. In this study, we asked whether mouse apoE vs human apoE4 differentially 1) lead to either parenchymal plaque formation versus CAA and 2) co-aggregate with Aβ in plaques and CAA by quantifying their degree of co-localization with plaques and CAA when they are expressed in the same brain at the same level.
Here we utilized APP Tg mice carrying one copy of endogenous mouse apoE and one copy of apoE4 (APP/ apoE m/4 ). In this model, different apoE proteins interact with Aβ under an identical in vivo microenvironment. Therefore, the interactions between apoE and Aβ will depend on the intrinsic properties to each apoE isoform.
We first quantified the amyloid plaque and CAA load in 8-10 month old 5XFAD/apoE m/m , 5XFAD/apoE m/4 and 5XFAD/apoE 4/4 mice and verified that apoE4 strongly facilitated amyloid deposition in CAA, and that mouse apoE facilitated parenchymal plaque deposition. We then assessed 5XFAD/apoE m/4 mice and found that in the presence of both mouse apoE and human apoE4, there was an intermediate level of CAA as compared to either 5XFAD/ apoE m/m or 5XFAD/apoE 4/4 mice. In 5XFAD/apoE m/4 mice, mouse apoE co-localized with parenchymal plaques to a significantly greater extent while apoE4 co-localized with CAA to a significantly greater extent. Further, in 85 day old APPPS1-21/apoE m/4 mice, which have plaques without CAA, insoluble Aβ was strongly correlated with insoluble mouse apoE but not apoE4. The data suggest that the type of apoE dictates whether apoE will lead to greater plaque versus CAA and that differences in apoE sequence and co-aggregation with plaques versus CAA likely leads to this difference. Understanding the molecular basis for this difference will lead to insights into disease pathogenesis that may have future treatment implications.
Materials and methods
Animals 5XFAD mice, line Tg7031 on a C57/B6XSJL background (gift from Dr. Robert Vassar at Northwestern University) co-express the KM670/671NL, I716V, and V717I mutations in human APP (695), as well as the M146L and L286V mutations in human PS1 under control of the mouse Thy1 promoter [23]. APPPS1-21 mice on a C57BL/ 6 J background (gift from Dr. Mathias Jucker at Hertie-Institute for Clinical Brain Research) co-express human APP with a Swedish mutation (KM670/671NL) and mutant PS1 with the L166P mutation under the control of a Thy1 promoter [25]. ApoE4 knockin mice express apoE ε4 under control of the endogenous mouse regulatory elements on a C57BL/6 J background (apoE 4/4 ) [34]. 5XFAD mice carrying one copy (5XFAD/apoE m/4 ) or two copies (5XFAD/apoE 4/4 ) of apoE4 were generated by breeding 5XFAD/apoE m/m with apoE 4/4 mice. APPPS1-21/apoE m/4 mice were generated by breeding APPPS1-21/apoE m/m with apoE 4/4 mice. Age-matched non-APP/apoE m/4 mice were littermates of the corresponding APP mice. All experimental protocols were approved by the Animal Studies Committee at Washington University.
Tissue harvesting
The mice were perfused with ice cold PBS containing 0.3 % heparin. One hemibrain was dissected and flashfrozen on dry ice and then stored at -80°C for biochemical assays. The other hemibrain was fixed in 4 % paraformaldehyde for histological study. Before staining, serial coronal sections at 50 μm thickness were collected using a freezing sliding microtome (Leica).
X-34 staining
Quantitative analysis of fibrillar amyloid deposition was performed on 8-10 month old 5XFAD/apoE m/m , 5XFAD/ apoE m/4 and 5XFAD/apoE 4/4 mice as previously described [18]. Briefly, three sections per mouse (Bregma, -1.4 mm caudal to Bregma, -2.0 mm caudal to Bregma) were stained with X-34 and then scanned using a Nanozoomer slide scanner (Hamamatsu Photonics). Images were exported with NDP viewer (Hamamatsu Photonics), converted to grayscale, thresholded to highlight positive staining of plaques or CAA, and analyzed using ImageJ (National Institutes of Health). The average area covered by X-34 from the 3 sections/mouse was used to represent each mouse. The quantification was performed by an investigator who was blinded for the genotype of the animals.
Immunohistochemistry
Brain sections from 10-month-old 5XFAD/apoE m/4 or 85-day-old APPPS1-21/apoE m/4 mice were co-stained for fibrillar amyloid, mouse apoE, and apoE4 using X-34, anti-mouse apoE monoclonal antibody HJ6.3-Alexa Fluor 568 (generated in-house) and anti-human apoE monoclonal antibody HJ15.7-Alexa Fluor 488 (generated in-house), respectively. For quantification of apoE/CAA or apoE/ plaque co-staining in 10-month-old 5XFAD/apoE m/4 mice, brain sections were imaged using a Zeiss LSM 5 PASCAL system coupled to a Zeiss Axiovert 200 M confocal microscope. Three sections from each mouse were used and five fields containing CAA and five fields containing plaques on each section were imaged. Images were thresholded for corresponding colors and the degree of co-localization was analyzed with ImageJ. Percent area of CAA or plaque covered by mouse apoE or apoE4 was calculated and the average from the 3 sections/mouse was used to represent each mouse. For quantification of apoE/plaques in 85-day-old APPPS1-21/apoE m/4 mice, which were at the initiation of plaque deposition, all the plaques in the cortical area were counted under Nikon Eclipse 80i fluorescent microscope and the apoE co-staining status for each individual X-34 plaque was recorded. The average of 3 sections from each mouse was used to represent each mouse.
Tissue lysate ELISA
Brain cortices from 85-day-old APPPS1-21/apoE m/4 and apoE m/4 mice were sequentially homogenized with cold PBS, 1 % triton-X 100, and 5 M guanidine buffer in the presence of 1X protease inhibitor mixture (Roche). Aβ 40 , Aβ 42 , mouse apoE, and apoE4 in each fraction were measured by ELISA. For Aβ 40 or Aβ 42 ELISA, anti-Aβ [35][36][37][38][39][40] HJ2 (generated in-house) or anti-Aβ 37-42 HJ7.4 (generated in-house) was used as the capture antibody, and anti-Aβ 13-28 HJ5.1-biotin (generated in-house) as the detecting antibody [4]. For mouse apoE ELISA, plates were coated with HJ6.2 (in-house generated) at a concentration of 10 μg/ml in carbonate coating buffer at 4°C overnight. After blocking with 2 % BSA in PBS at 37°C for 1 h, the samples were loaded on the plates and incubated overnight at 4°C. Then the plates were incubated in 300 ng/ml HJ6.8-biotin (generated in-house) at 37°C for 1.5 h. Followed by a incubation in 1:10000 Streptavidin Poly-HRP40 Conjugate (Fitzgerald) at room temperature for 1.5 h, the plates were developed using Super Slow ELISA TMB (Sigma) and read on a Bio-Tek Synergy 2 plate reader at 650 nm. The standard curve was mouse apoE purified from mouse astrocytes conditioned medium using a polyclonal antibody (Calbiochem). Human apoE ELISA shared the same protocol as mouse apoE ELISA except for the antibodies were different. For human apoE ELISA, HJ15.6 (generated in-house) at a concentration of 10 μg/ml was used as the capture antibody and 150 ng/ml HJ15.4-biotin (generated in-house) was used as detecting antibody. Recombinant apoE4 (Leinco) was used as the standard for human apoE ELISA.
Data analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software, version 5.0). All data were analyzed using ANOVA, One-way ANOVA with repeated measures, student t-test or paired ttest as indicated in the respective figure legends. Sample sizes were specified in the respective figure legends. Data were expressed as mean ± S.E.M. unless otherwise specified.
Results
ApoE4 shifts parenchymal Aβ deposition from plaques to CAA in the 5XFAD mice Plaque deposition begins in 5XFAD (line 7031) on mouse apoE background (5XFAD/apoE m/m ) at the age of 4 months. When 5XFAD mice were bred onto an apoE4 background (5XFAD/apoE 4/4 ), plaque deposition began at the age of 5 months (Additional file 1: Figure S1). To further determine the effect of replacing mouse apoE with apoE4 on Aβ plaques and CAA, we stained fibrillar amyloid with X-34 in 8-10 month old 5XFAD/apoE m/m , 5XFAD/apoE m/4 and 5XFAD/apoE 4/4 mice (Fig. 1a). The amount of CAA on the blood vessels within the brain parenchyma (parenchymal CAA) and amyloid plaques were quantified. There is minimal parenchymal CAA compared to amyloid plaques in 5XFAD/apoE m/m animals (Fig. 1b, c). With the introduction of one copy of apoE4 (5XFAD/apoE m/4 ), the plaque load did not change while the CAA tended to increase (Fig. 1b, c). With two copies of apoE4 (5XFAD/ apoE 4/4 ), there was a significant reduction of plaque load (Fig. 1b) and a significant elevation of CAA (Fig. 1c) as compared to 5XFAD/apoE m/m mice.
Plaques contain more mouse apoE while parenchymal CAA contain more apoE4 in 5XFAD/apoE m/4 mice To determine whether the mouse apoE and apoE4 facilitate plaque or CAA differently via differential coaggregation with Aβ in plaques and CAA, we examined the amount of apoE4 or mouse apoE co-localized with plaques and CAA. We co-stained amyloid deposition with an anti-mouse apoE specific antibody HJ6.3 (Additional file 2: Figure S2) and an anti-human apoE specific antibody HJ15.7 (Additional file 2: Figure S2) in 10-month-old 5XFAD/apoE m/4 brains (Fig. 2). We first quantified colocalization of each form of apoE in relation to plaques and CAA. Within brain parenchyma, both apoE4 (3.33 ± 1.04 % in CAA vs 7.49 ± 0.71 % in plaques, n = 9, p < 0.05, paired t-test) and mouse apoE (1.23 ± 0.63 % in CAA vs 19.89 ± 1.30 % in plaques, n = 9, p < 0.001, paired t-test) exhibited less co-localization in parenchymal CAA as compared to plaques. However, the amount of each form of apoE localized in the same lesion was different. Parenchymal plaques contained more mouse apoE immunoreactivity than apoE4 (Fig. 2a, b), whereas parenchymal CAA contained more apoE4 immunoreactivity than mouse apoE (Fig. 2c, d).
Next, we compared the co-localization of different apoE within parenchymal CAA (Fig. 2c) and the CAA on leptomeningeal vessels (leptomeningeal CAA, Fig. 3a). Interestingly, both mouse apoE (Fig. 3c) and apoE4 (Fig. 3d) exhibited significantly more co-localization with leptomeningeal CAA as compared to parenchymal CAA. In addition, while apoE4 co-localized more than mouse apoE within parenchymal CAA (Fig. 2d), leptomeningeal CAA co-localized with less apoE4 as compared to mouse apoE (Fig. 3b). For the mouse apoE to human apoE4 ratio, the ratios were similar in plaques and leptomeningeal CAA while parenchymal CAA had the lowest ratio (Additional file 3: Figure S3).
To assess the level of mouse and human apoE in cortical tissue lysates of the same APPPS1-21/apoE m/4 mice, we developed both a mouse and a human apoE Fig. 1 ApoE4 shifted parenchymal Aβ deposition from plaques to parenchymal CAA in the 5XFAD mice. 8~10 months old 5XFAD/apoE m/m , 5XFAD/apoE m/4 and 5XFAD/apoE 4/4 mice were stained with X-34. a Representative brain sections with CAA (empty arrows) and plaques (solid arrows). Scale bar, 1 mm. The right panel is the high power magnification of the area labeled in the squares in the corresponding left-side images. b The % area covered by parenchymal fibrillar plaques in the cortex. c The % area covered by parenchymal CAA quantified in the cortex (n = 3-9/group; *p < 0.05, One-way ANOVA followed by Tukey post-test) specific ELISA (Additional file 4: Figure S4). We then performed a 3-step sequential extraction of APPPS1-21/apoE m/4 cortex using PBS, 1 % Triton X-100 and 5 M guanidine, and measured apoE levels in these three fractions. Tissue from age-matched non-APP transgenic apoE m/4 mice were also analyzed as controls to see the basal levels of apoE in the absence of amyloid plaques. In the non-APP transgenic, apoE m/4 mice, we found that the mouse apoE and apoE4 levels were similar in the sum of the three fractions (Fig. 5a). However, the fractional distributions of different apoE were very different. For mouse apoE, 50.9 ± 1.1 % was present in the PBS soluble fraction and only 4.7 ± 0.4 % was present in the insoluble (guanidine) fraction (Fig. 5b). ApoE4 was more equally distributed in 3 fractions (Fig. 5b). The PBS soluble fraction contained significantly more (p < 0.001) mouse apoE than apoE4 (Fig. 5a), while the insoluble fraction contained significantly more (p < 0.001) apoE4 than mouse apoE (Fig. 5a).
In APPPS1-21/apoE m/4 cortex which contained amyloid plaques, apoE4 absolute levels (Fig. 5c) or fractional distribution (Fig. 5d) were unaltered as compared to that in apoE m/4 cortices. For mouse apoE, the absolute levels were unaltered in the PBS and 1 % Triton fraction of APPPS1-21/apoE m/4 cortices as compared to that in apoE m/4 . However, the mouse apoE in the insoluble fraction of APPPS1-21/apoE m/4 mice was significantly higher than that in apoE m/4 mice (117.3 ± 21.7 vs 48.8 ± 4.4 ng/ 100 mg tissue for APPPS1-21/apoE m/4 and apoE m/4 , respectively; p < 0.01, Student t-test). As a result, the percentage of insoluble mouse apoE was increased in APPPS1-21/apoE m/4 mice as compared to that in apoE m/4 mice (10.0 ± 1.6 % vs 4.7 ± 0.4 % for APPPS1-21/apoE m/4 and apoE m/4 , respectively; p < 0.01, Student t-test; Fig. 5b, d). The increase of insoluble mouse apoE is very likely due to co-aggregation of mouse apoE with Aβ in the plaques, which did not occur to a significant extent with apoE4. To verify this possibility, we performed correlation analysis of Fig. 2 Co-localization of mouse apoE and apoE4 in CAA or plaques within the same brain parenchyma in 5XFAD/apoE m/4 mice. 10-month-old 5XFAD/apoE m/4 mice were co-stained with HJ6.3-Alexa 568 for mouse apoE, HJ15.7-Alexa 488 for apoE4, and X-34 for fibrillar amyloid. a-b Representative images of co-staining for mouse apoE and apoE4 in the plaques and % area of plaque covered by different apoE. c-d Representative images of co-staining for mouse apoE and apoE4 in parenchymal CAA and % area of parenchymal CAA covered by different apoE. Values connected by lines were measured from the same animals (n = 9/group; *p < 0.05, ***p < 0.001, paired t-test) Fig. 3 Comparison between apoE co-localization in parenchymal and leptomeningeal CAA in the same 5XFAD/apoE m/4 brains. Brain sections from 10 months old 5XFAD/apoE m/4 animals were co-stained with HJ6.3-Alexa 568 for mouse apoE, HJ15.7-Alexa 488 for human apoE4, and X-34 for fibrillar amyloid. a-b Representative images of co-staining for mouse apoE and apoE4 in the leptomeningeal CAA and % area of leptomeningeal CAA covered by different apoE (n = 9/group). Scale bars, 50 μm. c % area of plaque, parenchymal and leptomeningeal CAA covered by mouse apoE. d % area of plaque, parenchymal and leptomeningeal CAA covered by human apoE4. Values connected by lines were measured from the same animals (n = 8/group; **p < 0.01, ***p < 0.001, One-way ANOVA repeated measures) Fig. 4 Co-localization of mouse apoE and apoE4 in plaques within the same brain parenchyma in APPPS1-21/apoE m/4 mice. Brain sections from 85-day-old APPPS1-21/apoE m/4 animals (n = 7) were co-stained with HJ6.3-Alexa 568 for mouse apoE, HJ15.7-Alexa 488 for human apoE4, and X-34 for fibrillar amyloid. a Representative images and the % of plaques containing both mouse apoE and apoE4 (scale bar, 20 μm). b Representative images and the % of plaques containing only mouse apoE (scale bar, 20 μm) insoluble Aβ and apoE levels in the same sample in the cortices of these APPPS1-21/apoE m/4 mice. Mouse apoE demonstrated a strong correlation with insoluble Aβ 42 (Fig. 5e), in agreement with the observation that mouse apoE highly co-localized with plaques by co-staining. In contrast, apoE4, which is poorly co-localized with plaques, demonstrated no correlation with Aβ 42 (Fig. 5f). As expected, insoluble Aβ 40 strongly correlated with insoluble Aβ 42 in APPPS1-21/apoE m/4 mice (Fig. 5 g).
Discussion
APOE genotype is the strongest genetic risk factor for late-onset AD and CAA. ApoE4 is associated with increases in both plaque burden and CAA in humans relative to the other human apoE isoforms [17]. ApoE likely influences deposition of Aβ in plaques and CAA through some common mechanisms affecting Aβ clearance and aggregation. Interestingly, previous studies in mouse models of amyloid deposition have shown that murine apoE is significantly more amyloidogenic than human apoE isoforms, including apoE4 [9,41]. Despite the fact that it is more amyloidogenic, previous studies showed that apoE4 led to greater CAA than mouse apoE. In the current study, we showed that mouse apoE and human apoE4 were each dominant when present in the same brain ( Fig. 1): mouse apoE promoted plaques while apoE4 promoted CAA. Overall, these findings suggest that differences in the inherent properties/structures of each form interact with Aβ in different ways leading to differential co-aggregation of Aβ in parenchymal plaque versus CAA. In addition, we found that 5XFAD/apoE m/4 have an intermediate level of CAA as compared to either 5XFAD/apoE m/m or 5XFAD/ apoE 4/4 mice. This is important as it is consistent with human data in which it has been found that apoE4, in a dose-dependent fashion, is associated with greater parenchymal CAA [22,30,42].
To investigate the co-aggregation of different apoE in plaques and CAA, we assessed co-localization of apoE and amyloid in these two lesions in the same brain. Our data clearly demonstrated that within the brain parenchyma, the degree to which apoE co-localizes with Aβ in plaques or CAA was associated with its ability to facilitate the corresponding lesion. Aβ plaques contained more mouse apoE which facilitates plaque deposition (Fig. 2b), while parenchymal CAA contained more apoE4 which facilitates the formation of parenchymal CAA (Fig. 2d). In addition, the fact that parenchymal plaques contained more apoE (regardless of whether it is mouse apoE or apoE4) also suggested that apoE interacted with Aβ in plaques and CAA differently.
While most apoE present in physiological fluids such as CSF may not complex with monomeric Aβ [36], there is no question that once Aβ aggregates in the brain parenchyma or in CAA in the form of fibrils, apoE is then found co-aggregated with amyloid. However, data quantifying the amount of individual apoE isoforms co-depositing with Aβ in plaques or CAA are lacking. Although in PDAPP/apoE 4/4 mice, the co-localization of apoE with Aβ plaques was higher than that in PDAPP/apoE 3/3 and PDAPP/apoE 2/2 mice [1], the different Aβ concentration and oligomerization states in individual mice with different apoE genotypes could influence the results. In the current study, the co-localization of different forms of apoE with different amyloid lesions within brain parenchyma was compared in the same mice carrying one copy of each apoE isoform and expressing each copy under the same regulatory elements at the same level (5XFAD/apoE m/4 ). Thus, the Aβ environment was identical for both apoE isoforms and the data provided definitive in vivo confirmation that mouse apoE and human apoE4 differentially facilitate specific types of amyloid deposition at least in part by co-aggregating with them differentially. The different % area covered by mouse apoE vs. human apoE4 observed here is likely not an artifact caused by the performance of apoE antibodies since plaques co-localized more with mouse apoE while parenchymal CAA co-localized more with apoE4. Furthermore, this co-localization of apoE and Aβ in plaques was verified by correlating Aβ with different apoE in the insoluble fraction of APPPS1-21/ apoE m/4 mice. Interestingly, while our current data and previous studies using APP transgenic mice generating normal human Aβ [10,24] suggested that apoE4 redistributed Aβ deposition to CAA as compared to mouse apoE, a previous study using mice expressing human Dutch/Iowa (E22Q/D23N) mutant Aβ showed an opposite pattern [38]. In transgenic mice (Tg-SwDI) that accumulate human Dutch/Iowa (E22Q/D23N) mutant Aβ, both human apoE3 and apoE4 strongly shifted the Aβ deposition from CAA into plaques [38]. Unlike in general AD populations where apoE4 is strongly associated with increased plaques and CAA, in humans with the rare Dutch mutation, apoE4 genotype was not correlated with plaques or CAA [5]. The Dutch mutation resides within amino acids 12-28 of Aβ peptide, a domain which appears to be required for interaction with apoE [33]. Taken together, our data and previous studies suggested that apoE modifies Aβ pathology through interaction with Aβ aggregates.
Since mouse apoE and apoE4 in 5XFAD/apoE m/4 brains were exposed to identical Aβ conditions, the co-aggregation of different apoE with Aβ in plaques or CAA was determined by the intrinsic properties of different apoE such as their binding preference to a certain species of Aβ or their concentration. CAA contains a higher Aβ 40 /Aβ 42 ratio than do plaques [14] although Aβ 42 is required to "seed" CAA [19]. The observation that apoE4 better colocalized with parenchymal CAA than plaques was not likely due to its higher binding preference to Aβ 40 over Aβ 42 . If this were the case, we should expect to see even more apoE4 present than mouse apoE in the leptomeningeal CAA since the ratio of Aβ 40 /Aβ 42 is even higher in leptomeningeal CAA than that in parenchymal CAA [26]. However in leptomeningeal CAA, we observed less apoE4 than mouse apoE. To see whether apoE levels play a role in the different co-aggregation, we measured apoE levels in cortical lysates of apoE m/4 and APPPS1-21/apoE m/4 mice. The total levels of mouse apoE and apoE4 in brain lysates were similar (Fig. 5) in apoE m/4 mice but their fractional distribution was different. In general there was more mouse apoE in the PBS soluble fraction while there was more human apoE4 in the insoluble fraction in the non-APP/apoE m/4 mice. This suggests an inherent difference in the biochemical properties of mouse apoE vs. human apoE4. It is possible that the conformation of mouse apoE in the PBS soluble fraction localized in parenchyma in such a way to interact with Aβ seeds that forms plaques as compared to apoE4 resulting in its precipitation into plaques to a greater extent. It is also possible that the structure of apoE4 may result in its localization to a greater extent in the vasculature, enabling it to interact to a greater extent with Aβ seeds that form CAA.
When leptomeningeal CAA and parenchymal CAA were compared, we found that 1) leptomeningeal CAA contained much higher apoE than did parenchymal CAA, and 2) leptomeningeal CAA contained more mouse apoE while parenchymal CAA contained more apoE4. These observations suggested that apoE might be differently involved during the formation of CAA in blood vessels of different locations.
Conclusion
Understanding how apoE influences the development of parenchymal plaques versus CAA is important. While this study does not provide the molecular basis for why mouse apoE and human apoE4 result in differential plaques versus CAA, it demonstrates that apoE is a major determinant of where Aβ deposits given that the Aβ in the in vivo microenvironment is the same. Therefore, studying the differences in sequence (30 % difference in sequence) and structure between mouse and human apoE that result in these differences could provide important insights. For example, understanding the structural variations could provide new insight into how to block or influence the apoE/Aβ interaction. In addition, treatment with some anti-Aβ antibodies has resulted in humans in the complication of amyloid-related imaging abnormalities either with edema or hemorrhage. This complication is far more frequent in apoE4 positive individuals [31,32]. It was also observed that Aβ immunotherapy was associated with redistribution of apoE from cortical plaques to cerebral vessel walls, mirroring the altered distribution of Aβ [28]. Understanding the basis for the differential effects of apoE on plaques vs. CAA might provide important insights into this phenomenon that could lead to ways to understand and prevent it. | v3-fos-license |
2019-03-08T14:11:39.953Z | 2019-02-07T00:00:00.000 | 73451594 | {
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} | pes2o/s2orc | Retractile lysyl-tRNA synthetase-AIMP2 assembly in the human multi-aminoacyl-tRNA synthetase complex
Multi-aminoacyl-tRNA synthetase complex (MSC) is the second largest machinery for protein synthesis in human cells and also regulates multiple nontranslational functions through its components. Previous studies have shown that the MSC can respond to external signals by releasing its components to function outside it. The internal assembly is fundamental to MSC regulation. Here, using crystal structural analyses (at 1.88 Å resolution) along with molecular modeling, gel-filtration chromatography, and co-immunoprecipitation, we report that human lysyl-tRNA synthetase (LysRS) forms a tighter assembly with the scaffold protein aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) than previously observed. We found that two AIMP2 N-terminal peptides form an antiparallel scaffold and hold two LysRS dimers through four binding motifs and additional interactions. Of note, the four catalytic subunits of LysRS in the tightly assembled complex were all accessible for tRNA recognition. We further noted that two recently reported human disease-associated mutations conflict with this tighter assembly, cause LysRS release from the MSC, and inactivate the enzyme. These findings reveal a previously unknown dimension of MSC subcomplex assembly and suggest that the retractility of this complex may be critical for its physiological functions.
Assembly or disassembly of the complex is essential to regulate the functions of involved MSC components in cellular homeostasis. For example, GluProRS was reported to have roles in INF-␥-related inflammation induced gene-specific silencing of translation and antiviral immunity when released from MSC (7,8). IFN-␥ induces sequential phosphorylation of Ser 886 and Ser 999 in the noncatalytic linker of GluProRS, which triggers GluProRS release from the MSC. Phosphorylation of GluProRS is also required for the interaction with NS1-associated protein-1 (NSAP1), ribosomal protein L13a, and glyceraldehyde-3-phosphate dehydrogenase to form functional GAIT complex (9). The GAIT complex binds GAIT element in specific mRNA and eventually represses translation (10). Upon DNA damage response, the general control nonrepressed-2 (GCN2) kinase phosphorylates MetRS at Ser 662 . The phosphorylation induces a conformational change of MetRS and releases the scaffold protein AIMP3, resulting in AIMP3 translocation to nucleus to activate ataxia telangiectasia mutated/ATM-and Rad3-related pathways (11). Another scaffold protein AIMP1 has several proteolytic forms, including EMAP II and p43ARF (12)(13)(14)(15). EMAP II, p43ARF, and the full-length AIMP1 can all be secreted out of cells to play cytokine functions, including procoagulant, proinflammatory, proapoptotic, and angiogenic activities (12, 16 -18). Outside of MSC, AIMP2 binds and regulates the protein stability of far upstream element-binding protein 1 (FBP1) through the central region of AIMP2. AIMP2 promotes FBP1 degradation by Smurf 2-dependent post-translational ubiquitination and decreases the transcription level of c-myc (19).
Human LysRS plays noncanonical functions in several cellular processes, including HIV reverse transcription, viral packaging, neuropathy, immune response, cancer metastasis, etc. (20). Upon cro ARTICLE could be selectively packed into the HIV-1 virion. LysRS, tRNA Lys3 , viral precursor proteins Gag, and GagPol form a cytoplasmic nucleoprotein complex to facilitate the HIV viral reverse transcription after infecting new host cells (21)(22)(23). In mast cells, IgE-antigen stimulation activates specific phosphorylation of LysRS on a Ser 207 residue (24). The Ser 207 phosphorylation causes a structural opening of LysRS that disrupts its interaction with AIMP2 and leads to its dissociation from the MSC. The phosphorylated LysRS further enters into the nuclear, binds a transcription factor MITF, and eventually activates the MITF-targeted genes transcription (25). Human LysRS also has a cytokine activity (26). When secreted from macrophage-like differentiated THP-1 cells, LysRS mediates the transduction of inflammatory signals in the Shiga toxinproducing Escherichia coli-infected host cell (27). In addition, through a different structural mechanism, LysRS binds to cell membrane through the laminin receptor 67LR to promote epithelial cell migration (28,29).
LysRS's recruitment to fulfill noncanonical functions involves its dissociation from MSC and association with new binding partners. Characterization of LysRS assembly within the MSC helps to understand these cellular mechanisms. Our previous studies showed one N terminus of the scaffold protein AIMP2 was able to hold one LysRS dimer in MSC (25,30), so that two LysRS dimers and two AIMP2 molecules per MSC exist in redundancy (25,30,31). The two LysRS dimers and the two copies of AIMP2 are packed with a V shape in solution (V form) (Fig. S1). In this form, each of the two LysRS dimers is connected by the N-terminal 32 residues from one AIMP2, so that two LysRS dimers do not directly contact. In this loose form, each LysRS dimer could move flexibly in solution, function for aminoacylation, and diffuse from AIMP2 for functions outside of MSC (30). Here we report a new crystal structure of LysRS-AIMP2 subcomplex, in which the two LysRS dimers are retracted by AIMP2 into a tighter assembly. Two human disease-related mutations disturbed LysRS's incorporation into MSC in cells and are conflicted with this tight assembly. This finding reveals a previously unknown dimension of MSC subcomplex assembly and suggests that the retractility of the complex may be critical for its diverse physiological functions.
Overall structure of the LysRS-AIMP2 subcomplex in a tight assembly
We had previously determined a LysRS-AIMP2 complex at a resolution of 2.86 Å and discovered that one AIMP2 N terminus was able to bind one LysRS dimer through two LysRSbinding motifs (25,30). Recently, we solved a second LysRS-AIMP2 complex structure with 1.88 Å resolution in a different condition (Table 1 and Fig. S2). In the new crystal, only motif 1 (MYQVKPYH) of AIMP2 interacts with LysRS in the same asymmetric unit (ASU1) (Fig. 1A), and motif 2 (MYRLPNVH) extends to a nearby asymmetric unit (ASU2), where it interacts with another LysRS dimer (Fig. 1, B and C). Symmetrically, motif 2 from the AIMP2 in the ASU2 extends back to ASU1 and interacts with the other AIMP2-binding pocket on LysRS dimer 1. Thus, two nearby ASUs form one biological unit that each of the AIMP2s concatenates two LysRS dimers (Fig. 1, B and C). This new crystal structure reveals a tightly packed X-shaped assembly of the LysRS-AIMP2 subcomplex (X form) (Fig. 1C), which is different from the previously discovered V-form assembly (Fig. S1). Four tRNA molecules can be docked on the X-form complex at the same time without any clash with other
Retractile LysRS-AIMP2 assembly in human MSC
protein or tRNA atoms (Fig. 1D), indicating that all four LysRS catalytic subunits are functional in this form. It implies that the X-form complex might not only exist in crystal but also reflect physiological assembly.
LysRS-AIMP2 interactions in the X-form complex
Consistent with previous reports, motifs 1 and 2 of AIMP2 contribute to the major interactions between AIMP2 and LysRS ( In addition, the two LysRS dimers in the X-from complex are mainly held by the two AIMP2s. There are only a few direct interactions between the two LysRS dimers, such as a hydrophobic interaction between Pro 390 and His 348 or a van der Waals interaction between Thr 388 and Ser 478 (Fig. S4).
Presence of X-form complex in human cells
To confirm the X-form complex exists in human cells, we designed an MSC incorporation assay based on gel-filtration chromatography. In this assay, we fused the LysRS binding region of AIMP2 (amino acids 1-36) to the N-terminal end of eGFP and expressed the fusion protein in HEK293T cells. Dis-tinct from the V form, only the X-form assembly allows two AIMP2 N-terminal peptides to bind corporately across two LysRS dimers. If the X form indeed exists in cells, the AIMP2N-eGFP protein can be incorporated into the MSC by forming a heterogeneous X-form complex together with one endogenous AIMP2 and two LysRS dimers (Fig. 3A). In the V-form complex, one AIMP2 N-terminal peptide binds one LysRS dimer. Competitive binding of AIMP2N-eGFP with the endogenous AIMP2 will lead to release of LysRS from the complex (Fig. 3B). As a result, when we loaded the AIMP2N-eGFP-expressing cell lysate onto a Superose 6 gel-filtration column, the AIMP2N-fused protein was detected in both low-molecularweight fractions and high-molecular-weight fractions (Ͼ1 MDa). Although the control eGFP only existed in low-molecular-weight fractions (Fig. 3C). Therefore, the presence of AIMP2N-eGFP in the high-molecular-weight fractions implies the existence of X-form complex in human cells. In addition, more LysRS in low-molecular-weight fractions was found from the AIMP2N-eGFP-expressing cells compared to the eGFP control cells (Fig. 3D), suggesting that the X and V forms may co-exist in HEK293T cells.
Two human disease-related mutations conflict with the X-form complex
A 14-year-old girl patient harboring two novel biallelic mutations in LysRS (L350H and P390R) was reported recently (33). The L350H mutation was inherited from her mother, whereas the P390R mutation was a de novo mutation. The patient manifests a severe form of cardiomyopathy associated with lactic acidosis, mild myopathy, and intellectual disability (33). The pathogenesis is unclear. We therefore analyzed the two mutations on the X-form complex. Leu 350 locates on the bottom side of LysRS, which is opposite to the tRNA-binding side, and is
Retractile LysRS-AIMP2 assembly in human MSC
ϳ6.5 Å away from the Met 3 residue of AIMP2 (Fig. 4A). The side chain of Leu 350 makes hydrophobic interactions with LysRS residues Phe 340 , Met 342 , Ala 345 , and Ala 545 (Fig. 4A). Among these residues, Met 342 and Ala 345 have direct hydrophobic interaction with Met 3 residue of AIMP2 (Fig. 4A). When Leu 350 is mutated to a bigger histidine residue, the Met 342 and Ala 345 residues are slightly pushed away for ϳ2.0 and 0.6 Å, which is negative for AIMP2 binding (Fig. 4B, and Fig. S5). The Pro 390 residue locates 13 Å away from AIMP2 (Fig. 4C); thus, the P390R mutation should not affect the AIMP2 binding directly. However, Pro 390 closely faces the other LysRS dimer in the X-form complex and makes hydrophobic interaction with His 348 from the other LysRS dimer (Fig. S4). The P390R mutation causes clashes with the LysRS dimer 2 at residues such as Tyr 347 , His 348 , Met 351 , and Arg 393 (Fig. 4D). Because of the crystallographic symmetry, the P390R mutation from LysRS dimer 2 causes same clash with LysRS dimer 1. This indicates that the P390R mutation disturbs the X-form complex. On the other side, the two LysRS dimers in the previous V-form complex are distant from each other; thus, the P390R mutation might not affect the V-form complex assembly.
Mutations related to human disease intervene in MSC assembly and enzyme activity
To confirm the effect of the mutations upon MSC assembly in cell, we performed co-immunoprecipitation experiment to examine the ability of MSC association of LysRS WT and mutant proteins. L350H mutant protein co-precipitated with AIMP2 and MetRS as WT protein (Fig. 5A and Fig. S6), indicating that a single L350H mutation did not significantly affect the MSC association, consistent with the subtle change in the L350H crystal structure and the normal phenotype of the patient's mother. The P390R mutation was also able to interact with AIMP2 and MetRS as normal, whereas the L350H/P390R double mutant significantly lost the ability to interact with AIMP2 and MetRS in the co-immunoprecipitation experiment ( Fig. 5A and Fig. S6). These results indicate that the two mutations L350H and P390R might each weakly affect the association of LysRS within MSC. However, the dual mutations can aggravate the interruption of MSC assembly.
We then examined how the two mutants might affect the enzyme activity for supporting essential protein translation using a functional replacement assay in Saccharomyces cerevi-
Retractile LysRS-AIMP2 assembly in human MSC
siae yeast (25). WT LysRS could substitute for the yeast cytoplasmic LysRS (which is controlled by a tetracycline-induced promoter and can be suppressed by doxycycline) and sustain normal cell growth (Fig. 5B). The inactive LysRS S207D mutant was used as a negative control (25). Both L350H and P390R single mutants were as active as WT in supporting cell growth. However, the L350H/P390R double mutant was completely inactive (Fig. 5B). These results show the two mutations have a synergetic effect in disrupting both the MSC assembly and the enzyme activity.
Notably, Leu 350 locates at the ␣-helix (amino acids 346 -366) right below the seven-strand central -sheet of the active center, and Pro 390 locates at a long loop connecting the same helix to the 3 of the seven-strand -sheet (Fig. S7). The double mutation might have a synergistic affect in altering the local conformation of the helix (amino acids 346 -366) and the central -sheet of the enzyme, which disturbed both X-form complex assembly and enzyme activity. Either of the dual defects or both might play a role in the pathogenesis.
A pseudo-disulfide bond stabilizes the X-form complex
The two AIMP2 N-terminal peptides bind antiparallelly across the two LysRS dimers (Fig. 6A). Interestingly, Cys 23 next to motif 2 formed a disulfide bond in the crystal structure, which stabilizes the compact X-form complex (Fig. 6B and Fig. S8). Following the LysRS binding sequence, AIMP2 has a leucine-zipper region at residues 48 -81 and a GST domain at the C terminus. The leucine-zipper region dimerizes AIMP2 and interacts with AIMP1, ArgRS, and GlnRS, whereas the GST domain interacts with AspRS and GluProRS (34,35). The distance between the two His 31 residues of AIMP2 in the X-form complex is ϳ41 Å (Fig. 6C), agreeable to the dimerization of the C-terminal part of the protein for further assembly of the whole MSC complex (Fig. 6D). If the disulfide bond formed in vivo, it would stabilize not only the LysRS-AIMP2 subcomplex but also the whole MSC.
Interestingly, the C23S mutant of the AIMP2N-eGFP protein was still capable of forming X-form complex with endogenous AIMP2 and LysRS (Fig. 3C), suggesting that the disulfide bond is not a prerequisite of the X-form assembly. On the other side, the Cys 23 together with residues involved in the X-form complex formation (such as Leu 16 -Thr 22 ) are strictly conserved from zebrafish to human (Fig. S9). In addition, LysRS from the cells expressing AIMP2N-eGFP C23S mutant showed slightly different distribution comparing to the cells expressing WT fusion protein (Fig. 3D). These results imply the Cys 23 might play a role in controlling the MSC assembly at certain physiological conditions, for example, when intracellular redox potential fails under oxidative stress conditions (Fig. 6E).
Potential advantage for the retractile assembly of MSC subcomplex
From our previously solved LysRS-AIMP2 crystal structure, one LysRS dimer forms two symmetric AIMP2-binding pockets by both N-terminal anticodon binding domain and C-terminal catalytic domain (25). Phosphorylation of Ser 207 on the N-C domain interface triggers significant conformational change to LysRS and disrupts the AIMP2-binding pocket, thus releasing LysRS from MSC for nontranslational function in mast cell activation (25). In the current crystal structure, we show that LysRS could also form tight assembly with AIMP2 ( Fig. 1). In this assembly, the major interactions between LysRS and AIMP2 are consistent with the previous result (Fig. 2), which double ensures the molecular mechanisms for the phosphorylation triggered LysRS release from MSC (25). These two complex forms could both represent the assembly of two LysRS dimers and two AIMP2 proteins in the human MSC. The two forms might co-exist in an equilibrium in cells.
Complementary to the previously solved V-form complex, the X-form setup may have a merit of orderliness. The more organized status can avoid potential clash of tRNAs and ensure efficient aminoacylation catalysis (Fig. 7A), although the V-form assembly has an advantage in release one LysRS dimer from the scaffold for nontranslational functions upon cellular stimuli while retaining the other LysRS dimer for fundamental protein translation (Fig. 7, B and C). The two forms of assembly may reflect different stages of LysRS function (Fig. 7). The mechanisms for controlling the switch of the two forms yet need to be explored through further research.
In summary, this work solved a tighter LysRS-AIMP2 subcomplex in a compact X form. The study reveals a previously
Retractile LysRS-AIMP2 assembly in human MSC
unknown dimension of MSC subcomplex assembly and suggests that the retractility of the complex may be critical for its diverse physiological functions.
Structure determination
The LysRS-AIMP2 1-36 complex crystal diffraction data were obtained from Beamline LS-CAT at Advanced Photon Source of Argonne National Laboratory. The LysRS L350H crystals diffraction data were obtained from Beamline 17U1 at Shanghai Synchrotron Radiation Facility (36). All data sets were processed with HKL2000 (37). The structures were solved by molecular replacement using human LysRS structure (Protein Data Bank code 3BJU) with the program MOLREP (38). Iterative model building and refinement were performed using Coot and Phenix (39,40). The data collection and refinement statistics are given in Table 1.
Modeling LysRS-AIMP2-tRNA complex structure
Crystal structure of the AspRS-tRNA asp complex (Protein Data Bank code 1IL2) was used to generate the LysRS-AIMP2-tRNA structure model used in Fig. 1D, because no LysRS-tRNA crystal structure is currently available. The chain A (AspRS) and chain C (tRNA asp ) were extracted from 1IL2 and aligned onto the LysRS-AIMP2 complex structure based on the protein C␣ atoms in PyMOL (root mean square deviation, 2.317 Å). The tRNA molecules could then be merged into the LysRS-AIMP2 structure with reasonable coordinates. The LysRS-AIMP2-tRNA model is generated by repeating this operation for all four LysRS subunits in the X-form complex.
Yeast viability assay
The cDNA sequences encoding full-length human LysRS and its mutations were constructed in the p413GPD vector multicloning site. The plasmids were then transformed into the yeast Tet-Promoters Hughes Collection (yTHC) mutant strain from Open Biosystems (Dharmacon). The endogenous promoter of yeast cytoplasmic LysRS gene (krs1) has been replaced with a TET-titratable promoter in the yTHC genome. Thus, the expression of the gene can be switched off by the addition of doxycycline to the growth medium. 10-fold serial dilutions of freshly grown yeast cells were spotted onto selective medium SCM-HIS with or without doxycycline. The plates were incubated at 30°C for 3 days and then photographed.
MSC incorporation assay
Human AIMP2/p38 (amino acids 1-36) and the C23S mutant were fused to the N terminus of eGFP, and expressed in HEK293T cells using a pMSCV-puro vector. EGFP alone was also constructed in the same vector and expressed in HEK293T cells as control. After 48 h of transfection, the cells were collected and lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% Nonidet P-40) and then loaded onto a Superose 6 gel-filtration column (GE Healthcare, 10/300 GL) with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl. The fractions A, the compact and ordered X-form complex ensures efficient tRNA aminoacylation. B, Two LysRS dimers are held by the two AIMP2 N terminus separately in a V-form assembly, which is ready to release one LysRS dimer for nontranslational functions. C, a potential third assembly form of LysRS-AIMP2 complex, in which one LysRS dimer was released for nontranslational function and one LysRS dimer was retained for fundamental translational function. | v3-fos-license |
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} | pes2o/s2orc | Novel Multiarm Polyethylene glycol-Dihydroartemisinin Conjugates Enhancing Therapeutic Efficacy in Non-Small-Cell Lung Cancer
The clinical application of dihydroartemisinin (DHA) has been hampered due to its poor water-solubility. To overcome this hurdle, we devised a novel polymer-drug conjugate, multiarm polyethylene glycol-dihydroartemisinin (PEG-DHA), made by linking DHA with multiarm polyethylene glycol. Herein, we investigated PEG-DHA on chemical structure, hydrolysis, solubility, hemolysis, cell cytotoxicity in vitro, and efficacy in vivo. The PEG-DHA conjugates have showed moderate drug loadings (2.82 ~ 8.14 wt%), significantly good water-solubilities (82- ~ 163-fold of DHA), excellent in vitro anticancer activities (at concentrations ≥8 μg/ml, showed only 15–20% cell viability) with potency similar to that of native DHA, and long blood circulation half-time (5.75- ~ 16.75-fold of DHA). Subsequent tumor xenograft assays demonstrated a superior therapeutic effect of PEG-DHA on inhibition of tumor growth compared with native DHA. The novel PEG-DHA conjugates can not only improve the solubility and efficacy of DHA but also show the potential of scale-up production and clinical application.
The clinical application of dihydroartemisinin (DHA) has been hampered due to its poor water-solubility. To overcome this hurdle, we devised a novel polymer-drug conjugate, multiarm polyethylene glycol-dihydroartemisinin (PEG-DHA), made by linking DHA with multiarm polyethylene glycol. Herein, we investigated PEG-DHA on chemical structure, hydrolysis, solubility, hemolysis, cell cytotoxicity in vitro, and efficacy in vivo. The PEG-DHA conjugates have showed moderate drug loadings (2.82 , 8.14 wt%), significantly good water-solubilities (82-, 163-fold of DHA), excellent in vitro anticancer activities (at concentrations $8 mg/ml, showed only 15-20% cell viability) with potency similar to that of native DHA, and long blood circulation half-time ( L ung cancer is the most common cause of cancer-related morbidity and mortality, resulting in .1.1 million deaths per year worldwide 1,2 . Non-small-cell lung cancer (NSCLC) represents more than 80% of lung cancer diagnoses, and has an overall 5-year survival rate of approximately 16% which decreases precipitously among patients diagnosed with latestage disease 3,4 . In this situation, chemotherapy is still a mainstay treatment.
Dihydroartemisinin (DHA) is a derivative of artemisinin, the active principle of the Chinese medicinal herb Artemisia annua 5,6 . Recommended by the WHO, the drug has been used for treating more than 2 million cases of malarial infection, mainly in Africa and Asia 7 . Artemisinin and its analogs are considered as safe drugs with no obvious adverse reactions or noticeable side effects 8 . Recently, it has been found that DHA has antiproliferative effects on various tumor cell lines including cancers of the breast, colon, pancreas, liver, lung, ovary and prostate [9][10][11][12] . Moreover, some research has also shown that DHA can significantly reduce the level of c-MYC protein, which leads to cell cycle arrest and apoptosis in tumors cells 13 . Although DHA has demonstrated an anticancer activity, it still has some drawbacks, such as low bioavailability, caused by its poor solubility in solution and blood, an initial burst release effect, and high peak plasma concentration from its rapid metabolism [14][15][16] . Therefore, increasing the solubility of DHA is necessary to achieve the above-mentioned therapeutic or anticancer effects 17 .
Many attempts to increase the solubility of DHA have focused on stable substitutions at hydroxyl radical 18 , but the substitutions have no effect on the half-time in vivo. PEGylation with high solubility in water, minimal side effects, and well defined molar mass may provide a solution to these problems. An alternative strategy is to modify the lactone moiety reversibly through macromolecules in order to create a water-soluble ''prodrug'' capable of releasing active-lactone DHA via hydrolysis processes. ''Simple'' polyethylene glycol (PEG) carrier can substantially enhance the properties of the drugs. The most obvious effect of PEGylation in this system is to increase the solubility and sustained release of macromolecular drugs, which in turn increases the cellular drug availability, decreases toxicity and enhances specific activity [19][20][21][22] . However, a traditional linear PEG has only two functional groups available for the conjugating one or two drug molecules though covalent bonds, so the linear structure of PEG limits the loading capacity for small molecule drugs 23,24 . In addition, some investigators claim that the PEG with a comparatively low molecular weight may have toxic effects, and a large molecule impede the release of drugs with small molecular weights, so that drugs do not reach therapeutic concentrations at the target sites 25,26 .
To overcome these potential shortcomings, in this study, we proposed to synthesize a novel prodrug system for insoluble anticancer drug DHA by using multiarm PEG (4 or 8 arms) with more functional groups and the appropriate molecular weight (20 KDa or 40 KDa, Figure 1a), in which the 10-hydroxy group of DHA was bound to carboxylic group of PEG using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride as coupling reagent and 4-dimethylaminopyridine as the organic base ( Figure 1b). The obtained multiarm polyethylene glycol-dihydroartemisinin (PEG-DHA) was characterized to show the physicochemical properties and in vitro release profiles. The cytotoxicity in vitro was investigated by human lung cancer cells (A549) and lewis lung carcinoma cells (LLC), and also the pharmaceutical effects of these prodrugs in vivo were assessed on the C57BL/6 mouse model.
Drug-to-carrier ratios. The absorbance spectra of DHA and PEG-DHA were measured by UV-Vis spectrophotometer (Figure 1d). From the standard plot of concentration vs absorbance, the coefficient (D) for DHA was calculated to be 251.18 mg/ml. The 4armPEG 40K -DHA, 8armPEG 20K -DHA, and 8armPEG 40K -DHA solutions were diluted into 3.5 mg/ml, 2.0 mg/ml, and 6.0 mg/ml, respectively; and then the UV absorbance of them at 238 nm was determined after pretreating solutions 30 . Using the absorbance value, and employing the coefficient D obtained from above, the concentration of DHA in the sample was determined. Thus, dividing the DHA concentration by the PEG-DHA provided the percentage of DHA in the conjugates ( Table 1). The mean mass ration of drug-to-carrier for 4armPEG 40K -DHA, 8armPEG 40K -DHA, and 8armPEG 20K -DHA was 2.82 6 0.16%, 4.70 6 0.13%, and 8.14 6 0.28%; and the molar ration of them was 3.29 6 0.23, 6.94 6 0.18, and 6.24 6 0.20, respectively. The mass/molar ration indicated that approximately 1 or 2 functional groups of multiarm PEG remained unconjugated.
Solubility study. Solubility studies were conducted to investigate the solubility of PEG-DHA conjugates in various solvents ( Figure 1e). As predicted, PEGylation has substantially increased the solubility of DHA, which is an insoluble molecule by itself (Table 1). DHA is almost insoluble in water (,0.1 mg/ml) which was referred by Pharmacopoeia of People's Republic of China 31 . All the PEG-DHA conjugates have 199.8 to 289.0 mg/ml solubility in water, which is equivalent to as high as 16.26 mg/ml of DHA. This highly increased solubility (82 , 163 times increase) makes it possible to systemically evaluate the therapeutic efficacy of DHA in vivo.
Stability study. As the PEG-DHA conjugates were synthesized through covalent bonds between the carboxylic groups of PEG and hydroxide radicals of DHA, we expected the hydrolytic cleavage of ester bonds to occur before the drug can exert a significant cytotoxic effect 32 . Hydrolysis studies demonstrated that native DHA is released from PEG-DHA conjugates in PBS and the hydrolysis rate is strongly dependent on pH. PEG-DHA conjugates were placed in aqueous solutions that simulated biological fluids to measure the rates of hydrolysis by UV-Vis analysis. The stabilities of PEG-DHA conjugates were determined in buffered solutions at pH 8.1 (Figure 2a), pH 7.4 (Figure 2b), and pH 6.1 (Figure 2c) at 37uC to simulate the blood environment 33,34 . In the preclinical studies, the release of DHA could be due to several mechanisms as previously proposed 35 . The resultant hydrolysis data is shown in Figure 2 and the half-lives for these curves are given in Table 1. All conjugates were much more stable at pH 6.1 than at pH 7.4 or 8.1, and the stability trend was the same as the trend of hydrolysis rates for the three conjugates (8armPEG 20K -DHA , 8armPEG 40K -DHA , 4armPEG 40K -DHA). The hydrolysis half-lives of the conjugates were increased 3-times and 5-times at pH 6.1 over pH 7.4 and pH 8.1 values, respectively.
Hemolysis study. The detrimental interactions between conjugates and blood constituents such as the red blood cells (RBCs) must be avoided when injecting the conjugates into the blood circulation as a carrier for drug delivery 35 . Erythrocytes were incubated with two concentrations of polymer, 1 mg/ml and 0.1 mg/ml, for 1 h at 37uC. Hemolysis was evaluated by measuring the amount of hemoglobin released in the supernatant at 541 nm (Figure 3), and Triton X-100 was used to induce full hemoglobin release. Each kind of PEG-DHA (4armPEG 40K -DHA, 8armPEG 20K -DHA and 8armPEG 40K -DHA) at 0.1 mg/ml and 1 mg/ml showed a comparable hemoglobin release to blank values (,2%), which was significantly lower than comparable concentrations of polyethylenimine (PEI 25K ), a cationic polymer known to have a significant hemolytic effect 26 . Moreover, DHA is not cytotoxic to the RBCs in a previous study 36 , suggesting the excellent safety of PEG-DHA conjugates.
In vitro cytotoxicity. To ensure the effectiveness of the prodrug before their entry into human application, in vitro toxicity should be considered upfront 37,38 . To examine the cytotoxicity of DHA and PEG-DHA conjugates, a CCK-8 assay was conducted after incubating cells with different treated. The response of two cell lines (A549 and LLC) was tested in vitro by seeding the cells and exposing them to various concentrations of PEG-DHA conjugates, native DHA, or PEG. Cells were exposed to drug for 24 h, 48 h or 72 h. The analysis of vitro cytotoxicity measurements showed that DHA applied at 8 mg/ml induced cell death that was dependent upon the length of incubation, as shown in Figure 4a and 4b. At a dose of 8 mg/ml PEG-DHA conjugates, the viability was even reduced by ,85% and ,90% for LLC and A549 cells, respectively. PEG-DHA conjugates were equipotent to native DHA.
To compare the potency of conjugates, a drug concentration corresponding to 50% death of the cells (IC 50 ) was estimated from survival curves in Figure 4c and 4d, obtained from replicate experiments. The IC 50 of PEG-DHA conjugates were slightly greater than free drug ( Table 2, column 1). All conjugates were sensitivity of cells with the trend in the IC 50 values for the samples remaining the same (8armPEG 20K -DHA . 8armPEG 40K -DHA . 4armPEG 40K -DHA . DHA). These values are virtually equivalent to that for the native drug, indicating that DHA is being released into the medium.
Given the significant inhibitory effect of DHA and PEG-DHA conjugates on the viability of LLC cells, whether DHA could inhibit cell cycle progression was further examined. LLC cells was exposed to 20 mM of DHA and PEG-DHA conjugates for 24 h, and then collected for flow cytometry analysis. Significant changes in cell cycle were noted in LLC cell lines ( Figure 5). As compared with the control group, the ratio of cells in G1 phase gradually increased. The ratio of cells in G1 phase are in the order DHA . 4armPEG 40K -DHA . 8armPEG 40K -DHA . 8armPEG 20K -DHA, which is the same to the trend of the conjugates stability. The results indicate that DHA and PEG-DHA conjugates could all induce G1 phase arrest in LLC cells, and the PEG-DHA conjugates also have the similar bioactivity to DHA. In addition, our results indicate that G1 phase arrest is correlated with the extent of DHA release.
The pharmacokinetics in mice. Long blood circulation half-time of a drug carrier is desired to improve the bioavailability of the drug. The determined drug concentration after hydrolysis under basic condition was actually the total DHA in plasma, the combination of both parent form and conjugate form. The plasma clearance curves of free DHA and PEG-DHA conjugates in mice were shown in Figure 6a, b, and c. Disappearance of DHA from the blood circulation after intravenous administration of DHA injection was very rapid with the plasma concentration below 10% of injected dose (Figure 7a, 7b, and 7g, Table 3). However, a single dose of DHA given at the 50mg/kg had no effect on tumor growth. Multiple-dose treatment of 8armPEG 40K -DHA caused 89.1% TGI, and by day 24, 83.3% of animals were survived. In contrast, multiple-dose DHA treatment resulted in 43.7% TGI (Figure 7d, 7e, and 7h, Table 4). No significant changes in body weight were noticed in all treatment groups compared to control group (Figure 7c and 7f).
Discussion
DHA is shown to inhibit the growth of some human cancer cell lines with low toxicity, leading to a promising prospect in clinical application, but DHA suffers from low aqueous solubility and bioavailability [14][15][16] . So far many investigators have developed drug delivery systems for entrapping or conjugating DHA to a drug carrier in order to increase drug residence time at the site of pain and inflammation, decrease systemic exposure of immunosuppressants, and improve pharmacokinetics and preclinical efficacy.
In this study, we developed a new kind of prodrug based on multiarm PEG-DHA. Linear PEG is the most widely used for the simple synthetic steps and good water solubility, but the linear structure with only two functional groups limits the drug loading capability of PEG. While, multiarm PEG with a special molecular structure can be a better choice for highly efficient incorporation of drugs on the polymer scaffold. It has been widely demonstrated that polymers with a molecular weight .40 KDa display reduced renal clearance (enhanced pharmacokinetic), and are able to migrate through open malignant neovasculature and accumulate in tumors via the enhanced permeability and retention (EPR) effect 39 . Here the multiarm PEG was selected with very appropriate molecular weight (,40 KDa) and more functional groups (four or eight). PEG- DHA conjugates were synthesized via an esterification reaction between the carboxy groups of multiarm PEG-COOH and the hydroxyl group of DHA by a one-step method (Figure 1a and 1b). The PEG-DHA we generated is a more soluble DHA variant, owing to the hydrophilic ether bonds of PEG and enabled facile conjugation of DHA to a carrier compounds with an ester bond susceptible to hydrolysis 40 . In vitro release study showed significantly slower release kinetics for the conjugates (Figure 2). Since the ester bonds between the PEG and DHA hydrolyze and release the intact DHA under basic conditions. As expected, when treated with buffers, the releasing rate of DHA increased with increasing the pH from 6.1 to 8.1 in phosphate buffer at 37uC (Figure 2, Table 1). As a result, all the PEG-DHA conjugates show a potential to break the ester bonds, which further suggests that the anticancer activity of DHA can be regenerated during the incubation with tumor cells.
In vitro, PEG-DHA conjugates showed potent effects on A549 and LLC cell lines; however, there is a little difference in sensitivity to different cell lines. This could be due to differences in either the rate of releasing, intracellular delivery of native DHA, or resistance of cells to native DHA. PEG-DHA conjugates were comparable to native DHA in antitumor activity with IC 50 in the low concentration range (Figure 4c and 4d). The IC 50 of the conjugates correlated with the hydrolytic stabilities of the compounds in PBS, suggesting that a little more conjugates were needed to kill an equivalent fraction of cells and more released DHA from the PEG-DHA conjugates may lead to more cytotoxicity. In vitro experiments the tumor cell culture does not capture the advantages of PEGylating DHA compared to native DHA, such as improved pharmacokinetics, and hence may underestimate the efficacy of PEG-DHA conjugates.
It was confirmed in vivo, as expected, that all PEG-DHA conjugates demonstrated long blood circulation half-time, excellent anticancer activities and showed improved therapeutic efficacy of PEG-DHA over DHA in a mouse tumor xenograft model. There was almost no body weight loss in all the cured mice. They all were superior to DHA since the treatment with DHA resulted in either little TGI (single dose) or partial TGI sustained for a short period of time (multiple-dose regimen). The much enhanced antitumor efficacy of the PEG-DHA conjugates with appropriate molecular weights could be partially attributed to the high bioavailability due to the excellent water solubility, the prolonged tumor residence time, and the passive tumor targeting effect due to the EPR effect 39 .
In conclusion, a conjugate of one multiarm PEG molecule with several DHA molecules can effectively solubilize DHA. DHA conjugated in this prodrug system shows a significantly slow DHA release kinetics. PEG-DHA conjugate well retains the biological activity of DHA, and the 8armPEG 40K -DHA and 4armPEG 40K -DHA are both more active in cytotoxicity than 8armPEG 20K -DHA in vitro. In vivo treatment using PEG-DHA conjugates was significantly more effective than DHA in the LLC xenograft model. Due to the excellent anticancer efficacy as well as the straightforward chem-istry with little or no inherent impurities generated during the transformations, high drug loading capability and appropriate molecular weight, the 8armPEG 40K -DHA was selected as the lead candidate for further preclinical development. dissolved in 100 ml of tetrahydrofuran (THF), and the crude product was precipitated with ethyl ether (500 ml). After filtration, the resulting solids were recrystallized with a mixture of N,N-dimethylformamide/isopropyl alcohol (DMF/ IPA) (120 ml/480 ml). Then, the solids were filtered, washed with ethyl ether (2 3 500 ml), and dried under vacuum at 40uC to give 8armPEG 40K -DHA. 4armPEG 40K -DHA and 8armPEG 20K -DHA were similarly synthesized and purified as that for 8armPEG 40K -DHA (Table 1). Samples were dissolved in deuterated chloroform (CDCl 3 ) for analysis by 1 H-NMR (Bruker DRX-600 Avance III spectrometer).
Synthesis
Drug-to-carrier ratio characterization. A mass and molar drug/carrier ratio for the PEG-DHA conjugate was determined as described here. DHA was detected by UV-Vis spectrophotometer as reported earlier 30 . Briefly, DHA were dissolved in 75 ml 60% (wt) of ethanol water solution, filter. 5 ml of the filter was mixed with 23 ml 2% (wt) of NaOH water solution and water bath heating at 60uC for 30 min. Then The UV absorbance of the derivative of DHA was determined at 238 nm for five different concentrations ranging from 40 to 200 mg/ml. The pretreatment of PEG-DHA conjugates was the same as native DHA. A mass or molar of PEG-DHA (mConjug or nConjug) were diluted in a known concentration, and absorbance at 238 nm and the concentration of DHA in the sample was used to obtain the mass and molar of DHA (mDHA, nDHA). The mass and molar drug/carrier ratios were thus reported as mDHA/mConjug and nDHA/nConjug.
Hydrolysis in buffers.
Hydrolysis profiles were obtained in phosphate buffer at pH 6.1, 7.4 and 8.1. PEG-DHA conjugates (20 mg/ml) were diluted into phosphate buffered saline and maintained at 37uC throughout the course of the hydrolysis study. Aliquots were removed at different time points, high-speed centrifuged to get supernatant, pretreatment as described previously, and then analyzed by UV-Vis at 238 nm. The percentage was calculated on the basis of the absorbance of the sample at 0 to 90 h vs the initial absorbance. Each stability profile represents the average of two independent runs with the same sampling schedules. The standard deviation of each point is typically 2% or less.
Solubility study. The solubility of the conjugate was investigated as reported earlier 41 . Briefly, excess amounts of PEG-DHA were added to the screw capped scintillation vials containing 10 ml of various solvents or purified water. The suspension was mixed at ambient temperature. An aliquot of the sample (5 ml) was taken at 24 and 48 h intervals. Each withdrawn sample was filtered using a 0.45 mm PTFE filter, and then analyzed by UV-Vis as described previously.
Hemolysis assay. The hemolytic activity of polymer solutions were evaluated as described previously 42,43 . Briefly, fresh blood samples were collected through cardiac puncture from rats. Ten milliliters of blood was added with EDTA-Na 2 immediately to prevent coagulation. The red blood cells (RBCs) were collected by centrifugation at 1500 rpm for 10 min at 4uC. After washing in ice-cold DPBS until the supernatant was clear, erythrocytes were diluted at a final concentration of 5 3 10 8 cells/ml in icecold DPBS. 1 ml PEG-DHA conjugates solution (1 mg/ml and 0.1 mg/ml) was mixed with 1 ml erythrocyte suspension. DPBS and 1% Triton X-100 in DPBS were used as controls for 0% lysis and 100% lysis, respectively. Samples were incubated for 1 h at 37uC under constant shaking. After centrifugation at 1500 rpm for 10 min at 4uC, supernatant was analyzed for hemoglobin release at 541 nm using an infinite M200 microplate spectrophotometer (Tecan, Switzerland). Hemoglobin release was calculated as (OD sample 2 OD negative control )/(OD positive control 2 OD negative control ) 3 100%. Hemolysis was determined from three independent experiments.
In vitro cell cytotoxicity. Human lung cancer cells (A549), murine Lewis lung carcinoma (LLC) cells were obtained from the Peking University Health Science Center (China) and were grown in the listed medium: A549 (RPMI 1640 with 10% FBS, 1% streptomycin-penicillin); LLC (DMEM with 10% FBS, 1% streptomycinpenicillin). All cell lines were maintained in an incubator supplied with 5% CO 2 /95% air humidified atmosphere at 37uC. CCK-8 assay was used for cell viability of different samples 44,45 . Briefly, A549 cells were seeded at a density of 4 3 10 3 cells/per well in 180 ml culture medium within a 96-well plate (Corning, USA) and incubated overnight. Then, the cells were treated with various samples (DHA, 4armPEG 40K -DHA, 8armPEG 40K -DHA, and 8armPEG 20K -DHA) at 37uC in a humidified incubator with 5% CO 2 for 24 h, 48 h, and 72 h, where the samples of the DHA and PEG-DHA conjugates were dissolved in dimethylsulfoxide (Merck, Darmstadt, Germany) and diluted into medium before assay and DHA dose ranged from 0.2 to 8 mg/ml. 20 ml of CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well of the plate and incubated for another 1 h at 37uC. The absorbance at 450 nm was measured an infinite M200 microplate spectrophotometer. Percent viability was normalized to cell viability in the absence of the samples. The IC 50 was calculated as polymer concentration which inhibits growth of 50% of cells relative to non-treated control cells according to Unger et al. 46 . IC 50 was calculated using the Boltzmann sigmoidal function from OriginH 8.6 (OriginLab, Northampton, USA). Data are representative of three independent experiments.
Cell cycle was assessed by flow cytometry (Attune Acoustic Focusing Cytometer, Applied Biosystems, USA). 2.0 3 10 4 LLC cells were seeded in 25 cm 2 flasks with 10 ml of nutrient medium and kept at 37uC, 5% CO 2 , for 24 h. Next day, DHA and PEG-DHA conjugates at their IC 80 values were added. Cells were treated during 24 h. The adherent cells and the supernatant were harvested, centrifuged (1,000 rpm, 3 min, 4uC) and the pellet was washed with DPBS. The cells were fixed with 1 ml ice cold 70% ethanol and the samples were kept at 4uC for 18 h prior to the analysis. Fixed cells were centrifuged and resuspended with 1 ml PBS. After further centrifugation, Pharmacokinetic experiments in mice. C57BL/6 female mice (6-8 weeks) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products. All animal experiments were performed in accordance with Guide for the Care and Use of Laboratory Animals, and approved by Experimental Animal Ethics Committee in Beijing. 24 tumor-free healthy C57BL/6 female mice were divided into four groups at random. Group 1 was treated with DHA injection, groups 2-4 with different PEG-DHA conjugates, respectively, via the tail vein. All groups were given a single dose of DHA (50 mg/kg) or PEG-DHA conjugates (equal to 50 mg/kg DHA). After intravenous administration, blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 5, 10, 24, 48, 72 h from the orbital plexus and centrifuged immediately at 3,000 rpm for 10 min at 4uC. The plasma was frozen at 220uC until assay. To determine the level of total DHA in each plasma sample, 100 ml of plasma was mixed with 50 ml of 0.1 N NaOH for 15 min in water bath at 37uC, allowing the hydrolysis of the conjugate. After that, 0.1 N HCl (50 ml) was added, followed by 100 ml methanol. After vortexed for 2 min, the mixture was sonicated for 5 min and centrifuged at 5,000 rpm for 5 min. The clear supernatant was dried under nitrogen, reconstituted by 100 ml methanol before HPLC analysis 47 . The HPLC employs a VYDAC 214TP54 (C18, 5 mm, 4.6 3 250 mm) with a UV detector, using a gradient of 60% of acetonitrile in 0.05% TFA at a flow rate of 1 ml/min. Blood circulation data were plotted as the blood DHA or PEG-DHA conjugates levels with the unit of percentage of injected dose per gram (% ID/g) against time after injection.
In vivo efficacy study. LLC cells (1 3 10 6 cells/200 ml DMEM media) were subcutaneously inoculated into the shaved right lateral flank of C57BL/6 female mice 24 h post-irradiation. Treatments were started when tumor volume reached 100-150 mm 3 , and this day was designated as day 0. Non-tumor-bearing C57BL/6 female mice were injected intravenous with either DHA or PEG-DHA conjugates as a single dose of 50 mg/kg or multiple 10 mg/kg doses (every 2d, q2d 3 5). It is important to note that the doses or concentrations of PEG-DHA conjugates in this study refer to DHA equivalents. For example, a dose of 50 mg/kg of 8armPEG 40K -DHA means that the dose contains 50 mg/kg of DHA and 1063 mg/kg (21-fold higher) of whole conjugate, here the loading of DHA in the whole 8armPEG 40K -DHA is 4.70%. The mice were randomly divided into the following groups: PBS, DHA, 4armPEG 40K -DHA, 8armPEG 20K -DHA, and 8armPEG 40K -DHA were administrated via tail intravenous injection. The corresponding tumor volume data were collected by measuring tumor diameter with an electronic caliper every day. Tumor volume was calculated using the formula: (L 3 W 2 )/2, where L is the longest and W is the shortest tumor diameter (millimeter). Relative tumor volume (RTV) was calculated at each measurement time point (where RTV was equal to the tumor volume at a given time point divided by the tumor volume prior to initial treatment). To monitor potential toxicity, we measured the weight of each mouse. For humane reasons, animals were killed and regarded as dead if the implanted tumor volume reached 5,000 mm 3 . To further evaluate the hematological toxicity of different PTX formulations, we collected 200 ml of blood of each mouse after final administration. Obtained blood was immediately evaluated by a blood cell analyzer (MEK-7222K, Japan).
Statistical. All experiments in this study were performed at least three times, and the data were expressed as the mean 6 standard deviation (SD). Statistical analyses were performed by analysis of variance (ANOVA). All statistical analyses were performed using a 95% confidence interval (p , 0.05). | v3-fos-license |
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} | pes2o/s2orc | The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor
The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR’s C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with “biased agonist” properties.
INTRODUCTION
Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are peptide hormones released from the gut postprandially and act primarily to potentiate glucose-induced insulin secretion (Kim and Egan, 2008). Together these peptides mediate the incretin effect (i.e. the larger insulin response elicited by oral administration of glucose compared to intravenous administration, under comparable plasma glucose levels) by binding to their receptors (GIPR and GLP-1R respectively) expressed on pancreatic b cells (Mcintyre et al., 1964;Nauck et al., 1986). An early characteristic of type 2 diabetes mellitus is an impairment of the incretin effect (Holst et al., 2011). However, at pharmacological concentrations GLP-1 is insulinotropic in patients with T2DM whereas GIP does not appear to be (Nauck et al., 1993). As a result, several GLP-1 analogs are currently used clinically to treat T2DM, but to date, this is not the case for GIPR agonists (Andersen et al., 2018). Interestingly, peptide agonists that target both receptors have been investigated and appear to show greater efficacy in terms of glycemic control and weight loss and fewer adverse effects than agonists that target GLP-1R alone (Frias et al., 2018).
GIPR and GLP-1R are closely related members of the secretin family of G protein-coupled receptors (GPCRs) and share a high degree of sequence homology (Mayo et al., 2003;De Graaf et al., 2016). When activated, both receptors couple through Gs, resulting in an increase in intracellular cAMP (Yabe and Seino, 2011;Al-Sabah, 2016). GLP-1R has also been reported to couple to other G proteins, including Gq, which has been shown to mediate receptor internalization (Montrose-Rafizadeh et al., 1999;Thompson and Kanamarlapudi, 2015). Activation of GLP-1R also results in the rapid recruitment of arrestin to the receptor (Jorgensen et al., 2005). However, this remains a controversial subject with regards to GIPR, with some groups showing no interaction between GIPR and arrestin and others who do (Al-Sabah et al., 2014;Ismail et al., 2015;Gabe et al., 2018). Non-visual arrestins were first identified for their role in the desensitization, internalization and recycling of GPCRs (Lefkowitz, 2005;Reiter and Lefkowitz, 2006). Subsequently, arrestins have been shown to also operate as scaffolding proteins allowing GPCRs to signal in a G protein-independent manner, such as via ERK1/2 MAPKs (Luttrell et al., 2001;Xiao et al., 2010). This has led to the concept of biased agonism or functional selectivity, where ligands can favor a G proteindependent pathway or an arrestin-dependent pathway (Koole et al., 2010;Reiter et al., 2017). This could potentially lead to new therapeutics with greater efficacy and fewer adverse effects (Violin et al., 2014). While there is evidence that arrestins are involved in mediating GLP-1's insulinotropic effects on pancreatic b-cells, G protein biased GLP-1R agonists have been shown to produce greater long-term insulin release and less nausea than more balanced agonists (Sonoda et al., 2008;Jones et al., 2018).
Arrestin recruitment to GPCRs can be monitored by coimmunoprecipitation or visualization of fluorescently labeled arrestin to the plasma membrane by confocal microscopy. These techniques however are not without their drawbacks. For example, coimmunoprecipitation requires the use of a cross linking agent and neither technique is particularly amenable to quantification. More recently several resonance energy transfer techniques such fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) have been developed to study various aspects of GPCR functionality (Krasel et al., 2005;Pfleger et al., 2006). Both intermolecular FRET and BRET require the receptor and arrestin to be fused to a suitable donor/acceptor pair which allows the quantification of rate of arrestin association/dissociation to the receptor and the potency of agonists to recruit arrestin to the receptor (Krasel et al., 2004;Ayoub, 2016). Functional affinity or efficacy of an agonist for a given pathway are required to calculate a ligands' bias (Jaeger et al., 2014).
In this study we investigate how assay configuration, especially modifying receptor cell surface expression and altering the linker-region between the receptor and fluorescent protein, can dramatically affect the result of both FRET and BRET-based arrestin recruitment assays, potentially leading to false-positive results.
Construction of cDNA
cDNA encoding the following constructs have been previously described; wild-type and C-terminally enhanced yellow fluorescent protein (eYFP)-labeled human GLP-1R and GIPR (GLP-1R-eYFP, GIPR-eYFP) (Al-Sabah et al., 2014), arrestin3cyan fluorescent protein (Arr3-CFP) (Krasel et al., 2005), G protein-coupled receptor kinase 2 (GRK2) (Krasel et al., 2001). GIPR-and GLP-1R-labeled at the C-terminus with super yellow fluorescent protein 2 (SYFP2) (Kremers et al., 2006) were generated by amplifying the open reading frame of human GLP-1R and GIPR with primers which added a HindIII restriction site ahead of the start codon and replaced the stop codon with an XbaI site. The start codon of SYFP2 was replaced by PCR with an XbaI site, and a NotI site was inserted behind the stop codon of SYFP2. The resulting fusion of GIPR-XbaI-SYFP2 was cloned in pcDNA3. Subsequent mutations of the linker region between receptor and SYFP2 were performed by sitedirected mutagenesis (Q5 site-directed mutagenesis kit, New England Biolabs, USA). GIPR and GLP-1R both possess a putative N-terminal signal peptide that is cleaved during receptor processing and trafficking (Huang et al., 2010;Whitaker et al., 2012). Hence in order to N-terminally label the receptors, a FLAG-tag was introduced immediately downstream of the predicted signal peptide. This was achieved by replacing the myc-tag of N-terminally labeled GIPR and GLP-1R with a FLAG-tag (DYKDDDDK) by site-directed mutagenesis. Myc-tagged GIPR and GLP-1R constructs (previously described (Al-Sabah et al., 2014)) encode the influenza hemagglutinin signal peptide (MKTIIALSYIFCLVFAA) in place of the native signal peptide.
Nluc-Arr3 has been previously described (Al-Zamel et al., 2019). The whole construct was subsequently cloned into pcDNA5-FRT (Invitrogen) in order to generate a stable isogenic cell line. mCherry-CAAX was cloned by PCR attaching the codons for the last 18 amino acids of the small GTPase H-ras to mCherry (which encode the H-ras palmitoylation site) in the process. The PCR product was cloned into pcDNA3 using HindIII and NotI. All constructs were verified through sequencing.
Cell Culture and Transfection
HEK-293 and Flp-In HEK-293 cells (Invitrogen) were cultured in Dulbecco's modified Eagle's media supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37°C in a humidified environment containing 5% CO 2 . HEK-293 cells were transiently transfected using Effectene (Qiagen, Hilden, Germany), following the manufacturer's protocol. In order to generate stable cell lines Flp-In HEK-293 cells were transfected with the pcDNA5.FRT vector and pOG44 using Effectene. Stable isogenic clones were selected by the addition of hygromycin at a concentration of 100 µg/ml.
Luciferase Assay
Activation of various GIPR constructs was assessed by a luciferase reporter gene assay using a previously described protocol (Al-Sabah et al., 2014). Briefly, Flp-In HEK-293 cells were transiently transfected with cDNA encoding either GLP-1 or GIP receptor constructs and a reporter gene construct consisting of a cAMP-response element fused to a reporter gene encoding firefly luciferase (Cre-luc) using Effectene (Qiagen, Hilden, Germany), following the manufacturer's protocol. Twenty-four hours after transfection, the cells were seeded into white 96-well plates (Thermo Scientific, Roskilde, Denmark) at a density of 10,000 cells/well. Twenty-four hours later, the cells were incubated for 3 h in media containing peptide ligand and then lysed. Luciferase activity was quantified using LucLite reagent (PerkinElmer Life and Analytic Sciences, Wellesley, MA, USA).
Confocal Microscopy
Flp-In HEK-293 cells transiently expressing SYFP2-labeled receptors and mCherry-CAAX were plated on to a poly-Dlysin-coated coverslip and mounted on to an "Attofluor" holder (Molecular Probes, Leiden, The Netherlands). The cellular location of the labeled receptors was monitored by live cell confocal microscopy performed on a Zeiss LSM 800 meta system (Carl Zeiss, Oberkochen, Germany). Zeiss Zen Blue 2 software (2.1) was used for data acquisition and analysis. Images were taken with an oil-immersion 63× lens using the factory settings for mCherry and YFP.
BRET Assays
Flp-INFlp-In HEK-293 cells stably expressing Arrestin3-Nluc were transiently transfected with SYFP2-labeled receptor as previously described. For BRET saturation assays increasing amounts of SYFP2-labeled receptor DNA were transfected (0-2 µg). Forty-eight hours posttransfection cells were detached and washed with Hank's Balance Salt Solution (HBSS). Cells were resuspended in HBBS and plated on to white 96-well plates (PerkinElmer) in suspension at a density of 180,000 cells/well. Cell were incubated with agonist for 10 min and BRET measurements were taken using a Victor X4 (PerkinElmer) plate reader immediately after the addition of coelenterazine h (final conc. 5 µM). Nluc emission was measured through a 460/ 40 nm filter and the resulting SYFP2 emission was read through a 535/25-nm filter. Expression levels of Nluc and SYFP2-labeled constructs were monitored by measuring luminescence and fluorescence respectively. Luminescence was measured using a Victor X4 and factory settings for luminescence. For fluorescence measurements, cells from the same transfection were plated on to black 96-well plates and after 1-h incubation in darkness, total fluorescence was measured with excitation 490/6 nm and an emission filter at 535/25 nm. For BRET saturation assays raw data was corrected by subtracting the BRET ratio determined from cells expressing Nluc only. Data was then plotted as BRET ratio vs. fluorescence/luminescence and curves were fitted using "one site-specific binding" function (GraphPad 7.0).
FRET Measurements
HEK-293 cells were cotransfected with either GLP-1R-YFP or GIPR-YFP and Arr3-CFP. At 24 h posttransfection, the cells were plated on poly-D-lysine-coated coverslips (25-mm diameter) in six-well plates. After 24 h, FRET measurements were performed as previously described (Zindel et al., 2015) with two modifications: first, the light source was an LED excitation system (pE-2, CoolLED, Andover, UK); second, ligands were always applied in FRET buffer supplemented with 0.1% bovine serum albumin. The fluorescence signal at 535 nm is the sum of the YFP fluorescence and bleed through of CFP fluorescence into the YFP channel (approx. 40% of the fluorescence at 480 nm); therefore the "real" YFP fluorescence was calculated by subtracting the CFP bleed through from the F 535 signal. FRET was calculated as F YFP /F 480 . To get information about the expression of YFP-tagged receptors, cells were excited directly with 500 nm light and the fluorescence intensity was measured (dYFP, d for "direct excitation"). The CFP signal before the start of the experiment is a good indication for the expression level of the arrestin (as there is no FRET at the beginning of the experiment), and therefore the ratio F dYFP /F CFP can be used as an approximation of the stoichiometry between the receptor and arrestin.
Data Analyses
Dose-response data were fitted to a sigmoidal curve and BRET saturation experiments were fitted to one-site specific binding curve using GraphPad 7.0 (GraphPad, San Diego, CA). The values are expressed as the mean ± standard error of the mean; n = number of independent experiments. Statistical analysis of significance was calculated with GraphPad 7.0 using a two-tailed, unpaired Student's t-test or ANOVA where appropriate.
RESULTS AND DISCUSSION
Arrestin3-Nluc Recruitment to eYFP Labeled GIPR and GLP-1R Using a FRET-based assay we have previously reported that activated GLP-1R tagged at the C-terminus with eYFP and expressed in HEK-293 cells interacts robustly with both CFPtagged GRK2 and arrestin3 (b-arrestin2) whereas similarly tagged GIPR does not (Al-Sabah et al., 2014). Ismail et al. have also reported that GIPR does not significantly interact with either arrestin2 (b-arrestin1) or arrestin3 (Ismail et al., 2015). However, a recent study by Gabe et al., shows that stimulated GIPR can recruit both isoforms of arrestin (Gabe et al., 2018). Both groups used a BRET-based arrestin recruitment assay albeit using different configurations. In order to investigate this issue further we measured agonist stimulated recruitment to GIPR and GLP-1R using a BRET-based assay. Using the same receptor constructs as in our previous FRET-based experiments (GIPR-eYFP and GLP-1R-eYFP, C-terminal tail sequence shown in Figure 1), Flp-In HEK-293 cells stably expressing Arr3-Nluc were transfected with either GIPR-eYFP or GLP-1R-eYFP and stimulated with their corresponding peptide. GLP-1 stimulated Arr3 recruitment to GLP-1R-eYFP in a dose-dependent manner ( Figure 2A) with a pEC 50 value of 7.1 (± 0.3) whereas there was no significant difference in BRET ratio between unstimulated cells expressing GIPR-eYFP and those stimulated with 1 µM GIP ( Figure 2B), confirming our previous results from FRET experiments.
FLAG-GIPR-SR-SYFP2 Can Recruit Arr3
The GIPR-eYFP construct used in the previous RET experiments contains a 10 amino acid linker between the end of the receptor's C-terminal tail and the XbaI restriction-site immediately upstream of the eYFP (Figure 1). In subsequent FRET experiments a GIPR construct was employed that used a brighter version of YFP (SYFP2) and did not include the 10 amino acid linker found in the original construct (GIPR-SR-SYFP2 Figure 1). This receptor was also tagged at its N-terminus with a FLAG epitope and the native signal peptide was replaced with the influenza hemagglutinin signal peptide (FLAG-SR-GIPR-SYFP2). HEK-293 cells transiently expressing FLAG-GIPR-SR-SYFP2 and either GRK2-CFP or Arr3-CFP were observed by FRET microscopy. Stimulation with 1 µM GIP resulted in an increase in FRET ratio indicating that this GIPR construct could recruit both GRK2 and arrestin3 ( Figures 3A, B). There are two requirements for arrestin recruitment to GPCRs; an agonist-induced change in receptor conformation and phosphorylation of the agonist-occupied receptor by G proteincoupled receptor kinases (GRKs) (Krasel et al., 2005). It has also been shown that the affinity of arrestins for a GPCR can be increased by modifying the receptor so that the number of phosphorylation sites in the C-terminal tail is increased (Zindel FIGURE 1 | Sequence of the C-terminal region of GLP-1R and GIPR constructs used in the present study. GIPR-eYFP contains a 10-amino acid linker (shown in green) between the end of the C-terminal region and the XbaI site (shown in cyan), eYFP is shown in yellow. There is no linker between the end of the C-terminal tail and the XbaI site in GIPR-SR-SYFP2. However, this results in the introduction of a serine residue (S) at the end of GIPR's C-terminal region which could potentially be a phosphorylation site. The SR was subsequently substituted with GG to give GIPR-GG-SYFP2. . By removing the 10 amino acid linker and moving the XbaI restriction site, which codes for serine/arginine (SR), to the distal end of GIPR's C-terminal tail an additional potential phosphorylation site may have been introduced to GIPR. When comparing the sequence of GIPR and GLP-1R's C-terminal tail it can be seen that not only is GIPR's C-terminal tail longer but that it also contains fewer serine and threonine residues. Furthermore, GLP-1R ends on a serine residue (Figure 1). To test if this recruitment of arrestin to GIPR was due to the presence of a potential phosphorylation site, the serine/arginine coded for by the XbaI site was substituted with glycine/glycine. The resulting construct, FLAG-GIPR-GG-SYFP, showed a significantly (P<0.01) reduced ability to recruit arrestin3 with 1 µM GIP in the FRET assay ( Figure 3B) but nonetheless could still recruit arrestin3. Importantly there was no significant difference in the stoichiometry between YFP and CFP fluorescence for arrestin recruitment assays performed with either FLAG-GIPR-SR-SYFP or FLAG-GIPR-GG-SYFP ( Figure 3C). Therefore, the amplitude of the FRET signal may be compared.
GLP-1R-SYFP2, but Not GIPR-SR-SYFPR, Recruits Arrestin in a BRET Saturation Assay
To further investigate arrestin recruitment to the incretin receptors we performed BRET saturation assays. In these experiments receptors were labeled with SYFP2 at the Cterminus (Figure 1) but were not tagged at the N-terminus and retain their native signal peptides. Flp-In HEK-293 cells stably expressing Arr3-Nluc were transfected with increasing amounts of either GIPR-SR-SYFP2 or GLP-1R-SYFP2 and stimulated with 1 µM of their corresponding agonist. Data were fitted to a single binding-site equation by non-linear regression. Stimulation with 1µM resulted in a BRET signal between GLP-1R-SYFP2 and Arr3-Nluc that reached saturation (BRETmax 0.22 ± 0.01) whereas the BRET signal between nonstimulated GLP-1R-SYFP2 and both stimulated and unstimulated GIPR-SR-SYFP2 and Arr-NLuc increased in a quasi-linear fashion, suggesting a non-specific interaction between receptor and arrestin ( Figure 4). These results suggest that an additional potential phosphorylation site at the distal C-terminus is not sufficient to permit arrestin recruitment to GIPR, at least to an extent detectable in a BRET saturation assay.
Addition of an N-Terminal FLAG-Epitope and Cotransfection of GRK2 Improve Agonist Stimulated Arrestin Recruitment to GIPR-SR-SYFP2
As our FRET experiments demonstrated that FLAG-GIPR-SR-SYFP2 could recruit arrestin upon stimulation we used the same construct in a BRET saturation assay ( Figure 5A). Although we observed an increase in BRETmax with agonist stimulation this data set had a poor R square value (0.65) when fitted to a single binding-site equation and was rejected. However, when additional GRK2 (200 ng) was cotransfected with the receptor we observed a BRET signal that was best described by a one-site specific-binding model ( Figure 5B). Furthermore, when the potential phosphorylation site (SR) was substituted with (GG) agoniststimulated arrestin recruitment was abolished. The results of these BRET saturation experiments are in agreement with the results of our FRET experiments for FLAG-GIPR-SR-SYFP2. However, whereas we were unable to detect arrestin recruitment to FLAG-GIPR-GG-SYFP2 in the BRET saturation assay, we were able to in the FRET assay. This discrepancy may be due to the FRET assay's greater sensitivity or possibly because of the kinetic nature of the FRET assay. The BRET assay is an end point assay measured after a 10 min incubation with agonist whereas the FRET assay shows a peak in amplitude approximately 3 min after stimulation. Together these data demonstrate that GIPR can be engineered to interact with arrestin under certain assay conditions. To achieve this, an additional serine residue is required at the very end of GIPR's C-terminal tail. This was inadvertently achieved by placing an XbaI restriction site directly between the receptor and the fluorescent protein. However, the presence of an engineered Cterminal serine residue was not sufficient to observe arrestin recruitment to GIPR as demonstrated in our initial BRET saturation experiments (Figure 4). It was only with the inclusion of an N-terminal FLAG epitope and replacement of the signal sequence with that of hemagglutinin that we began to observe arrestin recruitment ( Figure 5A) and even then the BRET signal could only reliably be fitted to a one-site specific binding curve with the cotransfection of additional GRK2 ( Figure 5B). There are five non-visual members of the GRK family that are now understood to regulate cell signaling independent of GPCRs as well as phosphorylating GPCRs (Gurevich and Gurevich, 2019). The present study could be extended by investigating the effect of overexpression of different members of the GRK family on arrestin recruitment to both GLP-1R and GIPR. It would be interesting to investigate how the rate of arrestin association to the receptor is influenced by different GRKs.
A B
FIGURE 5 | Bioluminescence resonance energy transfer (BRET) saturation experiments for agonist-induced interaction between FLAG-GIPR-SYFP2 and arrestin3. Increasing amounts of FLAG-GIPR-SYFP2 receptors were transiently expressed in Flp-In HEK-293 stably expressing Arr3-NLuc. (A) Stimulation of FLAG-GIPR-SR-SYFP2 with 1 µM GIP results in an increase in BRET ratio that appears to produce an exponential curve. However, this data set has a poor R square value (0.65) when fitted to a single binding-site equation. (B) Cotransfection of 200 ng GRK2 with the receptor results in a BRET ratio that is best described by a one-site specific-binding model when FLAG-GIPR-SR-SYFP2 is stimulated with 1 µM GIP consistent with a specific agonist-induced interaction between the receptor and arrestin3. Substitution of the SR linker with GG (FLAG-GIPR-GG-YFP2) abolishes arrestin recruitment. Data are pooled results from at least 3 independent experiments performed in triplicate.
Substitution of GIPR's Native Signal Peptide With the Influenza Hemagglutinin Signal Peptide Increase Receptor Expression at the Plasma Membrane
The influenza hemagglutinin signal peptide has been shown to enhance the surface expression of the b 2 -adrenergic receptor and is often used for this purpose when studying GPCRs in a recombinant system (Guan et al., 1992). When expressed in adipocytes GIPR has been shown to be constitutively trafficked between the plasma membrane and intracellular compartments with less than half of the receptors being expressed at the cell membrane (Mohammad et al., 2014). We investigated the effect of replacing the native signal peptide with that of the hemagglutinin signal peptide on GIPR's cell surface expression further. HEK-293 cells transiently expressing the YFP2-labeled receptor and a membrane-targeted red fluorescent protein (mCherry) were observed by confocal microscopy. GLP-1R-SYFP2 appeared to be expressed predominantly at the plasma membrane whereas GIPR-SYFP2 appeared to also be located in intracellular compartments. The addition of a N-terminal FLAG-epitope and substitution of the native signal peptide with the influenza hemagglutinin signal peptide appears to enhance the trafficking of GIPR to the plasma membrane ( Figure 6A) and significantly increased (P<0.001) the receptor's colocalization with membranetargeted mCherry ( Figure 6B). This observation may be more accurately quantified by ligand binding studies. Nonetheless, the data from the confocal microscopy experiments demonstrate that the proportion of expressed GIPR that colocalizes with plasma membrane-targeted mCherry-CAAX is increased when the native signal peptide is replaced with the hemagglutinin signal peptide.
To test if the addition of the YFP variants to the C-terminus affects the ability of GIPR to signal through Gs, dose response curves were generated using a cAMP-response element-linked reporter gene assay (Table 1). GIP displayed a similar potency at all constructs used in the present study except GIPR-SYFP2 (SR) where GIP was significantly less potent than at wild type (P<0.01) and FLAG-GIPR-SYFP2 (GG) (P<0.05). This could possibly be explained by the absence of the hemagglutinin signal peptide and reduced receptor surface expression. Nonetheless, all receptor constructs retained their ability to interact with Gs including those that showed no interaction with arrestin3 (GIPR-eYFP, FLAG-GIPR-GG-SYFP2 and GIPR-SR-SYFP2).
CONCLUSION
GPCRs such as the b3-adrenergic receptor (b3AR) and the gonadotropin-releasing hormone (GnRHR) are known not to interact with arrestin (Cao et al., 2000;Jensen et al., 2013). While GLP-1R has been shown to robustly recruit arrestin, the A B FIGURE 6 | Visualization of the cellular location of SYFP2-labeled receptors transiently expressed in HEK-293 cells by confocal microscopy. (A) Representative live cell images of HEK-293 cells transiently cotransfected with plasma membrane targeted mCherry-CAAX (red) and SYFP2-labeled receptor (yellow). GLP-1R-SYFP2 appears to be expressed predominantly at the plasma membrane whereas GIPR-SYFP2 appears to be located not only at the plasma membrane but also in intracellular compartments. The addition of a N-terminal FLAG-epitope and substitution of the native signal peptide with the influenza hemagglutinin signal peptide appears to enhance the trafficking of GIPR to the plasma membrane. The images are representative of at least 3 independent experiments. Scale bar = 10 µm.
(B) Colocalization of the SYFP2-labeled receptors with plasma membrane-targeted mCherry. Replacement of GIPR's native signal peptide with the influenza hemagglutinin signal peptide significantly (****P < 0.001) increases the receptor's colocalization with membrane-targeted mCherry. GLP-1R-SYFP2 with its native signal peptide colocalizes with membrane-targeted mCherry to a significantly (****P < 0.001) greater extent than GIPR-SYFP2 with its native signal peptide. Data are the mean ± SEM from values measured in n = 11.
literature regarding GIPR's ability to bind arrestin is contradictory (Al-Sabah et al., 2014;Ismail et al., 2015;Gabe et al., 2018). The results presented here demonstrate how arrestin recruitment assay configuration can dramatically alter the result. Substitution of the native signal peptide with the influenza hemagglutinin signal peptide influences the outcome of the experiment, most likely by enhancing cell surface expression of the receptor. Our results also highlight the importance of the linker between receptor and fluorescent protein in BRET and FRET-based arrestin recruitment assays. The use of an XbaI restriction site may introduce an additional potential phosphorylation site, resulting in false positive results. GPCRs are also commonly tagged at their Cterminal tails with a 1D4 epitope (TETSQVAPA) (Cai et al., 2017). This epitope also adds potential phosphorylation sites to the receptors' C-terminal tail and may also affect the results of arrestin recruitment assays. With the advent of the concept of "biased agonism" and "functional selectivity" these observations become pertinent to drug discovery programs.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. | v3-fos-license |
2015-09-18T23:22:04.000Z | 2015-08-01T00:00:00.000 | 8679357 | {
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} | pes2o/s2orc | Determination of Free Radical Scavenging, Antioxidative DNA Damage Activities and Phytochemical Components of Active Fractions from Lansium domesticum Corr. Fruit
Lansium domesticum Corr. or “long-kong” is one of the most popular fruits in Thailand. Its peel (skin, SK) and seeds (SD) become waste unless recycled or applied for use. This study was undertaken to determine the bioactivity and phytochemical components of L. domesticum (LD) skin and seed extracts. Following various extraction and fractionation procedures, 12 fractions were obtained. All fractions were tested for antioxidant capacity against O2−• and OH•. It was found that the peel of L. domesticum fruits exhibited higher O2−• and OH• scavenging activity than seeds. High potential antioxidant activity was found in two fractions of 50% ethanol extract of peel followed by ethyl acetate (EA) fractionation (LDSK50-EA) and its aqueous phase (LDSK50-H2O). Therefore, these two active fractions were selected for further studies on their antioxidative activity against DNA damage by hydrogen peroxide (H2O2) in human TK6 cells using comet assay. The comet results revealed DNA-protective activity of both LDSK50-EA and LDSK50-H2O fractions when TK6 human lymphoblast cells were pre-treated at 25, 50, 100, and 200 μg/mL for 24 h prior to H2O2 exposure. The phytochemical analysis illustrated the presence of phenolic substances, mainly scopoletin, rutin, and chlorogenic acid, in these two active fractions. This study generates new information on the biological activity of L. domesticum. It will promote and strengthen the utilization of L. domesticum by-products.
Introduction
Thailand has a variety of fruits; however, only some of them are widely consumed. Among these is the fruit of Lansium domesticum Corr. which is known in Thai as "long-kong". It has been very popular in Thailand and surrounding countries in Southeast Asia. It belongs to the Meliaceae family and is known by numerous common names. In Indonesia, it is known mainly as langsat, duku, or kokosan while in Malaysia it is known as langsat, lansa, langseh, or langsep. In the Philippines, it is known as lansones and it is known as bòn-bon in Vietnam [1,2]. The well-known and economic fruit long-kong is largely cultivated in peninsular Thailand, especially in the southern region. Long-kong develops between 15 and 25 fruits per bunch with little non-sticky sap on the skin. The appearance of long-kong fruit is globular in shape with an average size of 1.2-2.4 inches in diameter ( Figure 1). It has a brittle and rough skin. It is almost seedless, with five segments of white translucent flesh [3]. The bark of L. domesticum is used traditionally as an anti-malarial remedy by the native people of Borneo [4]. The leaves have been used by indigenous people in the Philippines for the control of mosquitoes [5]. Previous phytochemical studies on the peels and seeds of L. domesticum found several types of triterpenoids [6,7]. The peel of this fruit is traditionally known to be toxic to domestic animals. Phytochemical investigations of the peels revealed the presence of triterpene glycosides and seco-onoceranoids such as lansic acid [8].
Over-production of free radical leads to "oxidative stress" that can be defined as the state of imbalance between the high production of reactive oxygen species (ROS) and the low amount of antioxidant defense systems [9]. This imbalance can cause damage to cells, contributing to cellular dysfunction and leading to chronic degenerative diseases such as atherosclerosis, diabetes, cancer, neurodegeneration, The bark of L. domesticum is used traditionally as an anti-malarial remedy by the native people of Borneo [4]. The leaves have been used by indigenous people in the Philippines for the control of mosquitoes [5]. Previous phytochemical studies on the peels and seeds of L. domesticum found several types of triterpenoids [6,7]. The peel of this fruit is traditionally known to be toxic to domestic animals. Phytochemical investigations of the peels revealed the presence of triterpene glycosides and seco-onoceranoids such as lansic acid [8].
Over-production of free radical leads to "oxidative stress" that can be defined as the state of imbalance between the high production of reactive oxygen species (ROS) and the low amount of antioxidant defense systems [9]. This imbalance can cause damage to cells, contributing to cellular dysfunction and leading to chronic degenerative diseases such as atherosclerosis, diabetes, cancer, neurodegeneration, and cardiovascular diseases [10,11]. Therefore, the balance of free radical production and a sufficient level of antioxidants are essential for health [12]. Most antioxidants found in foods and supplements are of the non-enzymatic type. They boost the human enzymatic antioxidant defense system and prevent the depletion of our enzymatic antioxidants. Epidemiological evidence has supported that antioxidants have a role in the prevention of several chronic diseases including cardiovascular disease, cancer, and diabetes [13,14]. Fruits, vegetables, and medicinal herbs are the richest sources of antioxidant compounds. They are loaded with key antioxidants such as vitamin A, C, E, β-carotene, and important minerals, including selenium and zinc [15]. Moreover, the natural flavonoids (e.g., catechin, quercetin) or other phenolic (e.g., ferulic acid) or polyphenolic compounds (e.g., resveratrol) found in fruits also exert significant antioxidative ability [16].
Nowadays, the trend of using natural antioxidants has markedly increased due to the concern about the safety of synthetic antioxidants. Consequently, fruit is considered to be an important source of natural antioxidants, especially the peels and seeds which become waste unless recycled or applied to use. Even though Thailand has a variety of fruits, only some of them are widely consumed. Among these, the fruits of long-kong have been very popular in Thailand and countries in Southeast Asia. However, there is little information concerning the biological activity, particularly antioxidant activity, of peels and seeds of long-kong fruits. Therefore, this study was undertaken on long-kong to investigate the biological activities, particularly antioxidant mechanisms, using both cell-based (antioxidative DNA damage activity) and non-cell-based (ROS scavenging property) systems. Also, the phytochemical components of active fractions from L. domesticum Corr. fruit extracts were investigated.
Sample Preparation and Extraction
Mature L. domesticum (long-kong) fruits were purchased from Talad-Thai market in Prathumthani, Thailand. After washing, the peel or skin (SK) and seeds (SD) of the fruits were separated and air-dried at 50˝C in hot air oven for 1-2 days until weight constant. Each dried sample was then ground with an electrical grinder. The grounded samples were extracted with 50% or 95% (v/v) ethanol by maceration method. Firstly, each 100 g of fine air-dried peel and seeds was mixed with 300 mL of 50% or 95% (v/v) ethanol and left overnight at room temperature. Then, supernatant of each sample was kept and added to 50% or 95% (v/v) ethanol. This step was performed 12 times to reach completion of extraction. All 12 extracts were pooled, then filtered using Whattman No.1 filter paper before being evaporated by a rotary evaporator at 45˝C to get rid of ethanol. The aqueous phase residues were further fractionated with 100 mL ethyl acetate (EA) 5 times as well as dichloromethane (DCM) of similar volume and time. All fractions were then concentrated by a rotary evaporator at 45˝C. The obtained 12 semisolid fractions were named as shown in Table 1. They were stored at 4˝C in dark conditions until utilization. The antioxidant capacity of 12 above-mentioned fractions of the peel and seeds of long-kong fruits was determined using PHOTOCHEM (Analytik Jena, Thuringia, Germany), whose principle is based upon measurement of PCL. Briefly, superoxide anion radicals (O 2´b ullet ) were generated in the system by optical excitation of luminol, which was a photosensitizer substance. The antioxidant capacity of samples was measured by their inhibitory effect on luminescence generation compared with the standard antioxidant (constructed a calibration curve). The results were presented in equivalent units (nmol) of ascorbic acid for the antioxidative capacity of the water-soluble substances (ACW) system or trolox (synthetic vitamin E) units for antioxidative capacity of the lipid-soluble substances (ACL) system. For the measurement, the L. domesticum fractions were prepared by weighing 10 mg of each sample fraction and dissolved it in 1 mL of dilution reagent (reagent 1) supplied with the ACL or ACW reagent kits. The solution was sonicated for 10 min at room temperature to facilitate complete solubility. The supernatants were filtered through 0.45 µm syringe filter. The reaction was initiated by adding 10 µL of standard antioxidant compound (ascorbic acid and trolox) or test samples (long-kong fractions) to the mixture of 2300 µL of dilution reagent (reagent 1), 200 µL of reaction buffer (reagent 2), and 25 µL of protosensitizer (reagent 3). All samples were conducted and measured in triplicate.
Deoxyribose Assay
Deoxyribose assay was performed to evaluate hydroxyl radical (OH bullet ) scavenging activity of the 12 fractions. The method was based on the determination of malondialdehyde (MDA) pink chromogen which was a degraded product of 2-deoxyribose (2-DR) damaged by OH bullet . All sample fractions were prepared as previously mentioned in PCL assay except using distilled water as solvent. Typical reactions were started by the addition of 50 µM FeCl 3 to solutions (0.5 mL final volume) containing 5 mM 2-DR, 100 µM ethylenediaminetetraacetic acid (EDTA), 10 mM phosphate buffer (pH 7.2), 0.5 mM H 2 O 2, and various concentrations of sample fractions in presence of 100 µM ascorbic acid (reducing agent) for starting the reaction and generated OH bullet . Reactions were carried out for 10 min at room temperature and stopped by the addition of 0.5 mL 2.8% trichloroacetic acid (TCA), followed by the addition of 0.5 mL thiobarbituric acid (TBA) solution. After boiling for 15 min, solutions were allowed to cool at room temperature. The absorbance of reaction mixture was measured to determine MDA pink chromogen at 532 nm in micro-plate reader system (GENios Plus, TECAN , Port Melbourne, Victoria, Australia). All samples were tested in triplicate.
Cell Culture and Preparation
The TK6 human lymphoblasts (ATCC CRL-8015, Rockville, MD, USA) were cultured in RPMI-1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% heat-activated horse serum and 1% (v/v) penicillin-streptomycin in tissue culture flask. They were maintained at 37˝C in humidified atmosphere containing 5% CO 2 as exponential growing phase prior to the experiment. Cell density at 2ˆ10 5 cells/mL was employed for each comet assay experiment. All treatments resulted in a minimum of 70% viable cells, a level sufficient for avoiding cytotoxicity artifacts in the comet assay [17].
Cell Treatment
After overnight culture, TK6 cells were centrifuged and the pellets were adjusted to 2ˆ10 5 cell/mL in fresh medium. One milliliter of cell suspension was added to 1 mL volumes of complete medium contained 25, 50, 100, or 200 µg/mL of LDSK50-EA (fraction of 50% ethanol extract of peel followed by ethyl acetate (EA) fractionation) or LDSK50-H 2 O (aqueous phase of 50% ethanol extract of peel followed by EA) in a 12 well-plate and incubated at 37˝C in 5% CO 2 incubator for 24 h.
Hydrogen Peroxide Treatment
After treatment, the chemical-containing medium was removed by centrifugation at 3500 rpm for 3 min. Cells were washed twice with cold phosphate buffered saline (PBS) before being collected by centrifugation at 3500 rpm for 3 min. The cells were resuspended in 1 mL of fresh medium containing 50 µM H 2 O 2 and incubated at 4˝C for 5 min to produce oxidative DNA damage to the cells. At the end of incubation period, the H 2 O 2 treated cells were washed twice with cold PBS and resuspended in cold PBS prior to subjection to comet assay.
Comet Slide Preparation
The procedure for slide preparation performed using the standard technique was described by Singh et al. [18] with some modifications. Comet slides were prepared by pre-coating clean regular microscope slides with 0.75% (w/v) normal melting point (NMP) agarose. Slides were allowed to dry for 1-2 h at room temperature. The second or cell-containing layer was generally prepared from mixing 25 µL of treated cells with 75 µL of 0.5% (w/v) low melting point (LMP) agarose at 37˝C and the cell suspension was rapidly spread onto a pre-coated slide. The slides were gently covered with the coverslips and placed on a cold flat surface to allow the agarose to solidify for about 5 min. The coverslips were gently removed by sliding them sideways from the slides, and 80 µL of 0.5% LMP agarose was spread on glass slides, recovered with the coverslips and left on cold surface for agarose to solidify. At least two slides were made for each treatment.
Lysing, Unwinding, and Electrophoresis
The coverslips were gently removed and slides were submerged into freshly prepared lysis solution (2.5M NaCl, 100 mM EDTA, 10 mM Tris, 10% dimethyl sulfoxide (DMSO), 1% Triton X-100, pH 10; (4˝C)) for 2 h. After lysis, the slides were equilibrated in the freshly prepared electrophoresis buffer containing alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13 at 4˝C) to allow unwinding of double-stranded DNA for approximately 20 min. The slides were then transferred into an electrophoresis unit with the same buffer and subjected to an electrophoretic field at 300 mA and 25 V at 4˝C for 20 min. The level of the electrophoresis buffer was adjusted in order to achieve 300 mA.
Neutralization and DNA Staining
Following electrophoresis, the slides were neutralized in 0.4 M Tris (pH 7.5) for 5 min three times. After removing the neutralization buffer, the slides were washed with cold water and allowed to dry at room temperature. The DNA was stained with 50 µL of 0.2% ethidium bromide.
Comet Cell Scoring
From each slide, fifty comet cells were randomly selected for comet analysis. The comet images were scored using the fluorescence microscope (at 200ˆmagnification) connected with charge coupled device (CCD) camera. The camera was linked to a personal computer containing an automatic comet image analysis software (Comet Assay III, Perceptive Instruments, Haverhill, UK). The two parameters selected as indicator of DNA damage were tail length (TL, the distance of DNA migration measured from the center of the nucleus towards the end of the tail, µm) and tail moment (TM, a measure of the distance between the center of the tail and the center of the head, multiplied by the percentage of DNA in the tail, %).
Statistical Analysis
The mean values of 50 comet cells of all experiments were analyzed. All experiments were repeated on three separate occasions. The homogeneity of variance between concentration levels was determined using Levene's test. The statistical significance of the results was determined by means of one-way analysis of variance (one-way ANOVA). When the results were significant, pair-wise comparisons of data from treated cultures with the controls were conducted using Tukey multiple comparisons. A result was considered statistically significant when the p-value ď 0.05. All analyses were performed using the SPSS statistics version 17.0 (IBM, Chicago, IL, USA).
Thin Layer Chromatography (TLC)
Stock solution containing 100 mg/mL of LDSK50-EA and 10 mg/mL of each standard was prepared by dissolving in absolute ethanol. Then, approximately 10-20 µL of LDSK50-EA stock solution and standard phytochemicals of interest (e.g., rutin, chlorogenic acid, scopoletin) were spotted on silica gel F 254 plates Alufolien (Darmstadt, Merck, Germany). The TLC plate was developed with various solvents to select the suitable system for separation and identification.
Total Phenolic Content (TPC) Determination
The total phenolic contents were determined by using Folin-Ciocalteu method [19,20]. The reaction mixture contained 100 µL of 2 mg/mL LDSK50-EA in ethanol, 500 µL of the Folin-Ciocalteu reagent, and 1 mL of 20% sodium carbonate. The final volume was made up to 10 mL with pure water. After 1 h incubation, the absorbance at 760 nm was measured and used to calculate the phenolic contents using gallic acid as standards. Total polyphenol contents were expressed as mg gallic acid equivalents (GAE) per mg sample extract (mg GAE/mg extract). Triplicate reactions were conducted. Data were reported as mean˘standard deviation (SD).
Total Flavonoid Content (TFC) Determination
The total flavonoid content was determined using the aluminum chloride colorimetric method [21] with some modification. Briefly, 1 mL of the LDSK50-EA (2 mg/mL) or rutin standard solution was mixed with 5 mL of distilled water in a test tube, followed by addition of 300 µl of a 5% (w/v) sodium nitrite solution. After 5 min, 300 µl of a 10% (w/v) aluminium chloride solution was added and the mixture was allowed to stand for a further 1 min before 2 mL of 1 M NaOH was added. The mixture was made up to 10 mL with distilled water and mixed well. The absorbance was measured immediately at 510 nm. The results of triplicate analyses were expressed as mg of rutin equivalents (RE) per mg sample extract (mg RE/mg extract).
Hydroxyl Radical Scavenging Activity
The inhibitory effect of L. domesticum fractions on 2-DR degradation was determined by measuring the competition between 2-DR and sample fractions for the OH bullet generated from the Fe 3+ /ascorbate/EDTA/H 2 O 2 system. The antioxidant activity of OH bullet scavenging was expressed as % inhibition of 2-DR degradation for the test sample of 0.5, 1.0, and 2.0 mg/mL. As shown in Table 2, the results of deoxyribose assay exhibited a wide range of OH bullet scavenging activity, demonstrated from 0.50˘0.12 to 93.44˘0.84 in % inhibition of 2-DR degradation. Results were expressed as mean˘standard deviation (SD) The antioxidant capacity of 12 L. domesticum fractions determined by PCL and deoxyribose assays was summarized in Table 3. Regarding results demonstrated in Table 3, the L. domesticum fractions that exhibited the greatest antioxidant activity by PCL and deoxyribose assays were LDSK50-EA and LDSK50-H 2 O. These two fractions were classified as active fractions and selected for further study on their DNA-protective property against H 2 O 2 .
Antioxidative DNA Damage Activity of LDSK50-EA and LDSK50-H 2 O on TK6 Cells
To investigate the antioxidative activity of LDSK50-EA and LDSK50-H 2 O in protection of DNA damage, the TK6 cells were separately pre-treated with these two fractions at 25, 50, 100, and 200 µg/mL concentrations for 24 h prior to H 2 O 2 induction. Treatments of TK6 cells with LDSK50-EA and LDSK50-H 2 O at these assigned doses for 24 h did not exhibit an inhibitory effect on cell growth rates. Results demonstrated in Table 4 indicated the percentage of TK6 living cells prior to H 2 O 2 exposure (pre-H 2 O 2 ) and after H 2 O 2 exposure (post-H 2 O 2 ) with different concentrations of LDSK50-EA and LDSK50-H 2 O fractions. In this study, any concentrations that produced cell viability of less than 70% were discarded in order to distinguish the oxidative effect from the cytotoxic effect. Results detected by comet or SCGE assay revealed that treatment of 50µM H 2 O 2 for 5 min produced DNA damage (% TM, Figure 3 Results detected by comet or SCGE assay revealed that treatment of 50µM H2O2 for 5 min produced DNA damage (% TM, Figure 3) in TK6 cells at about 10-fold greater than untreated cells. Interestingly, this DNA damage could be prevented by pre-treating the TK6 cells with LDSK50-EA at 25, 50, 100, and 200 µg/mL for 24 h. The effect was found to be in a dose-dependent manner. The highest DNA preventive effect was found at 200 µg/mL concentration. In contrast, the LDSK50-H2O fraction exhibited a slight inhibitory effect on oxidative DNA damage when tested at similar concentration ranges. The DNA protective effect against H2O2 of LDSK50-H2O was indicated by a reduction in TL ( Figure 2) and TM (= distance between the centre of gravity of the head to the centre of gravity of the tail) × (tail DNA intensity/total comet DNA intensity) (Figure 3) damage parameters in comparison to cells treated with H2O2 alone. fractionation) and LDSK50-H2O (the aqueous phase product when the L. domesticum skin was extracted with 50% aqueous ethanol and partitioned with EA) fractions followed by H2O2 damage induction by comet assay. Results were expressed as means ± standard deviation (SD) (n = 3). * Significant difference was detected from 50 µM H2O2 treatment groups at p ≤ 0.05.
Fluorescence images of comet TK6 cells evaluated for the different treatment groups were demonstrated in Figure 5. (Table 5).
Fluorescence images of comet TK6 cells evaluated for the different treatment groups were demonstrated in Figure 5. Nutrients 2015, 7 13 Table 5. DNA damage parameters including tail length (TL) and tail moment (TM) and % inhibitory effect on DNA damage of LDSK50-EA and LDSK50-H2O in TK6 cells by comet assay.
Determination of Phytochemical Components in LDSK50-EA TLC
LDSK50-EA was dissolved in absolute ethanol at a concentration of 100 mg/mL, and spotted in 10-20 µL aliquots onto silica gel F254 plates. The developing solvents were System 1:
Determination of Phytochemical Components in LDSK50-EA TLC
LDSK50-EA was dissolved in absolute ethanol at a concentration of 100 mg/mL, and spotted in 10-20 µL aliquots onto silica gel F 254 plates. The developing solvents were System 1: toluene:ethyl acetate:formic acid (5:4:1) and System 2: ethyl acetate: formic acid: acetic acid: water (137: 11:11:26). After development, the plates were dried and sprayed with PEG reagent. Bands were visualized under ultraviolet (UV) detector at 366 nm and their R f values were recorded and compared with three standard phytochemicals including scopoletin, rutin, and chlorogenic acid. TLC analysis of LDSK50-EA was shown in Figure 6. Under the detecting condition used in this study, the results clearly revealed a presence of scopoletin (R f 0.44), rutin (R f 0.34), and chlorogenic acid (R f 0.49) in LDSK50-EA. toluene:ethyl acetate:formic acid (5:4:1) and System 2: ethyl acetate: formic acid: acetic acid: water (137: 11:11:26). After development, the plates were dried and sprayed with PEG reagent. Bands were visualized under ultraviolet (UV) detector at 366 nm and their Rf values were recorded and compared with three standard phytochemicals including scopoletin, rutin, and chlorogenic acid. TLC analysis of LDSK50-EA was shown in Figure 6. Under the detecting condition used in this study, the results clearly revealed a presence of scopoletin (Rf 0.44), rutin (Rf 0.34), and chlorogenic acid (Rf 0.49) in LDSK50-EA.
TPC Amount
The content of phenolic compounds was determined following the Folin-Ciocalteu method in comparison with standard gallic acid. The results are expressed in terms of mg GAE/mg sample extract. From our study, the TPC value for LDSK50-EA was 0.198 ± 0.001 mg GAE/mg extract.
TFC Amount
The content of flavoniod compounds was determined using the aluminum chloride colorimetric method in comparison with standard rutin and the results are expressed in terms of mg rutin equivalents (RE)/mg sample extract. This study showed that the TFC value of LDSK50-EA was 0.415 ± 0.005 mg RE/mg extract.
TPC Amount
The content of phenolic compounds was determined following the Folin-Ciocalteu method in comparison with standard gallic acid. The results are expressed in terms of mg GAE/mg sample extract. From our study, the TPC value for LDSK50-EA was 0.198˘0.001 mg GAE/mg extract.
TFC Amount
The content of flavoniod compounds was determined using the aluminum chloride colorimetric method in comparison with standard rutin and the results are expressed in terms of mg rutin equivalents (RE)/mg sample extract. This study showed that the TFC value of LDSK50-EA was 0.415˘0.005 mg RE/mg extract. [22]. In such conditions, the dietary intake of antioxidant compounds is needed to assist the body in neutralizing the free radicals and to remove the harmful effects of oxidative stress. Therefore, this study is aimed at evaluating the free radical scavenging activity of long-kong L. domesticum extracts. PCL measures the potential antioxidant property of L. domesticum fractions by two different protocols, e.g., ACW and ACL, that measure the antioxidant capacity of the water-and lipid-soluble components, respectively [23,24]. The antioxidant property of compounds is quantified and expressed in equivalent concentration units of ascorbic acid and trolox equivalents for water-and lipid-soluble systems, respectively [25]. Our study found that all 12 L. domesticum fractions exhibited O 2´b ullet scavenging activity at different degrees of activity for both ACL and ACW measurement systems. Results of the ACL demonstrated that the overall antioxidant capacity of the 12 fractions ranges from 0.380 to 6.625 nmol of trolox when all samples were tested at 10 µg/mL concentration. Among these, LDSK50-EA possessed the highest antioxidant activity with an equivalent to 6.625 nmol of trolox whereas other fractions exhibited slightly different antioxidant capacities. Interestingly, the antioxidant capacity of the ACW system indicated that the 50% ethanol extract of peel (LDSK50) still had a high antioxidant capacity. A wide range of antioxidant capacities of all fractions were found from´0.065 to 98.733 nmol of ascorbic acid. The highest antioxidant activity was found in the fraction of LDSK50-H 2 O (98.733 nmol of ascorbic acid), followed by LDSK50-EA (54.660 nmol of ascorbic acid).
PCL and Deoxyribose
Regarding the PCL results, they indicated that peels of L. domesticum fruits possessed higher O 2´b ullet scavenging activity than seeds, particularly when extracted with 50% aqueous ethanol and partitioned with ethyl acetate (LDSK50-EA), which had high potential of both hydrophilic and lipophilic antioxidants. The results of ACL and ACW suggested that the O 2´b ullet scavenger in LDSK50-EA fractions was of both polar and non-polar phytochemical groups. Furthermore, the OH bullet radical scavenging activity of L. domesticum was also determined by the deoxyribose assay, another cell-free radical generating system. This assay monitored an inhibitory effect of L. domesticum fractions on 2-DR degradation by measuring the competition between 2-DR and sample fractions for the OH bullet generated from the Fe 3+ /ascorbate/EDTA/H 2 O 2 system. OH bullet radicals formed in the solution were detected by their ability to degrade 2-DR into fragments that, on heating with TBA at a low pH, formed a pink chromogen [26,27]. The absorbance read at the end of the experiment was used for the calculation of the percentage inhibition of 2-DR degradation by the test samples [28,29]. When L. domesticum fractions were added to the reaction mixture, they removed OH bullet from the sugar and prevented their degradation. The scavenging effect of L. domesticum fractions on OH bullet was determined by monitoring the reduction of deoxyribose degradation. Results were expressed as % inhibition of 2-DR degradation. In the presence of L. domesticum fractions (0.5, 1.0, and 2.0 mg/mL concentration), a wide range of OH bullet scavenging activity was found from 0.50˘0.12 to 93.44˘0.84. The LDSK50-H 2 O fraction has clearly presented to be the most effective inhibitor of the OH bullet by exhibiting 93.44%˘0.84% inhibition on 2-DR degradation. However, the wide range of % inhibition values among various L. domesticum fractions was possibly caused by their solubility character in the water, which was the solvent mainly used in the deoxyribose assay.
Antioxidative DNA Damage Activity of Two Active Fractions, LDSK50-EA and LDSK50-H 2 O, on TK6 Cells by Comet Assay
In the last two decades, the comet assay or SCGE has swiftly become one of the most popular methods in genetic toxicology. Its advantage is based upon a relatively fast, simple, and sensitive technique for the analysis of single-strand break (SSB), double-strand break (DSB), alkali-labile site (ALS) of DNA, and incomplete excision repair sites in eukaryotic individual cells [30,31]. Moreover, the comet assay has been extensively used for the investigation of the effects of antioxidants [32][33][34]. Among underlying principles, the alkaline (pH > 13) version of comet assay is superior for evaluating a broad spectrum of DNA lesions, and maximizes sensitivity for the detection of low levels of damage. Thus, it has been chosen as a useful general tool for monitoring DNA damage [31,35].
In this study, comet assay on TK6 cells was performed with the aim to evaluate the antioxidative DNA damage mechanism of LDSK50-EA against H 2 O 2 induction. H 2 O 2 is a direct non-radical reactive oxygen species. Though H 2 O 2 itself is incapable of damaging DNA directly, it is the main source of OH bullet through the Haber-Weiss and Fenton reactions [36,37]. The analysis of results obtained from the comet assay results was based on two major DNA damage parameters, e.g., the tail length (TL, in µm) and tail moment (TM, in %). However, there are comments concerning the use of these parameters since TL would reach a plateau value after migrating a certain distance but would still grow in intensity. Therefore, TM is generally considered the main representation of DNA damage [38,39].
The results of the comet assay from this study revealed that the treatment of H 2 O 2 at 50 µM for 5 min produced DNA damage (% TM) in TK6 cells at about 10-fold greater than in untreated cells. This indicated that H 2 O 2 clearly played the important role of oxidative DNA damage in TK6 cells. The geno-protective activity of LDSK50-EA and LDSK50-H 2 O in TK6 cells was found when cells were pre-treated with one of these two active fractions (25, Interestingly, the H 2 O 2 -induced DNA damage in TK6 cells was prevented by LDSK50-EA pre-treatment at 25, 50, 100, and 200 µg/mL, in a dose-dependent manner. The highest DNA preventive effect was found at 200 µg/mL concentration with % DNA damage inhibition equal to 53.47˘1.99. However, the treatment of LDSK50-EA at a dose greater than 200 µg/mL (up to 250 µg/mL) caused a very little change in the % inhibitory effect, but induced high cytotoxicity. In contrast, the LDSK50-H 2 O fraction exhibited slight inhibitory oxidative DNA damage activity when tested at similar concentrations as LDSK50-EA. Nevertheless, the pre-treatment of cells with the highest dose (more than 1000 µg/mL) of LDSK50-EA did not induce a higher % inhibition effect.
Determination of Phytochemical Components in LDSK50-EA
TLC is a separation technique that has been generally used in chemistry to separate compounds in the mixture. It is generally agreed that TLC is most effective for the low-cost analysis of samples requiring minimal sample clean-up, or where TLC allows a reduction in the number of sample preparation steps.
In this study, the TLC technique was used to detect the presence of phytochemicals in the LDSK50-EA active fraction. Following chromatogram development, the TLC plates were sprayed with various reagents, such as PEG reagent, to detect the phenolic compounds [40].
Phenolic compounds are characteristic of plants and as a group they are usually found as esters or glycosides rather than as free compounds. Current classification divides the broad category of phenolics into polyphenols and simple phenols, based solely on the number of phenol subunits present. Polyphenols possess at least two phenol subunits, including flavonoids, and those compounds with three or more phenol subunits are referred to as tannins (hydrolyzable and non-hydrolyzable) [41,42].
Under the natural product-PEG detecting condition, the results clearly revealed the presence of scopoletin (R f 0.44), rutin (R f 0.34), and chlorogenic acid (R f 0.49) in LDSK50-EA. Subsequently, the TPC of LDSK50-EA was determined using Folin-Ciocalteu reagent to quantify the amount of phenolic compounds [20]. The results are expressed in terms of mg GAE/mg sample extract. From this study, the TPC value for LDSK50-EA was 0.198˘0.001 mg GAE/mg extract. At the same time, the TFC was determined using the aluminum chloride colorimetric method in comparison with standard rutin and the results are expressed in terms of mg RE/mg sample extract [43]. The results illustrated the TFC value of LDSK50-EA to be 0.415˘0.005 mg RE/mg extract. Overall, the results of determination of the phytochemical composition in the peel extract of L. domesticum fruits (LDSK50-EA) have shown that it was the major source of phenolic and flavonoid compounds. This finding was consistent with the earlier studies [44][45][46].
The data of this study warrant the good biological activities of LDSK50-EA, including antioxidant and antioxidative DNA damage activities. Its potent biological activities may be related to the occurrence of high potential phenolic and flavonoid substances. Many studies have demonstrated the antioxidant action of phenolic compounds, acting as terminators of free radical chains and as chelators of redox-active metal ions that are capable of catalyzing lipid peroxidation [47]. Similarly, the potent radical scavenging abilities of flavonoids could contribute by inhibiting lipid peroxidation and oxidation of the low density lipoprotein (LDL) [48].
A number of in vitro experiments have found that flavonoids exert a significant antioxidative ability due to the presence of the hydroxyl groups in the B ring of the basic flavonoid structure. It donates hydrogen atoms to radical reactions. The double-bond at position 2, 3 in conjugation with the 4-oxo-group in the C ring of the flavonoid structure, and the hydroxyl groups are capable of binding transition metal ions such as iron and copper. Hence, these contribute to the chelating ability of flavonoids. In the organism, the positive effect of flavonoids is exerted via several pathways. In addition to the antioxidative effect mentioned above, flavonoids also possess other antioxidative abilities, e.g., through the stimulation of antioxidative enzymes, and have vasodilating, anti-thrombotic, anti-inflammatory, and anti-apoptic effects. Moreover, flavonoids also exhibit anti-mutagenic abilities and can inhibit the bond of cancerogenic compounds to DNA [48,49].
Conclusions
The peel of L. domesticum fruits possessed higher O 2´b ullet and OH bullet scavenging activity than the seeds, particularly when extracted with 50% aqueous ethanol and partitioned with ethyl acetate (LDSK50-EA). This fraction had high potential of both hydrophilic and lipophilic antioxidants.
Moreover, LDSK50-EA had a geno-protective effect by reduction of the DNA damage induced by H 2 O 2 radicals, proven by comet assay in TK6 cells. This study generated new and updated information on the biological activity of extracts of long-kong fruits. It may lead to a discovery of new alternative sources of natural antioxidant and anti-genotoxic substances for the prophylaxis or treatment of free radical-related diseases as well as the development of the nutraceutical product industry. | v3-fos-license |
2020-03-05T10:39:16.000Z | 2020-02-01T00:00:00.000 | 216446014 | {
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} | pes2o/s2orc | A New Cofilin-Dependent Mechanism for the Regulation of Brain Mitochondria Biogenesis and Degradation
The aim Was to study the role of post-translational modifications of cofilin in the regulation of respiration and autophagy in murine brain mitochondria. Materials and Methods The experiments were performed with C57BL/6 mice. To obtain cytoplasmic and mitochondrial fractions of the brain tissue, differential centrifugation was used. Expressions of cofilin, phospho-cofilin, K48- and K63-associated chains of ubiquitin, and the autophagy marker LC3B were determined using electrophoresis, immunoprecipitation and Western blot methods. To study the processes of ubiquitination, we used PR619 — the inhibitor of deubiquitinating enzymes. Respiratory activity of brain mitochondria was evaluated using high-resolution fluorespirometry. Results Modification of cofilin by non-canonical K63 multiubiquitin chains in the cytoplasm and mitochondria from murine brain was demonstrated. Different levels of phospho-cofilin, cofilin, and its ubiquitinated proteoforms were found. PR619, the inhibitor of deubiquitinating enzymes, affects the expression of phosphorylated and ubiquitinated forms of cofilin in the mitochondria and cytoplasm, at the same time it changes the activity of tissue respiration and mitophagy. Conclusion The sensitivity of cofilin to the inhibitor of deubiquitinating enzymes indicates the existence of a new non-catabolic mechanism of cofilin modification, which may be involved in the regulation of mitochondrial functions, specifically, the mitochondrial respiration and autophagy. The data help understand the molecular mechanisms of mitochondrial function in normal and pathological conditions, which may be useful in developing novel methods for the treatment of diseases of the nervous system.
Introduction
Mitochondria play an important role in many intracellular processes: ATP synthesis, calcium metabolism, apoptosis, nucleotide synthesis, gene expression, and epigenetic changes. The synthesis of ATP occurs with the participation of the electron transport chain coupled with oxidative phosphorylation. The energy transformation in mitochondria is controlled by a number of signaling pathways. Among other factors, the functioning of mitochondria depends on their biogenesis and degradation balance. An important role in this balance is played by mitochondria autophagy (mitophagy), which is able to selectively remove damaged mitochondria and thus maintain normal cellular functions [1]. Cells are able to induce or stop mitophagy in accordance with their energy demands. Accumulation of mitochondria that are not capable of normal functioning may disrupt the respiratory activity and interfere with the expression of genes regulating the mitochondrial network [2]. Moreover, a number of studies showed that autophagy rather than mitophagy could play a crucial role in maintaining the integrity of mitochondrial DNA [3].
Another important pathway of degradation of cellular components is ubiquitination of proteins. Ubiquitination is the covalent attachment of ubiquitin to the accepting lysine exposed on the target protein surface. The ubiquitin-proteasome system is capable of recognizing the proteins that are destined to be destroyed and removed. Resulting from polymerization of several ubiquitin molecules, the so-called multiubiquitin chains are formed. It should be noted that ubiquitination is reversible; a variety of deubiquitinating enzymes are able to detach ubiquitin from its substrates and also cleave the multiubiquitin chains. When studying the process of ubiquitination, it is important to know the sites of the modification, since the binding of ubiquitin to specific lysine residues determines the fate of the target protein and its role in cellular processes. This ubiquitin-associated mechanism regulates most of the cell functions: proliferation and differentiation, DNA repair, intracellular signal transmission, apoptosis, and the immune response [4,5]. Disruption of the ubiquitin-proteasome system can cause cell damage, and eventually, give rise to malignant neoplasms and neurodegeneration.
It is known that protein degradation driven by the ubiquitin-proteasome system depends on ATP, which is needed for the ubiquitination and for the substrate attachment to the proteasome. In addition, some components of the ubiquitination and deubiquitination systems were identified among the proteins of the outer mitochondrial membrane, e.g., ubiquitin ligase [6]. Notably, a decrease in cellular ATP levels and mild oxidative stress can increase proteasome activity. In pathology, the processes of ubiquitination and subsequent degradation of proteins by the mitochondrial 26S proteasome are impaired.
Cofilin-Dependent Mechanism of Regulation in Brain Mitochondria
Mitophagy is also an ubiquitin-associated process. Recent studies have shown that PINK1 kinase (PTENinduced kinase 1) and E3-ubiquitin ligase (parkin) play an important role in mitophagy [7]. However, the signaling mechanisms of mitophagy are not fully understood. One of the stages of the PINK1-parkin signaling cascade is the remodeling of actin filaments with the participation of LIMK1 kinase and the actin-binding protein cofilin [8]. Parkin can bind to LIMK1 and inactivate it by ubiquitination, which leads to dephosphorylation and activation of cofilin.
Cofilin is known to be involved in various intracellular processes, including the reorganization of the actin cytoskeleton, regulation of gene expression, and triggering programmed cell death [9,10]. Changes in cofilin structure lead to changes in the respiratory function of cells [11]. At the same time, there is little data on post-translational modifications of cofilin by ubiquitination and its role in the regulation of mitochondrial functions. Nevertheless, the regulatory potential of ubiquitination exceeds all known posttranslational modifications and is comparable to the functional role of phosphorylation.
Currently, modification of the ubiquitin-proteasome and ubiquitin-like signaling systems is considered a new promising therapeutic strategy in neurodegenerative diseases.
Studying the role of cofilin and its ubiquitination can contribute in the understanding of the regulation of mitochondrial functions in normal and pathological conditions and the development of new treatments.
The aim was to study the role of post-translational modifications of cofilin in the regulation of respiration and autophagy of mouse brain mitochondria. Preparation of cytoplasmic and mitochondrial fractions of the brain tissue. The cytoplasmic and mitochondrial fractions were prepared using differential centrifugation method. After cervical dislocation and decapitation, the brain stem, cerebellum and olfactory components were cut off; the brain hemispheres were washed in an isolation medium (225 mM mannitol; 75 mM sucrose; 5 mM HEPES; 1 mM EGTA; 0.1% BSA; pH 7.4; 4°C) and placed in an incubation medium (110 mM sucrose; 60 mM lactobionic acid; 20 mM taurine; 20 mM HEPES; 10 mM KH 2 PO 4 ; 3 mM MgCl 2 ; 0.5 mM EGTA; 0.1% BSA; pH 7.2; 4ºС). The tissue was ground in a Potter-Elvehjem homogenizer. The resulting homogenate was centrifuged (100 g, 4°C, 10 min). The supernatant was centrifuged again at 9500 g, 4°C for 10 min to obtain the cytoplasmic fraction (supernatant) and mitochondrial fraction (sediment). The pellet was resuspended in the isolation medium without BSA.
Materials and Methods
The resulting mitochondrial and cytoplasmic fractions were incubated for 2 h with PBS alone in the intact group, 0.25% DMSO (dimethyl sulfoxide) in the control group, and 5 μM PR619 (a deubiquitinating enzyme inhibitor from Abcam, USA) in the experimental group and then used in subsequent experiments.
Immunoprecipitation, electrophoresis, and immunoblotting. After the incubation, the mitochondrial and cytoplasmic fractions of the brain tissue were resuspended in the RIPA lysis buffer at 4°C: 50 mM Tris-HCl; pH 8.0; 500 mM NaCl; 1% NP40; 0.1% SDS; 1 mM NEM; and 0.5 mM of the protease inhibitor cocktail (Sigma, USA) -and were treated with ultrasound. Then the samples were centrifuged at 14,000 g for 15 min at 4°C. Aliquots of each supernatant were used for subsequent experimentation. The protein concentration was made identical in all experimental samples. Immunoprecipitation was performed using anti-cofilin antibodies (Santa Cruz Biotechnology, USA) from the Immunoprecipitation Starter Pack (GE Healthcare, USA) in accordance with the attached instructions.
Protein-containing samples (20 μl/well) and molecular weight markers (ThermoFisher, USA) were loaded on a 12% SDS gel and run for 45 min at 20 mA in a working buffer (0.125 M Tris base; 0.96 M glycine; 0.5% SDS; pH 8.3). The samples were then transferred onto a PVDF membrane in an Immobilon-P system (Merk Millipore, Germany) using a semi-dry tank blot chamber (Bio-Rad Laboratories, USA) in accordance with the manufacturer's recommendations. Immunoblotting was performed using the SNAP i.d.® 2.0 system (Merk Millipore, USA). All Western blotting procedures were performed at room temperature with the exception of the transfer to PVDF membranes. The membranes were blocked with 1% BSA in PBST (0.02 M PBS containing 0.1% Tween-20; pH 7.4) and then incubated with primary antibodies in PBST with 1% BSA.
The following primary antibodies were used: rabbit polyclonal anti-ubiquitin (Dako, USA); mouse monoclonal anti-ubiquitin (linkage-specific K63) (Abcam, UK); mouse monoclonal anti-cofilin (Santa Cruz Biotechnology); mouse monoclonal anti-βactin (Santa Cruz Biotechnology); rabbit monoclonal anti-ubiquitin (linkage-specific K48) (Abcam); rabbit monoclonal anti-LC3B (Abcam). Excess primary antibodies were removed from the membranes by washing in PBST. Secondary anti-mouse or anti-rabbit antibodies conjugated to peroxidase (Sigma-Aldrich, Germany) were incubated with the membranes in PBST with 1% BSA for 10 min at room temperature. Excess secondary antibodies were removed by washing the membranes in PBST. Membranes were treated with the ECL reagent (GE Healthcare, USA) and exposed to Amersham Hyperfilm ECL film (Scientific Laboratory Supplies, UK). Detection and quantification of the signal were performed using the Gel Analyzer software.
Assessment of mitochondrial respiration. The rate of oxygen consumption was measured using high-resolution fluorespirometry with the help of an Oxygraph-2k respirometer (Oroboros Instruments, Austria). Incubation medium (2.1 ml) was added to the chambers. The device was calibrated at air saturation and zero oxygen concentration.
At the start of the experiment, mitochondrial respiration was recorded in the absence of exogenous substrates (routine respiration, R). After adding the respiration substrates (5 mM pyruvate, 2 mM malate) but in the absence of ADP (adenosine diphosphate), the respiration rate reflected the non-phosphorylating state of the respiratory chain associated with mitochondrial complex I. The rate of respiration during oxidative phosphorylation associated with complex I was measured after adding 1.25 mM ADP, and that associated with complexes I and IIafter adding 10 mM succinate (P). Then, the ATP synthase inhibitor oligomycin (2 ng/ml) was added to identify the respiration in the non-phosphorylating state with the participation of complexes I and II (L). The maximum respiration rate associated with complexes I and II was estimated after the sequential addition of the CCCP -the carbonyl cyanide m-chlorophenylhydrazone protonophore (E). The maximum activity of the respiratory chain associated with complex II was measured after the addition of 0.5 μM rotenone, an inhibitor of complex I (Rot). After adding 2.5 μM antimycin A (an inhibitor of complex III), the oxygen consumption rate was no more associated with the respiratory chain of mitochondria, but was due to other oxidative reactions. To assess the function and regulation of the respiratory chain of mitochondria, we calculated the following ratios: R/E, L/E, P/E, Rot/E, P/L (respiratory control). The primary data were processed using the DatLab 5.0 software and normalized per 1 mg of mitochondrial protein. The protein content was determined by the Bradford method. The rate of oxygen consumption (O 2 ) was expressed in picomoles per second/1 mg of mitochondrial protein.
Statistical analysis. Data are presented as mean ± standard error of the mean (M±SEM). The significance of differences between the experimental groups was determined using Student's t-test. Differences were considered statistically significant at p≤0.05.
Post-translational modifications of cofilin in the cytoplasm and mitochondria of the murine brain tissue E x p r e s s i o n o f p h o s p h o -c o f i l i n a n d c o f i l i n.
Western blotting was used to study the intracellular level of phospho-cofilin and cofilin. Figure 1 shows expression levels of phospho-cofilin and monomeric cofilin (17 kDa) in the mitochondrial and cytoplasmic fractions of mouse brain tissue. We found that the PR619, inhibitor of deubiquitinating enzymes, caused a decrease in the level of monomeric cofilin in the mitochondria (as compared to the intact and control groups), as well as a 1.3-fold increase in the level of cofilin in the cytoplasm (compared with the intact group). In the presence of PR619, expression of phospho-cofilin in the cytoplasm was significantly higher than the respective values in the intact and control groups; some increase was also found in the mitochondria. Incubation of mitochondria with PR619 led to a decrease in the cofilin/phospho-cofilin ratio in the experimental group, which was indicative of cofilin inactivation.
C o f i l i n u b i q u i t i n a t i o n i n t h e c y t o p l a s m a n d m i t o c h o n d r i a.
These processes were studied using immunoprecipitation and Western blot analysis. The Western blotting with anti-cofilin antibodies revealed the presence of monomeric (17 kDa) and mediummolecular weight forms (proteoforms) of cofilin in lysates of the cytoplasm and mitochondria, as well as in the immunoprecipitates.
At the next step, we studied the type of ubiquitin chains associated with cofilin in the cytoplasmic and mitochondrial fractions of murine brain tissue. To this end, we analyzed cofilin immunoprecipitates with antibodies against Lys48-and Lys63-associated ubiquitin chains (K63 ubiquitin). Lys48-modified cofilin proteoforms were detected in both the mitochondrial and cytoplasmic fractions. In addition, the medium-molecular weight forms of cofilin (30 and 70 kDa) were found to have specific cross-immunoreactivity with K63 chains of ubiquitin, thus indicating a modification of cofilin by K63 multiubiquitin chains (Figure 2).
Further quantification of the PR619 effect showed a 1.5-fold increase in the level of cofilin modified by K63 ubiquitin multichains (30 kDa) in the cytoplasm as compared to the intact group with no difference vs. the control group. At the same time, PR619 did not change the level of medium molecular weight proteoforms of cofilin in the mitochondria (Figure 3).
The study of autophagy in the cytoplasmic and mitochondrial fractions of brain tissue. We found that in the presence of PR619, the level of LC3B-I decreased and the level of LC3B-II increased in the mitochondrial fraction; as a result, the LC3B-II/ LC3B-I ratio increased in the mitochondria (Figure 4). In the cytoplasm, the ratio of LC3B-II/LC3B-I did not change.
Analysis of mitochondrial respiration.
To study the mitochondrial respiration, high-resolution fluorespirometry was used ( Figure 5).
The following estimated ratios were used to evaluate the function of the respiratory chain: R/E, L/E, P/E, R/P, P/L (respiratory control), and Rot/E (see the Table). In the presence of PR619, the R/E ratio increased about 1.4-fold as compared with the intact and control groups. A 2-fold increase in the ratio of the complexes I and II-associated respiration (with the mixture of substrates) to the maximum respiration (P/E) was detected in the presence of PR619; no changes were found in the intact or control groups.
Significantly lower ratios of the respiration rate with endogenous substrates to respiration with the substrate mixture (R/P) and a significant increase in the Rot/E ratio in mitochondria treated with PR619 were found.
Discussion
Changes in energy metabolism (the transition from oxidative phosphorylation to glycolysis and vice versa) depend on remodeling of the mitochondrial network, including the removal of damaged mitochondria and the biogenesis of new ones [12]. Moreover, disruption of the mitochondrial electron transport chain and ATP synthesis have an impact on autophagy and mitophagy.
In the present work, post-translational modifications of cofilin and its role in the mitochondrial functions were studied using murine brain mitochondria. We established the presence of both phosphorylated and dephosphorylated forms of cofilin in the cytoplasmic and mitochondrial fractions of brain cells. According to the results, the inactive phosphorylated form of cofilin predominates in the mitochondria of the intact group. Recent studies have shown that mitochondrial division and mitophagy depend on the assembly/disassembly of actin filaments, regulated by the actin-depolymerization activity of cofilin [13].
Phosphorylation -dephosphorylation is considered the main mechanism regulating the cofilin activity. Dephosphorylated cofilin can bind to G-actin and then translocate to mitochondria, which changes the actin cytoskeleton, causes mitochondrial dysfunction, provokes a release of cytochrome C, and apoptosis [14]. Cofilin-mediated Bax translocation is thought to be a key event in the excitotoxic death of neurons [15].
In the next step, we studied the process of cofilin ubiquitination. Ubiquitination is one of the most important mechanisms of post-translational modification of proteins. The regulatory potential of ubiquitination exceeds all known post-translational modifications and is comparable to phosphorylation as far as the control over cell function is concerned. Specifically, the Cofilin-Dependent Mechanism of Regulation in Brain Mitochondria Time ubiquitin system is involved in regulating the cell cycle, intracellular signal transmission, apoptosis, and DNA repair, whereas ubiquitination plays a crucial role in the pathogenesis of diseases [4,5].
The analysis of cofilin immunoprecipitates with antibodies against Lys48-and Lys63-associated ubiquitin chains indicated the presence of medium molecular weight cofilin proteoforms modified in the lysine 48 and 63 positions both in the mitochondria and cytoplasm of brain cells. In the present study, noncanonical modification of cofilin by K63 multiubiquitin chains has been demonstrated for the first time in the mitochondria of nerve cells. These results are consistent with data on cofilin modified by ubiquitin chains in epithelial cells [16].
The decrease in the cofilin/phospho-cofilin ratio in the murine brain mitochondria treated with PR619, the inhibitor of deubiquitinating enzymes, shows cofilin inactivation. Under the same PR619 treatment though, the level of cofilin modified by K63 multiubiquitin chains (30 kDa) increased in the cytoplasm (with some decrease in the mitochondria), which indicated a redistribution of the medium-molecular weight proteoforms of cofilin between the cytoplasm and mitochondria. The sensitivity of cofilin to PR619 may reflect a new non-catabolic mechanism of cofilin regulation.
The obtained data suggest that the intracellular deubiquitinating enzymes closely are related to the mechanism of control over cofilin proteoforms. It is possible that the modification of cofilin by K63 multiubiquitin chains can trigger additional intracellular processes. According to the literature, K63-associated ubiquitin chains can be a signal of degradation of ubiquitinated proteins by autophagy [17]. Specifically, the C-terminal ubiquitin-binding domain of the p62 protein can bind both Lys48-and Lys63-associated ubiquitin chains with a higher affinity for Lys63 chains. Both autophagy and the ubiquitin-proteosome system are the two main mechanisms for the degradation of intracellular components. In addition, it has been shown that Lys63-associated ubiquitin chains activate the NF-κB transcription factor, DNA repair, immune response, removal of damaged proteins and mitochondria, and splicing/translation of mRNA.
It is known that K63 ubiquitination plays a key role in the proteosome-associated protein degradation upon the formation of branched K48/K63 chains of ubiquitin. Notably, the deubiquitinating enzymes specific for the K63-associated chains of ubiquitin block this pathway. Our results showed an increase in the LC3B-II/LC3B-I ratio in the mitochondria, which might indicate an activation of the mitophagy process. The physiological role of deubiquitinating enzymes in the cell is to control the ubiquitination processes and maintain stability of proteins. Hyper-or hypofunction of these enzymes can lead to cell pathology [18]. For example, USP30 deubiquitinase is able to inhibit cell apoptosis and stop mitophagy mediated by the parkin protein (E3 ligase) [19].
According to Seiberlich et al. [20], inhibition of deubiquitinating enzymes leads to the accumulation of ubiquitinated proteins, the formation of protein aggregates and the subsequent inhibition of the ubiquitin-proteasome system. Using an oligodendroglial cell line (OLN-t40), the authors showed that inhibition of deubiquitinating enzymes led to activation of autophagy. Thus, the activation of mitophagy by PR619 may partially compensate for the malfunction of the ubiquitinproteasome system and indicate coordination between these degradation systems.
Based on the obtained data, it can be assumed that the protein ubiquitination correlates with mitochondrial dysfunction. Functional and structural damage to mitochondria in various diseases leads to impaired energy metabolism, decreased ATP levels, an increased production of reactive oxygen species and premature death of nerve cells. In this work, using the method of high-resolution respirometry, a change in mitochondrial respiration was detected in the presence of PR619 -the broadly specific inhibitor of deubiquitinating enzymes. The increase in the R/E ratio in the presence of PR619 indicated that the respiratory chain of mitochondria was functioning at a close to maximum velocity.
Previously, using confocal fluorescence microscopy and fluorescence lifetime imaging microscopy [21], we showed an increase in mitochondrial respiration (compared to the rate of glycolysis) in a murine brain hippocampal cell culture treated with PR619; the present results are consistent with the previous ones. The increase in the P/E ratio indicates the inability of mitochondria to use the substrates of complexes I and II for oxidative phosphorylation. Likewise, the low R/P ratio in the presence of PR619 also indicates an insufficient ATP synthesis activity. The increase in the Rot/E ratio indicates a dysfunction of the mitochondrial complex I in the presence of PR619. At the same time, the increased R/E ratio may reflect a decrease in the membrane potential of the mitochondria. It is known that a disturbance of the membrane potential can trigger the processes of mitophagy and autophagy.
Thus, the effect of deubiquitinating enzyme inhibitors on mitochondrial respiration and mitophagy suggests the involvement of the ubiquitin-proteasome system in the regulation of these two processes. Redistribution of cofilin proteoforms between the cytoplasm and mitochondria with a change in the mitochondrial functions indicates the involvement of cofilin and its post-translational forms in the regulation of mitochondrial biogenesis and degradation.
Conclusion
The biogenesis and degradation of mitochondria are significant factors of cell physiology and pathology. The sensitivity of cofilin to the deubiquitinating enzyme inhibitor suggests the existence of a new, non-catabolic mechanism that controls the structure and function of cofilin. Such a mechanism may be involved in the regulation of mitochondrial functions, respiration, and autophagy. By targeting the ubiquitin-proteasome system and ubiquitin-like signaling systems it is possible to find new therapeutic solutions. Understanding the mechanisms controlling the mitochondrial functions is important for developing novel strategies for the prevention and treatment of diseases of the nervous system.
Research funding. The study was supported by the Russian Science Foundation (project 17-75-10202) in the research on mitochondrial respiration and ubiquitination and by the Russian Foundation for Basic Research (project 18-34-00690) in the study of autophagy and post-translational cofilin modifications in mitochondria.
Conflict of interest. The authors have no conflict of interest. | v3-fos-license |
2018-09-16T06:22:59.805Z | 2018-09-01T00:00:00.000 | 52180773 | {
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"year": 2018
} | pes2o/s2orc | The Golden Activity of Lysinibacillus sphaericus: New Insights on Gold Accumulation and Possible Nanoparticles Biosynthesis
Power struggles surrounding the increasing economic development of gold mining give rise to severe environmental and social problems. Two new strains of Lysinibacillus sphaericus were isolated from an area of active alluvial gold mining exploitation at El Bagre, Antioquia. The absorption capacity of these strains and some of the L. sphaericus Microbiological Research Center (CIMIC) collection (CBAM5, OT4b.31, III(3)7) were evaluated by spectrophotometry according to a calibration gold curve of HAuCl4− with concentrations between 0 µg/mL and 100 µg/mL. Bioassays with living biomass were carried out with an initial gold concentration of 60 µg/mL. Their sorption capacity was evident, reaching percentages of gold removal between 25% and 85% in the first 2 h and 75% to 95% after 48 h. Biosynthesis of possible gold nanoparticles (AuNPs) in assays with living biomass was also observed. Metal sorption was evaluated using scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS) analysis. The sorption and fabrication capacity exhibited by the evaluated strains of L. sphaericus converts this microorganism into a potential alternative for biomining processes, especially those related to gold extraction.
Introduction
Mining is an important worldwide activity; indeed, it influences and involves economic, political, environmental, and social aspects in a complex matrix. The exploitation of metallic and non-metallic minerals can be carried out in soil, sub-soil, or even in riverbeds. Nowadays, gold mining is a controversial topic; it is associated with negative impacts such as the adverse effects in terms of environmental pollution and high health risk due to excessive exposure to mercury and cyanide, especially in developing countries where illegal and artisanal mining are present [1].
In Colombia, the gold industry plays an important role in the economic development of the country. Colombia is the fourth producer of gold in South America, and is among the 20 major producers in the global ranking, besides developed countries and world powers as China, the United States, Russia, and Australia [2]. Because gold is the only financial asset which is not controlled, it is a meaningful commodity not only for Colombia but worldwide. Unfortunately, this is also the reason why the gold mining industry is associated with violence, power struggles in the country, and an excessive presence of illegal mining [3]. In Colombia, the highest percentage of environmental licenses and, consequently, exploited hectares are in the departments of Choco and Antioquia [2]. The direct repercussions in these areas are the adverse environmental effect on water bodies, destruction of the diversity, and important health problems emerging in the surrounding communities [4,5].
It is true that areas with active mine exploitation represent challenging environments for microorganisms, due to their extreme conditions such as high pressure and temperature, elevated salt concentrations, a diverse range of acid and alkaline soil/water, and many others abiotic factors [6]. However, an impressive diversity of microorganisms has been found in these types of habitats including those associated with gold mining activity, where communities of Phylum Proteobacteria, Firmicutes and Actinobacteria, are predominant [7][8][9][10]. Despite the fact that the most abundant organisms are bacteria, representatives of the Archea and Eukarya domains have also been found [11][12][13]. The ample metabolic capacity of the organisms present in these areas is evident, and their ability to act as Fe, S, NH 3 , and CH 4 oxidants and SO 4 2− reducers has been proven [8,[14][15][16][17].
Microorganisms present great potential in sustainable mining, due to their metabolic capacities. For example, it has been demonstrated that some of them accelerate the process of sulfur oxidation and, as such, can be used in biomining and bioleaching [18]. Additionally, acidophilic microorganisms have allowed the development of different strategies for the remediation of important contamination problems in the exploitation of minerals; for example, the regulation and management of pH in the precipitation of iron in acid mine drainage (AMD) [19]. Furthermore, microorganisms, especially bacteria, have great potential in terms of the immobilization and accumulation of heavy metals such as Cu, Pb, Cr, and Fe, among others [20][21][22][23][24]. In this sense, they have the capacity to be used as an ideal alternative for remediation of contaminated sites, and an alternative to recover precious metals.
Gold exploitation is becoming increasingly important due to growing demand for the metal. This puts pressure on the industry to incorporate new methodologies and processes for efficient, less expensive, and ecologically sustainable extraction. In the biochemical cycle of the metal, secondary gold can be formed as a result of oxidation, solubilization, reduction, and precipitation of the ore under surface conditions due to microorganism activity [25]. Wide spectrums of organisms such as fungi, algae, and, in particular, bacteria have proved their potential for gold biomining at different stages of metal extraction and purification [26][27][28][29].
Lysinibacillus sphaericus, a sporulated gram-positive bacillus has become a focus of sustainable economic industry development because of its capacity for metal binding and remediation of contaminated matrices [30,31]. The first strain that exhibited metal binding capabilities was the JG-A12 (isolated from a uranium mining waste pile in Germany), which accumulated U, Cu, Pb, Al and Cd on its surface [32]. Others strains of the genus also manifest these sorption abilities with diverse metals such as Fe, Cr, Zn, among others [21,22,33].
The aim of the present study was to elucidate the ability of gold absorption by two new strains of L. sphaericus isolated from an area of active alluvial gold mining exploitation at El Bagre, Antioquia. The bioassays carried out showed efficiency in bioaccumulation and biosorption of gold in living cells, as well as the possible fabrication of nanoparticles.
Study Samples
Sampling was carried out in the exploitation territory of the company Mineros S.A., which specializes in alluvial gold mining in the Nechi River in the department of Antioquia, Colombia. This area is about 43,000 hectares with the following coordinates: 7 • 39 73 N, 74 • 47 22.73 O. Water samples were collected from different points throughout the gold exploitation process: Near to suction dredge, scoop dredge, moisture, and site of restoration at the initial phase. These samples were subjected to thermal shock to detect sporulated gram-positive bacilli. Five mL per sample was taken to be subjected to a temperature of 90 • C for 20 min. Dilutions from 10 −1 and 10 −2 were used. All cultures were carried out in duplicate and incubated for 48 h at 30 • C in nutrient agar (NA) [34].
L. sphaericus Strain Identification
For morphotypes identification, 16S rDNA gene amplification, sequencing, and phylogenetic analysis were carried out. Primers 27F (AGAGTTTGATCMTGGCTCAG) and 1493R (TACGGYTACCT TGTTACGACTT) [35] were used to amplify the 16S rDNA gene. For amplifications, 25-µL reactions were prepared containing 100 µM each dNTPs, 0.2 µM of each primer, 3 mM MgCl2, 2U Taq polymerase (Bioline), 1X PCR buffer, and 1 µL of crude extract from an overnight culture as the template DNA source. The amplification program consisted of a denaturing step of 94 • C for 3 min, 25 cycles of denaturing for 45 s at 94 • C, annealing for 45 s at 50 • C, and extension at 72 • C for 45 s and a final extension of 72 • C for 7 min. The PCR products were visualized in 1.0% agarose gels, then purified and sequenced by Macrogen Inc. (Seoul, Korea). Resulting sequences were compared with NCBI and RDP databases. The phylogenetic tree was constructed using sequences of L. sphaericus and Bacillus sp. reference strains obtained from GenBank. The phylogenetic tree choosen was the one that exhibited the minor Bayesian Information Criterion (BIC).
Additionally, characterization by the presence of S-layer protein and MTX and Bin toxins, and toxicology bioassays in larvae of Culex quinquefasciatus and Aedes aegypti were also performed [36]. The nucleotide sequences of the L. sphaericus strains from this study were deposited in the GenBank database under the accession numbers MH447519 and MH447518. A summary of the strains used in this study are shown in Table 1.
Calibration Curve for Gold Determination (HAuCl 4 )
For the calibration curve, 0.01 g of pure gold was dissolved in 20 mL of aqua regia and evaporated until the solution was near to dryness. Then, it was dissolved in approximately 2 mL of concentrated HCl and diluted with deionized water to 100 mL in a measuring flask [37]. Series of 20 mL for gold standard solutions between 0 and 100 µg/mL were used for the calibration curve. The absorption spectrum used was 313 nm and the linear regression equation obtained was A = 0.0251C-0.0101; R 2 = 0.9996.
Metal Sorption in L. sphaericus Strains
Sorption was evaluated by spectrophotometry of the biomass suspended in minimal salt medium (MSM) with sodium acetate at 0.5% [21]. Experiments were carried out in triplicate and with a HAuCl 4 − concentration of 60 µg/mL. The total volume was 15 mL in 50 mL flasks with a concentration between 10 6 and 10 7 UFC/mL. Gold within the cells was determined through supernatant measurements taken every 15 min for 2 h [21] and a final determination was made at 48 h for the possible formation of AuNPs.
The bioassays were carried out using CBAM5, OT4b.31 and III(3)7 strains, from the L. sphaericus CIMIC collection [30,38,39]. For these strains, the determinations were carried out using the mixture of the three strains and CBAM5 alone. The latter (CBAM5) was used for methods calibration due to its capacity to capture mercury [38]. This metal is highly likely to be found in the Nechi River, due to the presence of illegal and artisanal gold mining. Additionally, bioassays using the isolated strains of this study were also performed: L. sphaericus MCB1 and MCB2 as individual strains and as a mixture.
The light, oxygen, temperature, and agitation conditions were controlled, keeping the experiments in darkness with 150 rpm and a constant temperature of 30 • C [26].
SEM and EDS Analysis
The samples were fixed in 2.5% glutaraldehyde for 7 h followed by a process of dehydration through rinsing in an increasing ethanol gradient [40]. Modifications of previous protocol were carried out for the final steps. For sample mounting, briefly, a drop of approximately 5 uL of each sample was placed on an aluminum support and dried at room temperature for its posterior SEM observation and metal semi quantitation with energy-dispersive X-ray spectroscopy (EDS) using a JEOL JSM-6490LV (JEOL, Tokyo, Japan) scanning electron microscope equipped with an Oxford INCA PentaFetX3 EDS detector.
Samples were prepared for metal determination inside the cell, using a modification of previous protocols [41]. After glutaraldehyde fixation, a gradient between ethanol and resin was carried out until the sample was 100% embedded in just resin. Then, the sample was heated for 24 h at 56 • C. After polymerization, samples were cut using Leica ULTRACUT UC7 microtome to obtain 70 nm thick slices, which were placed on 200 mesh cupper grids. Uranyl acetate was used as contrast agent. Samples were observed using a Tescan LYRA 3 Scanning electron microscope (TESCAN, Brno, Czech Republic).
Sporulated Microorganisms
A total of 12 morphotypes were identified as sporulated microorganisms. Although the majority of them were gram variable or positive bacilli, gram-negative cocci and bacilli were also found in the samples. Only two morphotypes were identified as L. sphaericus through PCR of complete 16S and a posterior sequencing and analysis in NCBI and RDP databases. The phylogenetic tree was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model [42]. The tree with the highest log likelihood is shown (Figure 1). The percentage of replicate trees, in which the associated taxa clustered together in the bootstrap test (1000 replicates), are shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then the topology with superior log likelihood value was selected. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.0500)). Evolutionary analyses were conducted in MEGA X [43].
Additionally, tests were also carried out as confirmation probes. In both cases, the shape of the spore was terminal, the toxic bioassays show mortality for the larvae of C. quinquefasciatus and A. aegypti and the presence of the S-layer protein was evident ( Figure S1, Table S1). This protein is intrinsically associated with metal accumulation due to its characteristic morphology. It is the outermost layer of the microorganisms, composed of a single protein or a group of glycoprotein monomers and with a capability for self-assembly [44,45]. By virtue of this, microorganisms with S-layer are a potential tool for processes of bioremediation and biotechnology. Following these tests, the morphotypes were identified as L. sphaericus. Because they are new strains, their denotation in this study from now on will be MCB1 and MCB2 with the accession numbers MH447519 and MH447518, respectively, in the NCBI.
Metal Sorption in Living Cells of L. sphaericus Strains
The individual strain CBAM5, used as a calibration method, reached a percentage of recovery of 60% in the first 3 h of the bioassay and approximately 95% after 48 h (Figure 2a). The mixture of strains CBAM5, OT4B.31, and III(3)7 from the L. sphaericus CIMIC collection was the bioassay with the highest percentage of metal recovery, followed by CBAM5 alone (Figure 2a,b). Furthermore, CBAM5 had a similar percentage of metal recovery when is compared to the mixture between the new isolations of L. sphaericus MCB1 and MCB2, but this was reached in a shorter time, 30 min ( Figure 2b,c). On the other hand, the highest percentage of metal recovery was 24.27% for MCB1 and almost the double for MCB2 at 41.45% (Figure 2c,d). A mixture of MCB1 and MCB2 also demonstrated the microorganism's capability to capture this metal (Figure 2e). It is important to mention that the highest percentage of metal recovery (60.76%) was achieved in a shorter time when the strains were mixed than when they were used on their own (Figure 2c-e). Controls in all cases were above the absorbance for the treatments with L. sphaericus presence.
For all the bioassays performed, a similar pattern was detected. In the initial phase, a quick increment in the percentage of gold recovery was represented in a drastic fall of the absorbance. Subsequently, there were a series of oscillations until the end of the experiment ( Figure 2).
As mentioned earlier, the presence of oscillations in the last stages of the experiments can suggest the presence of efflux pumps. Behavior models of these pumps were similar to the previously described reports for the same species but for the heavy metals, chrome and lead [21,39].
Metal Sorption in Living Cells of L. sphaericus Strains
The individual strain CBAM5, used as a calibration method, reached a percentage of recovery of 60% in the first 3 h of the bioassay and approximately 95% after 48 h (Figure 2a). The mixture of strains CBAM5, OT4B.31, and III(3)7 from the L. sphaericus CIMIC collection was the bioassay with the highest percentage of metal recovery, followed by CBAM5 alone (Figure 2a,b). Furthermore, CBAM5 had a similar percentage of metal recovery when is compared to the mixture between the new isolations of L. sphaericus MCB1 and MCB2, but this was reached in a shorter time, 30 min (Figure 2b,c). On the other hand, the highest percentage of metal recovery was 24.27% for MCB1 and almost the double for MCB2 at 41.45% (Figure 2c,d). A mixture of MCB1 and MCB2 also demonstrated the microorganism's capability to capture this metal (Figure 2e). It is important to mention that the highest percentage of metal recovery (60.76%) was achieved in a shorter time when the strains were mixed than when they were used on their own (Figure 2c-e). Controls in all cases were above the absorbance for the treatments with L. sphaericus presence.
For all the bioassays performed, a similar pattern was detected. In the initial phase, a quick increment in the percentage of gold recovery was represented in a drastic fall of the absorbance. Subsequently, there were a series of oscillations until the end of the experiment ( Figure 2).
As mentioned earlier, the presence of oscillations in the last stages of the experiments can suggest the presence of efflux pumps. Behavior models of these pumps were similar to the previously described reports for the same species but for the heavy metals, chrome and lead [21,39].
Biofabrication of Gold Nanoparticles (AuNPs)
Possible biosynthesis of AuNPs in the strains evaluated was observed. After 48 h, the treatment flasks with the bacteria turned to a strong red-purple color (Figure 3). In the biological formation of AuNPs this color is very characteristic and in the majority of cases, it is a process that takes place several hours after the contact between the microorganism and the metal [28,29,46,47].
Biofabrication of Gold Nanoparticles (AuNPs)
Possible biosynthesis of AuNPs in the strains evaluated was observed. After 48 h, the treatment flasks with the bacteria turned to a strong red-purple color (Figure 3). In the biological formation of AuNPs this color is very characteristic and in the majority of cases, it is a process that takes place several hours after the contact between the microorganism and the metal [28,29,46,47].
Biofabrication of Gold Nanoparticles (AuNPs)
Possible biosynthesis of AuNPs in the strains evaluated was observed. After 48 h, the treatment flasks with the bacteria turned to a strong red-purple color (Figure 3). In the biological formation of AuNPs this color is very characteristic and in the majority of cases, it is a process that takes place several hours after the contact between the microorganism and the metal [28,29,46,47]. For living cells, gold colloids did not have a specific formation around the cell or a clear pattern of accumulation; they were all over the surface (Figure 4). Additionally, some of the particles were not attached to the cells, but free in the area. These possible AuNPs varied in size from 50 nm to 100 nm. In general, the shape of all L. sphaericus strains were elongated and thin, possibly due to the pressure they were subjected to, with an acid pH solution and high gold concentrations. L. sphaericus CBAM5 shows clusters of possible, small AuNPs (Figure 4a,b). The possible AuNPs observed in the mixture between the strains from the CIMIC collection were not clumped, but rather dispersed throughout the surface covered by the cells (Figure 4c,d). MCB1 and MCB2 show bigger clumps of gold compared with the strains from the CIMIC collection (Figure 4e-h). Finally, the mixture of these strains also reveals dispersion all over the cell surface as in the mixture of the CBAM, OT4b.31, and III(3)7 strains (Figure 4i,j). For living cells, gold colloids did not have a specific formation around the cell or a clear pattern of accumulation; they were all over the surface (Figure 4). Additionally, some of the particles were not attached to the cells, but free in the area. These possible AuNPs varied in size from 50 nm to 100 nm. In general, the shape of all L. sphaericus strains were elongated and thin, possibly due to the pressure they were subjected to, with an acid pH solution and high gold concentrations. L. sphaericus CBAM5 shows clusters of possible, small AuNPs (Figure 4a,b). The possible AuNPs observed in the mixture between the strains from the CIMIC collection were not clumped, but rather dispersed throughout the surface covered by the cells (Figure 4c,d). MCB1 and MCB2 show bigger clumps of gold compared with the strains from the CIMIC collection (Figure 4e-h). Finally, the mixture of these strains also reveals dispersion all over the cell surface as in the mixture of the CBAM, OT4b.31, and III(3)7 strains (Figure 4i,j). For living cells, gold colloids did not have a specific formation around the cell or a clear pattern of accumulation; they were all over the surface (Figure 4). Additionally, some of the particles were not attached to the cells, but free in the area. These possible AuNPs varied in size from 50 nm to 100 nm. In general, the shape of all L. sphaericus strains were elongated and thin, possibly due to the pressure they were subjected to, with an acid pH solution and high gold concentrations. L. sphaericus CBAM5 shows clusters of possible, small AuNPs (Figure 4a,b). The possible AuNPs observed in the mixture between the strains from the CIMIC collection were not clumped, but rather dispersed throughout the surface covered by the cells (Figure 4c,d). MCB1 and MCB2 show bigger clumps of gold compared with the strains from the CIMIC collection (Figure 4e-h). Finally, the mixture of these strains also reveals dispersion all over the cell surface as in the mixture of the CBAM, OT4b.31, and III(3)7 strains (Figure 4i,j). (e) (f) On the other hand, it was evident for the samples immersed in resin, that the gold was also inside the cell ( Figure 5). The L. sphaericus strains MCB1 and MCB2 evaluated were able to adsorb and absorb gold, showing possible AuNPs inside and outside of the membrane. It was also remarkable that gold was also accumulated even when the cells were in the sporulation process (Figure 5a,b) or in vegetative stages (Figure 5c). The previous statement, suggests that the cycle stage of the microorganism is not an important factor that influences the biosynthesis or accumulation of the metal. On the other hand, it was evident for the samples immersed in resin, that the gold was also inside the cell ( Figure 5). The L. sphaericus strains MCB1 and MCB2 evaluated were able to adsorb and absorb gold, showing possible AuNPs inside and outside of the membrane. It was also remarkable that gold was also accumulated even when the cells were in the sporulation process (Figure 5a,b) or in vegetative stages (Figure 5c). The previous statement, suggests that the cycle stage of the microorganism is not an important factor that influences the biosynthesis or accumulation of the metal.
Discussion
The capability to bioabsorb gold of the two new strains of L. sphaericus MCB1 and MCB2 is indisputable, as well as of the strains of CIMIC collection. These abilities present great potential in the gold mining industry, particularly in the extraction process. Biological methodologies are generally low-cost techniques with high efficiencies, which make them attractive and adaptable to developing countries such as Colombia, where illegal and artisanal mining are present. These types of gold mining are characterized by the excessive use of mercury and cyanide, and the release of these heavy metals into the environment without any kind of pollution or health control [2,3]. The high efficiency shown by the strains, especially when in a mixture, makes L. sphaericus a promising candidate for gold biomining.
Bioassays using the mixtures had higher efficiencies than the ones with only one strain, displaying the benefits of mutualistic behavior by the microorganisms. It is also important to highlight the almost 100% recovery of gold by L. sphaericus MCB2, a native strain of the Nechi river at El Bagre, Colombia. This recovery was obtained in less than 50 h and with possible formation of gold nanoparticles or colloids.
Oscillations in the last stages of bioassays with live biomass suggest the presence of efflux pumps, an indirect mechanism of the cell for the introduction of divalent ions such as lead and chrome [21,40]. Also, this mechanism is proposed given that this metal does not bring any biological benefit to the cell. This strategy is known for other microorganisms because it is an evasion mechanism against metal accumulation and, consequently, it can confer resistance [48,49]. Additionally, L. sphaericus has a layer that surrounds the entire cell, known as the S-layer protein. The
Discussion
The capability to bioabsorb gold of the two new strains of L. sphaericus MCB1 and MCB2 is indisputable, as well as of the strains of CIMIC collection. These abilities present great potential in the gold mining industry, particularly in the extraction process. Biological methodologies are generally low-cost techniques with high efficiencies, which make them attractive and adaptable to developing countries such as Colombia, where illegal and artisanal mining are present. These types of gold mining are characterized by the excessive use of mercury and cyanide, and the release of these heavy metals into the environment without any kind of pollution or health control [2,3]. The high efficiency shown by the strains, especially when in a mixture, makes L. sphaericus a promising candidate for gold biomining.
Bioassays using the mixtures had higher efficiencies than the ones with only one strain, displaying the benefits of mutualistic behavior by the microorganisms. It is also important to highlight the almost 100% recovery of gold by L. sphaericus MCB2, a native strain of the Nechi river at El Bagre, Colombia. This recovery was obtained in less than 50 h and with possible formation of gold nanoparticles or colloids.
Oscillations in the last stages of bioassays with live biomass suggest the presence of efflux pumps, an indirect mechanism of the cell for the introduction of divalent ions such as lead and chrome [21,40]. Also, this mechanism is proposed given that this metal does not bring any biological benefit to the cell. This strategy is known for other microorganisms because it is an evasion mechanism against metal accumulation and, consequently, it can confer resistance [48,49]. Additionally, L. sphaericus has a layer that surrounds the entire cell, known as the S-layer protein. The presence of the protein can confer important advantages to the microorganisms. It is demonstrated that bacteria with this protein possess diverse mechanisms for metal interactions and biotransformation; this includes biosorption and accumulation, but also acts as a protective structure in environments that are harmful for the cells such as active mining zones [50,51].
The possible synthesis of gold nanoparticles has many benefits for medicine, agriculture, the cosmetic industry, drug delivery, and biochemical sensors [52,53]. For example, AuNPs synthesized by the fungi Candida albicans have been studied as a tool for the early detection of liver cancer [54]. The general mechanism for microbial synthesis of gold nanoparticles is the reduction of Au (III) ionic form to Au(0) or AuNPs [26]. The mechanism involves electrostatic interactions of the positive charge that the metal ions have, with the negative charge of the cell wall [52]. Many metabolic processes are proposed for the bio-mineralization of gold by the microbes such as the presence of organic phosphate compounds, ligninases, laccases, reductases (NADH/NADPH), amino acids (tyrosine, cysteine and tryptophan), the lack of specific metal transport systems, and extracellular complexation and efflux systems [51,52]. The production of AuNPs can be associated to detoxification in toxic environments, transforming metal ions to insoluble complexes. In this sense, the ability to form metal agglomerations can be used as an eco-friendly technique in the mining industry for different metals.
We propose that the mechanism by which L. sphaericus can synthesize gold is the presence of mer clusters. It is known that these genes confer Hg resistance, and are inducible in the presence of low concentrations of heavy metals in the environment. It suggests a cross-regulation of the Hg systems by Au(III) because of their physicochemical similarity [26]. The specific mechanism by which this occurs in L. sphaericus is still unknown and it will be the aim for future research.
To continue research in this field, it is important to evaluate the selectivity of the strains for the metal, due to the fact that the water effluent may contain other heavy metals such as mercury. Additionally, studies estimating the potential of the S-layer protein should also be performed, given its capacity of accumulate metal ions. Finally, the development of a bioreactor as a pilot could be an important approach to the real conditions that L. sphaericus is subject to in obtaining gold without mercury or cyanide.
Conclusions
The two new strains of L. sphaericus, as well as the ones from the CIMIC collection, show an elevated percentage of gold recovery, reaching 95% in less than 50 h. This bacterium can be a potential tool in the extraction of gold in Colombia due to its diverse advantages including efficiency, fast processing, the low cost of the technique, an environmentally friendly approach, health safety, and the use of native strains. These strains also have the ability to bio-produce possible gold nanoparticles in short time intervals, making them a more attractive alternative.
Conflicts of Interest:
The authors declare no conflicts of interest. | v3-fos-license |
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} | pes2o/s2orc | Taking the Hinge off: An Approach to Effector-Less Monoclonal Antibodies
A variety of Fc domain engineering approaches for abrogating the effector functions of mAbs exists. To address some of the limitations of the current Fc domain silencing approaches, we are exploring a less commonly considered option which relies on the deletion of the hinge. Removal of the hinge domain in humanized IgG1 and IgG4 mAbs obliterates their ability to bind to activating human Fc gamma receptors I and IIIA, while leaving their ability to engage their target antigen intact. Deletion of the hinge also reduces binding to the Fc neonatal receptor, although Fc engineering allows partial recovery of affinity. Engineering of the CH3 domain, stabilizes hinge deleted IgG4s and prevents Fab arm exchange. The faster clearing properties together with the pacified Fc make modality of the hinge deleted mAb an appealing solution for therapeutic and diagnostic applications.
Introduction
Gamma Immunoglobulins are key components of innate and acquired immunity. Their central role in controlling pathogen infections relies on two specialized functional modules: the antigen binding fragment (Fab) and the crystallizable fragment (Fc) [1]. The Fab is composed of the variable (Fv) and CH1 domains and is responsible for the recognition and binding to the target antigen via a set of six complementarity determining regions (CDRs) [1]. The Fc is composed of the CH2 and CH3 domains and mediates the interaction with the complement C1 complex and activating or inhibitory Fcγ receptors [1]. These Fc-mediated interactions enable the recruitment of effector cells and contribute to the potentiation and regulation of the immune response [2]. The Fc domain of immunoglobulins also binds to the neonatal Fc receptor (FcRn), an important component of the immunoglobulin endosome recycling process leading to prolonged half-lives [1]. Located between the Fab and the Fc domains, the hinge of human IgG1, 2 and 4 antibodies is a short linker peptide of 12 to 15 amino acids containing two to four inter heavy chain (HC) disulfide bridges [1]. The hinge domain of IgG3s is elongated and made of a repeat of four peptides homologous to that of the IgG1 hinge [1].
Small angle X-ray scattering, cryo-electron tomography and X-ray crystallography studies show that the hinge region of the four IgG subclasses is structurally disorganized and inherently flexible [3][4][5][6][7]. In a physiological context, hinge flexibility enables the antigen binding fragments to move freely relative to each other and to the Fc [6,7]. This critical feature facilitates the concomitant interaction of the antibody with up to two target antigens and the complement/Fc receptors [6,7]. The same structural
mAb Production and Purification
The recombinant humanized hinge deleted and full length IgG1 and IgG4 mAbs used in this study were produced transiently in Chinese Hamster Ovary cell and a standard fed batch fermentation process. Cell culture harvests were clarified by centrifugation and filtration followed by capture on a Protein A column. Elution from the protein A column was performed using a sodium acetate buffer as per standard industry practices. The mAbs were further purified as needed by a subsequent gel filtration chromatography polishing step to remove aggregate species.
Analytical Size Exclusion Chromatography with Multi-Angle Light Scattering Detection (SEC-MALS)
To determine average molar mass of mAb size variants, a Waters Acquity UPLC system was used to isocratically elute 5 µg of mAb at 0.1 mL/min on a Waters SEC 200 BEH column (4.6 mm ID × 300 mm), using a mobile phase consisting of 0.20 M potassium phosphate, 0.25 M potassium chloride, pH 6.0. The effluent was directed to Wyatt uDAWN and uTrex detectors, and a data analysis was performed on ASTRA v7 software.
SDS-Gel Capillary Electrophoresis (CE-SDS)
Molecular weight-based separation of mAb was performed on a Beckman PA800 plus using the IgG Purity and Heterogeneity Assay Kit. Samples were diluted with SDS solution and alkylated with 40 mM iodoacetamide, followed by 5 min incubation time at 70 degrees Celsius. The detection
mAb Production and Purification
The recombinant humanized hinge deleted and full length IgG1 and IgG4 mAbs used in this study were produced transiently in Chinese Hamster Ovary cell and a standard fed batch fermentation process. Cell culture harvests were clarified by centrifugation and filtration followed by capture on a Protein A column. Elution from the protein A column was performed using a sodium acetate buffer as per standard industry practices. The mAbs were further purified as needed by a subsequent gel filtration chromatography polishing step to remove aggregate species.
Analytical Size Exclusion Chromatography with Multi-Angle Light Scattering Detection (SEC-MALS)
To determine average molar mass of mAb size variants, a Waters Acquity UPLC system was used to isocratically elute 5 µg of mAb at 0.1 mL/min on a Waters SEC 200 BEH column (4.6 mm ID × 300 mm), using a mobile phase consisting of 0.20 M potassium phosphate, 0.25 M potassium chloride, pH 6.0. The effluent was directed to Wyatt uDAWN and uTrex detectors, and a data analysis was performed on ASTRA v7 software.
SDS-Gel Capillary Electrophoresis (CE-SDS)
Molecular weight-based separation of mAb was performed on a Beckman PA800 plus using the IgG Purity and Heterogeneity Assay Kit. Samples were diluted with SDS solution and alkylated with 40 mM iodoacetamide, followed by 5 min incubation time at 70 degrees Celsius. The detection Antibodies 2020, 9, 50 4 of 14 wavelength was set at 214 nm with a separation voltage and duration of 15 KV (480 V/cm) and 40 min, respectively.
Differential Scanning Fluorimetry
Differential scanning fluorimetry (DSF) was used to determine the conformational stability of the hinge deleted IgG4 and of the corresponding full length mAb. Molecules were diluted to 1.0 mg/mL in 20 mM Acetate pH 5.0. Each sample was placed into three separate capillaries and tested on the NanoTemper Prometheus NT.48 nanoDSF instrument. Samples were tested at 30% excitation power and the intrinsic fluorescence was measured from 20-95 • C at a rate of 1 • C per minute.
Intact and Reduced Mass Spectrometry
For identification by intact MS, samples were diluted to 0.2 mg/mL in 4 M guanidine with or without reduction with 50 mM 1,4-dithiothreitol Approximately 1 µg of each sample was injected onto a Waters BEH C4 column (1.7 µm, 2.1 × 150 mm) and eluted with a linear gradient of acetonitrile containing 0.1% Trifluoroacetic acid. The column effluent was directed to a Waters G2S TOF MS instrument and raw spectra were charge deconvoluted using MassLynx 4.1 software.
FcγRI Binding by ELISA
IgG4 hinge deleted and full-length molecules were assessed for FcγRI binding by ELISA. Samples, reference standard and control were serially diluted and added to Neutravidin-coated High Binding Capacity microtiter plates (Thermo Scientific 15507, Waltham, MA, USA) coated with recombinant human FcγRI protein (Avi-Tag Biotinylated, Sino Biological, Catalog Number: 10256-H27H-B, Wayne, NJ, USA). Antibody binding was detected with a goat anti-human antibody conjugated with horseradish peroxidase (Jackson Immunoresearch, Catalog Number: 109-036-097, West Grove, PA, USA) and fit to a 4 PL curve. Hinge deleted sample curves were compared to that of the reference standard.
FcγRI Binding by Competitive AlphaScreen ® Assay
IgG1 hinge deleted and full-length molecules were assessed for FcγRI binding by competitive AlphaScreen ® assay. Samples, reference standard and control were serially diluted and added to microtiter plates (Costar 3693) containing FcγRI protein (Avi-Tag Biotinylated, Sino Biological, Catalog Number: 10256-H27H-B, Wayne, NJ, USA) conjugated AlphaScreen streptavidin donor beads (Perkin Elmer Cat # 6760002, Waltham, MA, USA). After mixing and incubation, reference standard IgG1 mAb coupled to anti human F(ab')2goat IgG (Jackson Immunoresearch Cat # 109-006-097, West Grove, PA, USA) conjugated AlphaSceen acceptor beads (Perkin Elmer Cat # 6762001, Waltham, MA, USA) was added. After 30 min incubation, AlphaScreen ® signal was measured and fit to a 4 PL curve. Hinge deleted sample curves were compared to that of the reference standard.
FcγRIIIa Binding by ELISA
IgG1 hinge deleted and full-length molecules were assessed for FcγRIIIa V158 binding by ELISA. Samples, reference standard and control were serially diluted and added to Neutravidin-coated High Binding Capacity microtiter plates (Thermo Scientific 15507, Waltham, MA, USA) coated with recombinant human FcγRIIIa protein (Avi-Tag Biotinylated, Sino Biological, Catalog Number: 10389-H27H1b, Wayne, NJ, USA). Antibody binding was detected with a goat anti-human antibody conjugated with horseradish peroxidase (Jackson Immunoresearch, Catalog Number: 109-036-097, West Grove, PA, USA) and fit to a 4 PL curve. Hinge deleted sample curves were compared to that of the reference standard.
FcRn Binding by ELISA
IgG1 and IgG4 hinge deleted and full-length molecules were assessed for FcRn binding by ELISA. Samples, reference standard and control were serially diluted and added to Neutravidin-coated High Binding Capacity microtiter plates (Thermo Scientific 15507, Waltham, MA, USA) coated with recombinant human FcRn protein (Avi-Tag Biotinylated, Gilead Sciences, Foster City, CA, USA). An assay was conducted in PBS at pH 6.0. Antibody binding was detected with a goat anti-human antibody conjugated with horseradish peroxidase (Jackson Immunoresearch, Catalog Number: 109-036-097, West Grove, PA, USA) and fit to a 4 PL curve. Hinge deleted sample curves were compared to that of the reference standard.
Antigen Binding by Enzymatic Inhibition Assay
Relative potency of the IgG4 hinge deleted and full-length molecule samples was evaluated in a Mode of Action reflective enzymatic inhibition functional assay. All assays were carried out in a 96-well solid, black microplate (Corning, Tewksbury, MA, USA) at 24 • C. DQ TM Gelatin cleavage by MMP9 was monitored by measuring the increase in fluorescence (ex: 485 nm/em: 520 nm) on an Infinite M1000 Pro Reader (Tecan, Männedorf, Switzerland) over the course of 2 h. A fixed concentration of active MMP9 (0.5 nM MMP9-MYC-6HISAPMA, 5 nM MMP9-procatAPMA or 5 nM MMP9-cat) was mixed with increasing concentrations of DQ gelatin (0-5 µM) in a final volume of 100 µL of 50 mM Tris-HCl pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% (v/v) Brij-35 (buffer B). The cleavage of DQ gelatin was monitored by an increase in fluorescence, and initial rates were determined. Fluorescence increase was measured continuously over 2 h. Km and Vmax were determined by fitting data to the Michaelis-Menten equation with GraphPad Prism 6. To test for residual trypsin activity, various concentrations of active MMP9-cat (0-5 nM) were mixed with a fixed concentration of DQ gelatin (100 nM) and aprotinin (0.05 mg/mL) in a final volume of 100 µL buffer B. Sample activity was expressed relative to a qualified reference standard.
Antigen Binding by Cell based ELISA
The relative potency of the IgG1 hinge deleted and full-length molecule samples was evaluated in a MOA-reflective functional binding assay. Samples, reference standard and control were serially diluted and added to Poly-Lysine microtiter plates coated with gp120 antigen-presenting HEK293 cells. Antibody binding was detected by fluorescence with a biotinylated donkey anti-human antibody (Jackson Immunoresearch, Catalog Number: 709-066-149, West Grove, PA, USA) followed by a europium labeled streptavidin reagent (Perkin Elmer Cat #1244-360, Waltham, MA, USA). Fluorescence signal was read on an Envision spectrophotometer (Ex/Em = 340/615 nm). The percent of relative potency was calculated from a constrained 4 PL curve fit between the reference standard and the sample.
Fab Arm Exchange by Förster Resonance Energy Transfer
Fab arm exchange was examined using a plate-based Förster resonance energy transfer (FRET) assay. This approach was adapted from reference 24. The hinge variant samples were chemically labeled with Dylight488, while Natalizumab (Tysabri ® lot 1420037,) was labeled with Dylight594. A second sample of Natalizumab was labeled with Dylight488 as a positive control. Each sample was diluted to 20 µg/mL in PBS. The antibodies labeled with Dylight488 were mixed with Natalizumab in equal volumes. These antibody mixtures were then added to a 96-well plate in either the presence (reduced) or absence (nonreduced) of reduced glutathione (GSH) at a final concentration of 1 mM. Nonreduced samples demonstrated a lack of FRET signal (Ex/Em = 490/617 nm).
Fab Arm Exchange by Chromatographic Separation
Fab arm exchange was also evaluated by Chromatographic separation analysis and spectrophotometric/MALS detection. This approach was adapted from reference 23. The hinge-deleted samples and Natalizumab (Tysabri ® lot 1420037) were diluted to 1.0 mg/mL and mixed in equal volumes in the presence and absence of 1 mM GSH. These samples were then incubated at 37 • C for 5 h. Chrom-MALS was run with a 10 µL sample injection onto a Sepax Unix SEC-300 column using a 0.2 M Potassium Phosphate, 0.25 M Potassium Chloride pH 6.0 mobile phase at a flow rate of 0.10 mL/min. Positive and negative controls were also included in the analysis. The positive control was represented by recombinant human IgG4 kappa (HCA194, BioRad), while the negative control used was recombinant human IgG4 kappa mutant (HCA247, BioRad).
Construction and Purification of IgG1 and IgG4 Hinge Deleted Test Molecules
Two hinge deleted test molecules were built: an IgG1 with a lambda light chain and an IgG4 with a kappa light chain. The hinge sequences removed from each of the two heavy chain subtypes are shown in Figure 1. The hinge deleted molecules were expressed transiently in Chinese hamster ovary (CHO) and showed expression levels similar to that of their full-length control counterpart. All molecules were purified with a standard procedure involving a Protein A affinity step followed by gel filtration chromatography to remove aggregate species. The physicochemical and functional properties of the IgG1 and IgG4 hinge deleted mAbs and their controls were then explored.
Hinge Deleted Molecules Are Assembled as Monomers
The purified hinge deleted IgG1 and IgG4 mAbs were tested by size exclusion chromatography (SEC) with online UV and Multi Angle Laser Light Scattering (MALS) detectors. Under native conditions, results show the hinge truncated IgG4 molecule to have a retention time very similar to that of its full-length respective control sample (Figure 2a). The shift in retention time was more pronounced for the hinge truncated IgG1 molecule, reflecting a smaller hydrodynamic radius (4.2 vs. 5.1 nm) or possibly some interaction with the column (Figure 3a). Hinge-deleted IgG1 and IgG4 had molecular weights determined by the MALS of 140 kDa (IgG1) and 132 kDa (IgG4), consistent with a monomeric mAb quaternary structure. The two hinge deleted test molecules and their respective controls were further analyzed by nonreduced capillary electrophoresis (CE-SDS), a denaturing analytical method dissociating noncovalent protein-protein interactions (Figures 2b and 3b). The CE-SDS electropherograms of the hinge deleted samples showed the presence of the expected major peak, with a migration time consistent with that of an antibody half molecule (i.e., one covalently bound heavy and light chain); the control samples displaying a slower electrophoretic mobility typical of disulfide covalently bound inter and intraheavy and light chains (Figures 2b and 3b). The assignment of the molecular masses and structures to the major peaks detected in the CE-SDS electropherograms of the hinge deleted and control samples was confirmed by reversed phase and intact Mass Spectrometry ( Figure 4).
Thermodynamic Stability of the Hinge Deleted Molecules
The impact of the hinge deletion on the thermodynamic stability of the test molecules was explored using differential scanning fluorimetry (DSF). This analytical method monitors the temperature of unfolding of the individual domains based on intrinsic fluorescence. DSF thermograms showed a melting temperature decrease of 8 • C upon deletion of the hinge of the IgG4 mAb and about 5 • C in the context of an IgG1 molecule (Figures 2c and 3c). However, the melting temperature of the hinge deleted molecule was still well within the realm of thermodynamically stable proteins as the lowest temperature transition was greater than 50 • C for the IgG4 molecule and greater than 65 • C for the IgG1 (Figures 2c and 3c). Antibodies 2020, 9, x FOR PEER REVIEW 8 of 15
Deletion of the Hinge Does Not Affect the Fc N-glycan Distribution
Because of the impact of the hinge deletion on the Fc thermodynamic stability, we inquired whether deletion of the hinge domain would affect the Fc N-glycan profile. Mass spectrometry results showed that the N-Glycan profile of the hinge deleted molecule was nearly identical to that of the full-length reference IgG1 and IgG4 mAbs (Figures 2d and 3d). As for most mAbs expressed in CHO, the primary species attached to the hinge deleted antibody was G0F (Figures 2d and 3d).
Hinge Deletion Obliterates Binding to Activating Fcγ Receptors I and IIIA
The impact of the hinge deletion on the ability of the mAbs to engage effector receptors in vitro was explored next. Monomeric IgG1 and IgG4 antibody isotypes both bind to the activating FcγRI with high affinity [19]. The binding to FcγRI of the hinge deleted IgG1 and IgG4 relative to their full-length monomeric counterpart was determined using either an ELISA (IgG4) or AlphaScreen ® (IgG1) assay (Figures 5a and 6a). Binding to FcγRI was completely obliterated for both hinge deleted molecules (Figures 5a and 6a).
Thermodynamic Stability of the Hinge Deleted Molecules
The impact of the hinge deletion on the thermodynamic stability of the test molecules was explored using differential scanning fluorimetry (DSF). This analytical method monitors the temperature of unfolding of the individual domains based on intrinsic fluorescence. DSF thermograms showed a melting temperature decrease of 8 °C upon deletion of the hinge of the IgG4 mAb and about 5 °C in the context of an IgG1 molecule (Figures 2c and 3c). However, the melting temperature of the hinge deleted molecule was still well within the realm of thermodynamically stable proteins as the lowest temperature transition was greater than 50 °C for the IgG4 molecule and greater than 65 °C for the IgG1 (Figures 2c and 3c).
Deletion of the Hinge Does Not Affect the Fc N-glycan Distribution
Because of the impact of the hinge deletion on the Fc thermodynamic stability, we inquired whether deletion of the hinge domain would affect the Fc N-glycan profile. Mass spectrometry results showed that the N-Glycan profile of the hinge deleted molecule was nearly identical to that of the full-length reference IgG1 and IgG4 mAbs (Figures 2d and 3d). As for most mAbs expressed in CHO, the primary species attached to the hinge deleted antibody was G0F (Figures 2d and 3d). FcγRIIIa binds to monomeric IgG1 isotype antibodies in the sub micromolar range and with a 10-fold relative lower affinity to IgG4 antibodies in vitro [19]. We therefore tested the impact of the deletion on the ability of the hinge deleted IgG1 test molecule to engage FcγRIIIa in a direct binding ELISA (Figure 6b). The deletion of the IgG1 hinge abolished FcγRIIIa binding, in a manner similar to what was observed for FcγRI engagement. All sets of results confirmed that for both IgG1 and IgG4, the hinge is critical for the engagement of the FcγRs, and that hinge deleted mAbs have a deactivated effector Fc domain. The loss of effector function may be explained by two potentially conjointly contributing factors. The steric hindrance of the Fab may prevent access to the FcγR binding sites on the CH2 domain. Alternatively, in a manner similar to the loss of FcγR binding caused by the structural perturbation induced by the removal of the Fc N-glycans, the destabilization of the mAb noted by DSF may reflect a structural conformation of the binding site no longer conducive to the FcγRs productive engagement.
Hinge Deletion Reduces Binding to FcRn
The impact of the hinge deletion on the overall Fc domain was explored by measuring the ability of the test molecules to bind FcRn. The affinity to this receptor was measured by direct binding ELISA. For both IgG1 and IgG4 molecules, the deletion of the hinge resulted in a loss of binding to FcRn (Figures 5b and 6c). The lower affinity for FcRn is the most likely cause for the faster clearance of hinge deleted mAbs observed in vivo, relative to the intact molecule [18,20]. In a manner consistent with published literature, the incorporation of the M428L and N434S point mutations in the Fc of the IgG1 hinge deleted test molecules partially rescued the affinity loss (Figure 6c) [21].
Hinge Deletion Reduces Binding to FcRn
The impact of the hinge deletion on the overall Fc domain was explored by measuring the ability of the test molecules to bind FcRn. The affinity to this receptor was measured by direct binding ELISA. For both IgG1 and IgG4 molecules, the deletion of the hinge resulted in a loss of binding to FcRn (Figures 5b and 6c). The lower affinity for FcRn is the most likely cause for the faster clearance of hinge deleted mAbs observed in vivo, relative to the intact molecule [18,20]. In a manner consistent with published literature, the incorporation of the M428L and N434S point mutations in the Fc of the IgG1 hinge deleted test molecules partially rescued the affinity loss (Figure 6c) [21].
A point Mutation in the CH3 Domain Blocks Fab Arm Exchange in Hinge Deleted IgG4 Antibodies
IgG4 antibodies differ from their IgG1 counterpart in that they have the unique ability to form bispecific molecules in vivo via a process known as Fab arm exchange [1,22]. In native IgG4 molecules, the hinge disulfide bridges are in equilibrium between the inter-and intraheavy chain conformations [23]. The formation of the intraheavy chain disulfide isomer is a prerequisite for the exchange of the heavy-light chain units between two IgG4 antibodies. The molecular determinants for such a phenomenon are located in the IgG4 hinge and CH3 domain [1,8]. Modern IgG4 based therapeutic mAbs include the S228P point mutation, converting the hinge to that of an IgG1-like molecule and preventing the formation of bispecific molecules between the drug and endogenous human IgGs [1,24]. However, upon deletion of the hinge, the S228P mutation preventing FAE is removed. We therefore tested the ability of the hinge deleted IgG4 molecule to engage in FAE. This
A Point Mutation in the CH3 Domain Blocks Fab Arm Exchange in Hinge Deleted IgG4 Antibodies
IgG4 antibodies differ from their IgG1 counterpart in that they have the unique ability to form bispecific molecules in vivo via a process known as Fab arm exchange [1,22]. In native IgG4 molecules, the hinge disulfide bridges are in equilibrium between the inter-and intraheavy chain conformations [23]. The formation of the intraheavy chain disulfide isomer is a prerequisite for the exchange of the heavy-light chain units between two IgG4 antibodies. The molecular determinants for such a phenomenon are located in the IgG4 hinge and CH3 domain [1,8]. Modern IgG4 based therapeutic mAbs include the S228P point mutation, converting the hinge to that of an IgG1-like molecule and preventing the formation of bispecific molecules between the drug and endogenous human IgGs [1,24]. However, upon deletion of the hinge, the S228P mutation preventing FAE is removed. We therefore tested the ability of the hinge deleted IgG4 molecule to engage in FAE. This was achieved by two complementary approaches. We first relied on Förster resonance energy transfer (FRET) signal generated upon the formation of bispecific antibodies generated by the Fab arm exchange of parental antibodies labelled by two compatible fluorescent probes (Figure 7) [22,25]. Using this methodology, we tested the ability of the truncated IgG4 to exchange a half molecule (or Fab arm) with that of Natalizumab, a recombinant IgG4 with a native human IgG4 hinge. As the positive control, we conducted the exchange reaction using Natalizumab labelled with either of one of the two fluorescent probes. As the negative control, we tested the exchange of the S228P hinge stabilized full length IgG4 with Natalizumab. The results showed that the hinge deletion did enable FAE albeit to a lesser extent than when compared to the Natalizumab positive control (Figure 7). Incorporation of the stabilizing R409K mutation in the CH3 domain of the hinge deleted molecule reduced Fab arm exchange to about the same level as the negative control (Figure 7) [26]. We confirmed these observations by using a mixed mode chromatographic separation method with in line UV and MALS detectors ( Figure 8) [24]. This method allowed the chromatographic separation of the two parental antibodies and their Fab arm exchanged bispecific product: the in line MALS detector yielding the molecular mass of each species eluting the column. The different test mAbs were mixed with a native hinge mAb (Natalizumab) in the presence or absence of a mild reducing agent, and each individual reaction mixture was then chromatographed (Figure 8). The results showed the formation of a~140 kDa hinge deleted mAb-Natalizumab hybrid eluting between the two parental antibodies (Figure 8) [24]. As before, the R409K stabilization of the CH3 domain compensated for the absence of the S228P mutation in the hinge. In this assay format, positive and negative controls resulted in the expected formation of bispecific antibodies or their absence (Figure 8). were mixed with a native hinge mAb (Natalizumab) in the presence or absence of a mild reducing agent, and each individual reaction mixture was then chromatographed (Figure 8). The results showed the formation of a ~140 kDa hinge deleted mAb-Natalizumab hybrid eluting between the two parental antibodies (Figure 8) [24]. As before, the R409K stabilization of the CH3 domain compensated for the absence of the S228P mutation in the hinge. In this assay format, positive and negative controls resulted in the expected formation of bispecific antibodies or their absence ( Figure 8).
Hinge Deletion Does Not Alter Antigen Binding
Lastly, the impact of hinge deletion on antigen recognition and binding was explored by determining the relative potency of the hinge deleted test molecules. The IgG1 and IgG4 antibodies were tested against their respective target antigen. The hinge deleted IgG4 molecules were tested using an enzymatic inhibition functional assay, which showed the deletion of the hinge to be innocuous to the antibody Fab function (Figure 5c). Likewise, relative binding affinity of the hinge deleted IgG1 determined by cell-based ELISA showed no impact of the deletion on the ability to bind its cognate antigen (Figure 6d). Förster resonance energy transfer analysis (FAE) which was performed under 1 mM glutathione reducing conditions (a) or in the absence of reducing agent as the negative control (b). Förster resonance energy transfer (FRET) signal (ex 490 nm/Em 515 nm) was recorded for the positive control (Natalizumab x Natalizumab), the hinge deleted IgG4 (Natalizumab x hinge deleted IgG4), the hinge deleted IgG4 with R409K point mutation in the CH3 domain (Natalizumab x R409K hinge deleted IgG4), for the negative control (S228P full length IgG4 x Natalizumab) and for the blank background control. (c) Bar graph summary representing the propensity of each of the test molecules to engage in FAE with Natalizumab.
Discussions and Conclusions
The last twenty years have seen the full realization of IgG potential as therapeutic molecules to treat a wide array of ailments such as inflammation, infectious diseases and hematological and solid tumor cancers [27]. The new generation of therapeutic mAbs in clinical development incorporates molecular engineering derived from the most recent advances in the understanding of the interplay of immunoglobulins with the different receptors and interacting partners [14]. Molecular engineering is intended to tailor the function of these therapeutic mAbs to their specific mode of action and enhance their therapeutic index by increasing their efficacy or reducing undesirable safety signals.
A great deal of effort has been particularly devoted to addressing the potential safety liability associated with the Fc effector function when it is not required as part of a mAb mode of action. A number of Fc silencing strategies such as the removal of the Fc glycosylation or the introduction of point mutations at specific locations in the lower hinge of the Fc have been proposed [14]. These strategies, although clinically validated in some cases, may not offer a complete Fc passivation and may only affect the binding of some of the FcγRs or may create potentially immunogenic neo-epitopes.
The alternative Fc passivation strategy we propose in this report relies on the complete deletion of the hinge of a therapeutic mAb. Our results concur with earlier published data, establishing FcγRI, FcγRIIIA and C1q binding inactivation [16][17][18]. In addition to impacting FcγR binding, deletion of the hinge greatly reduces binding to FcRn, which results in a significant shortening of the molecule in vivo half-life [18,20]. The mechanism linking hinge deletion and impact on FcRn binding is unclear. However, the abrogation of FcγR binding and reduction in FcRn binding, which have different binding sites, suggest the alteration of the CH2 domain conformational stability or dynamics. While both hinge deletion and Fc aglycosylation result in the thermodynamic Fc destabilization of the same magnitude (~6-8 • C), the removal of Fc N-glycans does not appear to impact FcRn binding [28]. This potentially suggests a structural perturbation of a different nature in both cases.
The concomitant abrogation of the effector function and half-life reduction constitutes a particularly appealing proposition for the design of molecules with an improved safety profile and exposures intermediate between mAb fragments and full IgGs. The presence of an Fc domain prevents renal glomerular filtration and still ensures half-lives significantly longer than the smaller Fabs or single chain Fv constructs [20]. If deemed necessary, simple point mutations in the FcRn binding site allow for the modulation of the pharmacokinetic properties of the molecule to nearly match that of a standard mAb.
Owing to their particular functional and pharmaceutical properties, hingeless antibodies could find their place in the therapeutic arsenal to address a number of niche applications. The effectorless properties of a hinge deleted IgG4 mAb have recently been presented as a potent blocking agent for the treatment of neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys [18]. Beyond the more traditional blocking mode of action, the effectorless and shorter pharmacokinetics properties of recombinant mAbs could also be appealing for an array of different applications including the activation of cell surface receptors by Fc independent direct agonists when a pharmacodynamic effect does not need to be sustained via prolonged exposure or as an in vivo biologically inert diagnostic imaging agents with a clearance rate intermediate between those of a Fab and a full length monomeric mAb [29]. Hinge truncation also offers the opportunity of generating protease resistant mAb based therapeutics for the inactivation and clearance of toxins of bacterial origin during bacterial infections [30]. | v3-fos-license |
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} | pes2o/s2orc | Benefits of Combined All-Trans Retinoic Acid and Arsenic Trioxide Treatment of Acute Promyelocytic Leukemia Cells and Further Enhancement by Inhibition of Atypically Expressed Transglutaminase 2
Randomized trials in acute promyelocytic leukemia patients have shown that treatment with a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) is superior in efficacy to monotherapy, with significantly decreased mortality. So far, there are little data available to explain the success of the ATRA and ATO combination treatment in molecular terms. We showed that ATRA- and ATO-treated cells had the same capacity for superoxide production, which was reduced by two-thirds in the combined treatment. Secreted inflammatory biomarkers (monocyte chemoattractant protein-1 [MCP-1], interleukin-1 beta [IL-1β] and tumor necrosis factor-α [TNF-α]) were significantly decreased and were further reduced in a transglutaminase 2 (TG2) expression-dependent manner. The amount of secreted TNF-α in the supernatant of NB4 TG2 knockout cells was close to 50 times lower than in ATRA-treated differentiated wild-type NB4 cells. The irreversible inhibitor of TG2 NC9 not only decreased reactive oxygen species production 28-fold, but decreased the concentration of MCP-1, IL-1β and TNF-α 8-, 15- and 61-fold, respectively in the combined ATRA + ATO-treated wild-type NB4 cell culture. We propose that atypical expression of TG2 leads to the generation of inflammation, which thereby serves as a potential target for the prevention of differentiation syndrome.
Introduction
Acute promyelocytic leukemia (APL) is characterized by the presence of a chromosomal translocation between the retinoic acid receptor-alpha (RARα) gene on chromosome 17 and the promyelocytic leukemia protein (PML) gene on chromosome 15, resulting in the PML-RARα fusion gene. The PML-RARα oncoprotein acts as an abnormal retinoid receptor, repressing transcription and resulting in the suppression of retinoic acid-induced myeloid differentiation. All-trans retinoic acid in DS, we further investigated the role of TG2 in limiting toxicities arising from DS. NC9 is a TG2-selective irreversible inhibitor that blocks the enzyme active site and locks TG2 in an open conformation, thereby abrogating GTP binding as well. [11,17]. Our results showed that inhibition of TG2 by NC9 in ATRA + ATO combined treatment significantly reduces not just reactive oxygen species (ROS) but also proinflammatory cytokines and chemokine production that significantly increases the severity of DS.
ATO Alters the Cellular Morphology of Differentiating NB4 Cells
Several clinical studies have reported that the morphology of APL cells changed upon ATRA or ATO treatments. Apoptotic and necrotic cells appeared after ATO-induced cell death, exhibiting a variable size and quality of cytoplasm [26].
To determine the differentiation stages of NB4 cells, a variety of morphological changes were evaluated for the single and the combined treatments. In the case of ATRA treatment, the cells mainly represent differentiated and mitotic cells (blue and green triangles, Figure 1A). ATRA, ATO 0.5 µM and the combination treatment caused less apoptosis in NB4 WT and NB4 TG2-C (virus control) cells, such as TG2-deficient NB4 TG2-KD and NB4 TG2-KO cells (Figure 1(B1,B2), upper panels, green and black bars). Results showed that at day 5, arsenic trioxide induces a time-and dose-dependent cytotoxic effect on morphology, representing damaged and late apoptotic-necrotic phases. Higher concentrations of ATO were associated with an increased number of apoptotic and necrotic cells (Figure 1(B1,B2), lower panels, green and black bars). While the number of differentiated cells was low in NB4 WT and NB4 TG2-C cell lines, a higher apoptotic rate was seen in the ATRA + ATO 2.0 µM combined treatment compared to the ATRA + ATO 0.5 µM treatment of cells (Figure 1(B2), upper panels). There was a higher degree of apoptosis or necrosis in NB4 TG2-KD or NB4 TG2-KO cells (Figure 1(B2), upper panel, black and dark grey bars). ATO treatment alone resulted in minor differentiation, with ATO concentration-dependent apoptosis in the NB4 cell lines (Figure 1(B1,B2), lower panels).
ATRA + ATO Combined Treatment Decreases Differentiated NB4 Cells' Ability to Produce ROS
We previously reported that the atypical expression of TG2 greatly enhances neutrophil granulocytes' production of ROS by enhancing the expression of two important components of the NADPH-oxidase complex, NCF-2/P67PHOX and GP91PHOX. ATO treatment caused significant cellular changes in NB4 cell lines, which may affect the production of ROS. Because the NADPH-oxidase system is responsible for ROS production, we sought to determine the extent of ROS production after ATRA/ATO treatments. Both NCF-2/P67PHOX and GP91PHOX mRNA expression levels were measured at 1 µM ATRA, 0.5 µM, 2.0 µM ATO, respectively, and ATRA + ATO combined treatments at days 0, 3 and 5. While the levels of mRNS expression of both genes showed a similar pattern, especially on the fifth day, displaying a TG2-dependent expression after ATRA treatment, ATO treatments resulted in a magnitude of gene expression almost similar to that of ATRA generated in NB4 WT cells (Figure 2(A1,A2,B1,B2), left side). In combined treatments (ATRA + ATO, 0.5 and 2.0 µM), as a consequence both of the combined treatment and the extent of TG2 quantities, expression values remained low compared to ATRA or ATO treatments alone (Figure 2(A3,A4,B3,B4), right side). These expression values were also reflected in the production of ROS, especially in the ATRA + ATO 2.0 µM treatment, where a 1/3 ROS generating capacity was measured compared to the ROS production with ATRA or ATO treatment alone, depending on the amount of TG2 (Figure 2(C1-C4)).
Combined ATRA + ATO Treatment Markedly Reduces Inflammatory Biomarker Expression
DS, which can be fatal in 2.5-30% of cases in its moderate or severe forms, is characterized by the presence of a large number of inflammatory, differentiated leukemic cells in the bloodstream that synthesize and secrete chemokines and cytokines, triggering a so-called "cytokine storm." We previously showed that MCP-1, IL-1β and TNFα were secreted in a TG2-quantity-dependent manner in differentiated NB4 cell lines. MCP-1, IL-1β and TNFα were measured at the mRNA and protein levels in ATRA, ATRA + ATO 0.5 µM, and ATRA + ATO 2.0 µM differentiated NB4 WT, NB4 TG2-C, NB4 TG2-KD and NB4 TG2 KO-cell culture systems. Overall, at day 5, mRNA levels of MCP-1, IL-1β and TNFα were approximately 50% lower for the combined ATRA + ATO 2.0 µM treatment than for ATRA, but these values were further reduced in a TG2-quantity-dependent manner ( Figure 3(A1,B1,C1)). At day 5, we observed the effective inhibition of MCP-1, IL-1β and TNFα (with 5×, 10× and 20× lower values, respectively, than the controls) in the case of NB4 WT cells in combined therapy, especially ATRA + ATO 2.0 µM, with respect to proinflammatory cytokines and the chemokine content of cell culture supernatants. These values were further reduced in a TG2-quantity-dependent manner (Figure 3(A2,B2,C2), Table S1). Graphs are the representation of mean ± SD values normalized to 100 µg protein of cell lysate content. Statistical significance was determined via two-way analysis of variance (ANOVA; Bonferroni and Tukey post-hoc test; ATRA vs. ATRA+NC9 * p < 0.05, ** p < 0.01 and *** p < 0.001, **** p < 0.0001).
Normalised production in pg/mL
Discussion
ATRA-based therapy is frequently used in the clinical treatment of APL patients, which leads to the terminal differentiation of leukemic cells towards the neutrophil granulocyte stage. Hyperinflammatory reactions may constitute severe side effects of ATRA treatment, including the infiltration and damaging of soft tissues and organs, such as the lungs and heart, by differentiating APL cells that produce reactive oxygen species. ATRA-induced differentiation of APL cells could generate increased secretion of cytokines, chemokines and interleukins as well as cell adhesion and migration. ATRA-induced differentiation is also associated with the elevated expression of two components of the NADPH-oxidase complex, NCF-2/P67PHOX and GP91PHOX, resulting in the possibility of increased ROS production and, consequently, more severe organ damage.
It has previously been reported that the morphology of APL cells changed upon ATRA or ATO treatment. ATO treatment was associated with apoptotic or necrotic cell death, exhibiting variable size and quality of cytoplasm [26]. Notably, reduced or absent TG2 enhanced the sensitivity of NB4 cells to a combined ATRA + ATO 2.0 µM treatment, with significantly higher apoptotic and necrotic rates.
Oxidative stress caused by reactive oxygen species, a group of oxygen-based reactive molecules produced by ATO activated the NADPH-oxidase system, resulting in the disruption of mitochondrial membrane potential and subsequent apoptosis [27][28][29]. As published previously, the mRNA expression of NCF2/P67PHOX and GPPHOX91 were substantially higher in the presence of TG2 compared to NB4 TG2-KD and TG2-KO cells. Here we find that ATO alone can trigger an increase in the amount of mRNA of NCF2 and GPPHOX91 as well as the production of ROS, similar to ATRA in differentiated NB4 cell lines. So far, no study has shown a 2-fold decrease in ROS production in response to combined ATRA + ATO compared to single ATRA or ATO treatments. While amounts of atypically expressed TG2 in differentiated NB4 cell lines can enhance the function of the NAPDH-oxidase system, leading to high ROS production, TG2 deficiency in TG2-KD cells or TG2-KO cells is associated with significantly lower ROS production, which may be further reduced by the combined ATO treatments.
We have previously shown that the TG2 inhibitor NC9 decreases NF-κB translocation to the nucleus and NF-κB transcriptional activity as well as significantly reducing the production of inflammatory biomarkers, such as MCP-1, IL-1β and TNF-α [20]. Accordingly, our current study demonstrates that combined ATRA + ATO (2.0 µM) with an inhibition of atypically expressed TG2 by NC9 radically limits the expression and secretion concentration of three inflammatory biomarkers and radical oxygen species ( Figure 5(C1-C4)). ATRA + ATO (2µM) + NC9 treatment resulted in an 8-fold decrease in MCP-1, a 15-fold decrease in IL-1β levels and a 61-fold suppression of TNFα; there was also a 28-fold reduction in ROS production, compared to ATRA alone, at day 5.
Cytospin
Samples were taken from a homogeneous suspension of cell cultures. After pre-cleaning the slides (70% alcohol), 10 µL of a homogeneous sample together with 90 µL of 1x phosphate-buffered saline was applied to the Cytospin™ tube (Shandon CYTOSPIN II, 6511 Bunker Lake Blvd. Ramsey, MN 55303 USA), followed by centrifugation at 800 rpm for 3 min. Samples were then fixed at room temperature with methanol and were prepared for further staining.
May-Grünwald Giemsa Staining
May-Grünwald and Giemsa solutions were diluted with distilled water at a 1:10 ratio. Previously fixed samples were stained with a May-Grünwald solution for 10 min and were then rinsed with a diluted Giemsa solution for 5-30 min. Slides were washed and dried at room temperature. Light microscope images and documentation were obtained using a FLoid ® Cell Imaging Station instrument (Life Technologies) at a scale of 200 µm. The ratio of the undifferentiated, differentiated, apoptotic, necrotic and mitotic cells in NB4 cell lines was determined by morphological changes/features during the ATRA, ATO and combined ATRA + ATO treatments. Based on our morphological type evaluations, we classified the morphological types of ATRA-, ATO-and ATRA + ATO-differentiated NB4 cells into six groups: a, undifferentiated (unsegmented nuclear region and thin cytoplasmic region); b, differentiated (a segmented nuclear fraction with the white-grey higher proportion of the cytoplasmic region); c, apoptotic (well-defined membrane changes, with shrinkage) and d: necrotic (lack of nuclear fraction with severe destruction of the membrane structure). Other groups, such as: e, apoptotic-necrotic (strong blue nuclear remnants, disarrayed membrane structure) and f, mitotic (chromatin changes, round shape) were also defined from the ATRA and ATO combined treatment.
Gene Expression Analysis
For Q-PCR measurements, total RNA samples were isolated by TRIzol reagent, following the company's instructions. Total RNA was quantified by a NanoDrop 2000 Spectrophotometer (Thermo Fisher, Waltham, MA USA 02451). Each sample was diluted to 200 ng/µL concentration, followed by reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher) in a reaction of: 10 µL sample + 10 µL RT-Master mix. The assay and the PCR were performed according to the manufacturer's protocol. For the real-time Q-PCR reaction, the following TaqMan probes (ABI, Applied Biosystems, Waltham, MA USA 02451) were used: NCF2, GPPhox91, IL-1β, MCP-1, TNF-α and GAPDH. The analysis was carried out using the ABI Prism 7900 (ABI, Applied Biosystems). Relative mRNA expression levels were normalized to the level of GAPDH using the ∆∆Ct method.
Superoxide Anion Production
The amount of superoxide radicals was measured by luminol-chemiluminescence assay, using L-012 dye (Wako Pure Chemical Industries, Ltd., 1-2 Doshomachi 3-chome, Chuo-ku, Osaka, Japan) after PMA (50 nM) induction, in a reaction volume of 100 µL medium containing cells and L-012 (50 µM) dye. After 5 min, samples were measured in a Synergy Multimode Microplate Reader (BioTek Instruments, Inc, Winooski, VT, USA). Production of generated light by the reaction was recorded in relative luminescence units (RLUs) and was corrected with the protein concentration levels of the samples.
Conclusions
Together, these results suggest that the atypical expression level of TG2 in ATRA-induced differentiating APL cells is a crucial factor in developing inflammation and in the production of ROS. The lower expression of TG2 may effectively reduce the chance of inflammatory processes and organ damage.
In the combined ATRA + ATO treatment of differentiating APL cells, ATO can restrict ATRA-induced ROS and inflammatory biomarker production capacity in a dose-dependent manner. The irreversible inhibitor of TG2 NC9 not only decreased reactive oxygen species production 28-fold, but decreased the concentration of MCP-1, IL-1β and TNF-α 8-, 15-and 61-fold, respectively in the combined ATRA + ATO-treated wild-type NB4 cell culture.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/3/648/s1, Table S1: Protein content of supernatant of NB4 cell lines, Table S2: Protein content of supernatant of NB4 cell lines, Table S3: List of chemicals and reagents were used, Figure S1: Representative images of May-Grünwald-Giemsa staining, Figure S2: Production of ROS in vehicle treated NB4 cells, Figure S3: mRNA expression of MCP-1, IL-1β and TNFα inflammatory biomarkers, Figure S4: The protein content of the supernatants of NB4 cell lines was quantified by ELISA. | v3-fos-license |
2018-12-07T14:58:02.832Z | 2018-12-01T00:00:00.000 | 54446986 | {
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} | pes2o/s2orc | Irradiation-induced palladium-catalyzed decarboxylative desaturation enabled by a dual ligand system
Generation of alkenes through decarboxyolefination of alkane carboxylates has significant synthetic value in view of the easy availability of a variety of carboxylic acids and the synthetic versatility of alkenes. Herein we report that palladium catalysts under irradiation with blue LEDs (440 nm) catalyze decarboxylative desaturation of a variety of aliphatic carboxylates to generate aliphatic alkenes, styrenes, enol ethers, enamides, and peptide enamides under mild conditions. The selection of a dual phosphine ligand system is the key enabler for the successful development of this reaction. The Pd-catalyzed decarboxylative desaturation is utilized to achieve a three-step divergent synthesis of Chondriamide A and Chondriamide C in overall 68% yield from simple starting materials. Mechanistic studies suggest that, distinct from palladium catalysis under thermal condition, irradiation-induced palladium catalysis involves irradiation-induced single-electron transfer and dynamic ligand-dissociation/association process to allow two phosphine ligand to work synergistically.
P roduction of alkenes from carboxylic acids through extrusion of the carboxyl group is a highly significant process [1][2][3][4][5] considering the easy availability of carboxylic acids [6][7][8][9][10][11] and the synthetic versatility of alkene products 12 . The synthetic significance of this transformation is further enhanced when it is applicable to α-hydroxy acids and α-amino acids to produce enol ethers and enamides, which are important intermediates for synthesis 13,14 and privileged structures in bioactive compounds 15,16 . Reported methods for the transformation of aliphatic carboxylic acids into alkenes include transition-metalcatalyzed decarbonylative dehydration [17][18][19][20][21][22] , decarboxylative oxidation using Pb(IV) 23 , and enzymatic processes 24,25 . These methods require either harsh reaction conditions or toxic reagents, or lack generality for synthetic utility. Recently, Glorius and coworkers reported a decarboxylative desaturation reaction of aliphatic redox-active esters by using an organophotoredox catalyst merged with a copper catalyst 26 . With our interest in exploring palladium catalysis under visible-light irradiation [27][28][29][30][31][32] , and also inspired by the elegant achievements of Gevorgyan and coworkers on irradiation-induced palladium-catalyzed desaturation methodologies [33][34][35][36] , we conceived that a decarboxylative desaturation method should be feasible using irradiation-induced palladium catalysis through hybrid alkyl Pd(I) radical species 37 generated by single-electron transfer (SET) [27][28][29][30][31][32][33][34][35][36] activation and radical decarboxylation (Fig. 1). Herein, we report palladiumcatalyzed decarboxylative desaturation of various aliphatic carboxylates to generate aliphatic alkenes, styrenes, enol ethers, enamides, and peptide enamides under mild irradiation conditions. Distinct from palladium catalysis under thermal conditions, for which a single phosphine ligand is generally applied 38 , a dual phosphine ligand system containing a bidentate phosphine and a bulky monodentate phosphine is the key enabler to achieve this transformation. The reaction demonstrated herein not only provides access to olefins, enol ethers, and enamides from easily available carboxylates, which is of high synthetic value, but also reveals that palladium catalysis under irradiation excitation has distinct ligand requirements compared with traditional thermal systems, and that optimizing a dual ligand combination provides the opportunity to achieve new reactivity.
Results
Reaction optimization. The optimized reaction conditions are summarized in the equation in Table 1. A transparent Schlenk tube charged with palmitic acid-derived redox-active ester (0.2 mmol), PdCl 2 (2 mol%), 4,5-bis(diphenylphosphino)−9,9-dimethylxanthene (Xantphos, 3 mol%), 2-(dicyclohexylphosphino) biphenyl (Cy-JohnPhos, 4 mol%), and 2,4,6-collidine (0.2 mmol) in N,N-dimethylacetamide (DMA) solvent was exposed to irra-diation with 30 W blue LEDs at room temperature. After 15 h irradiation and aqueous workup, the desired decarboxylative elimination product 2 was obtained in 93% yield, and only trace amount of decarboxylative protonation by-product 3 was detected. The key results obtained by investigating the controlling parameters of this reaction are summarized in Table 1 (See Supplementary Tables 1-4 for details). Control experiments were conducted to examine the essential role of each parameter of the reaction (entries 1-4). The yield decreased sharply to 44% when Xantphos (3 mol%) was used alone, without Cy-JohnPhos (entry 1). The reaction did not proceed without Xantphos (entry 2). Using a combination of preformed Pd(Xantphos)Cl 2 complex (2 mol%) and Cy-JohnPhos (4 mol%), instead of adding PdCl 2 and each ligand alone, provided similar results. From these results, we can conclude that both Xantphos and Cy-JohnPhos play essential roles in controlling the reactivity of the palladium catalyst. The reaction did not proceed in the absence of irradiation (entry 3). 2,4,6-Collidine was added as base to promote the reaction by reacting with Pd-H formed after β-H elimination to regenerate Pd(0). The efficiency of the palladium catalyst decreased without the addition of 2,4,6-collidine (entry 4). Screening the palladium catalysts showed that PdCl 2 was the optimal catalyst; other palladium catalysts such as Pd(OAc) 2 , Pd(TFA) 2 , and Pd 2 (dba) 3 were ineffective (entries 5−7). Other bases, such as K 2 CO 3 , DMAP, and Et 3 N used instead of 2,4,6-collidine showed poor performance (entries 8−10). When the loading of palladium catalyst was increased to 3 mol%, the necessary amount of the 2,4,6-collidine could be reduced to 25 mol% to give comparable efficiency (entry 11).
The effect of the ligand was investigated, and representative examples are shown in Table 1. Testing Cy-JohnPhos in combination with other bidentate phosphine ligands revealed that the bite angle 39,40 and backbone structure of the bidentate ligand affects the outcome of the reaction significantly. Xantphos was the only effective bidentate ligand, probably because of its large bite angle and conjugated backbone structure. The superiority of Xantphos compared with other bidentate phosphine ligands was also observed in our previously reported alkyl Heck reaction 27 and decarboxylative alkyl Heck reaction 28 . The results of investigating various monodentate phosphine ligands were intriguing. When PPh 3 was used as monodentate ligand instead of Cy-JohnPhos, the reaction was almost completely suppressed. Applying P(1-Np) 3 (1-Np, 1-naphthyl) gave the desired product in 52% yield. Comparing the result using PPh 3 and P(1-Np) 3 , it is suggested that the cone angle and the steric bulk of the monodentate phosphine ligands control the reactivity. Guided by this hypothesis and consideration that electronic properties play important role, we further tested other monodentate alkyl phosphine ligands with large cone angles. Bulky monodentate phosphines such as PCy 3 and P(t-Bu) 3 effectively promoted the yields to 60 and 54%, respectively, but using the less bulky ligand PhPCy 2 gave the product in only 12% yield, which is consistent with our hypothesis. Our attention focused on Buchwald phosphine ligands 41 , which provide choices to screen ligands of different steric effects and electronic properties. After screening various Buchwald phosphine ligands, it was revealed that too much steric bulk is also detrimental for the reaction (comparing Xphos, Sphos, JohnPhos, and Cy-JohnPhos). Cy-JohnPhos appeared to be an excellent monodentate ligand to promote this reaction, working collaboratively with Xantphos. From the results of the ligand screening study, it is conclusive that both the structures of the ligands and the combination of monodentate and bidentate phosphine ligands determine the efficiency of the palladium catalyst. Substrates scope of the decarboxylative desaturation. The substrate scope of this reaction is demonstrated in Fig. 2. Besides primary aliphatic carboxylates, secondary and tertiary aliphatic carboxylates were also amenable substrates. The mild reaction conditions allowed various functional groups to be tolerated, including ether (5,11,17), alkyl chloride (6), tertiary amine (6), sulfonamide (8), carboxamide (9, 10), ketone (12,19), ester (18), and phenolic hydroxyl (18). To our delight, oleic acid containing a Z-double bond could be efficiently converted into the desired product without Z/E isomerization (4). Notably, allyl arenes were produced without isomerization of the double bond to form thermodynamically favored styryl isomers (5 and 6), even in the presence of Pd-H intermediates 42 . α-Aryl aliphatic carboxylates were efficiently converted into the corresponding styrene derivatives in excellent yields, without any byproducts of Heck reactions 28,[30][31][32] or oligomerization 43,44 (7, 11−14) being detected. The mild and redox neutral conditions allowed decarboxylative desaturation of mycophenolic acid, containing a phenolic hydroxyl group, to deliver a 1,3-diene (18). In addition, we have demonstrated that natural and pharmaceutical compounds, such as chlorambucil (6), gemfibrozil (17), and dehydrocholic acid (19), were all amenable, providing a useful way to prepare their boronic acid isosteres via hydroboration 45,46 . 1,3-diene and 1,3enyne could be produced in high yield with E-isomer as major isomer (20,21). When an alkyl carboxylate possessing two different eliminable β-H atoms was used, the reaction delivered a mixture of regioisomers with a certain degree of selectivity. High terminal/internal selectivity was achieved for a tertiary cyclic substrate (9). α-Amino acid-and α-hydroxy acid-derived redox-active esters were also amenable substrates (Fig. 3) to generate enol ether and enamide products. In a recent report on photoredox/Cu-catalyzed decarboxylative desaturation, the enol ether product was obtained in low yield (27%) and enamide was not demonstrated 26 . Both tertiary and secondary α-hydroxy carboxylates were suitable substrates, and aryl boronate functionality was well tolerated (22, Trace 3 Without irradiation 0 4 Without 2,4,6-collidine 51 5 Using Pd(OAc) 2 instead of PdCl 2 10 6 Using Pd(TFA) 2 instead of PdCl 2 <5 7 Using Pd 2 (dba) 3 instead of PdCl 2 <5 8 Using K 2 CO 3 instead of 2,4,6-collidine 30 9 Using DMAP instead of 2,4,6-collidine 43 10 Using Et 3 N instead of 2,4,6-collidine 40 11 Pd(Xantphos)Cl 2 (3 mol%), Cy-Johnphos (6 mol%) 23). 2,3-Dihydro-1-benzofuran-2-carboxylate underwent decarboxylative elimination to yield benzofuran in 82% (24). Natural α-amino acid-derived redox-active esters, including proline (26), alanine (27), phenylalanine (28), tryptophan (29), methionine (30), and glutamic acid (32), can be effectively converted into enamides of structural diversity with high E-selectivity. Lysine (33) derived redox-active ester delivered Boc-protected 2-aminopiperidine because of further intramolecular hydroamination of the enamide product. Protecting groups such as Boc- (28), Cbz- (26), and Phth- (31), used in peptide synthesis, are all compatible. The good performance for various N-protected αamino carboxylates encouraged us to further test the feasibility of applying this reaction to generate peptide enamides 47 . To our delight, dipeptide-and tripeptide-enamide 34, 35, and 36 were successfully synthesized. Considering the diverse transformations of enamide functionality, we believe our method will be useful for peptide modification [48][49][50] .
The synthetic utility of this decarboxylative desaturation is further highlighted by applying it as a key step to simplify the total synthesis of cytotoxic indole-enamide natural products Chondriamides A and C 51,52 . As shown in Fig. 4, Chondriamides A (37) and C (38) can be prepared at the same time in only three steps from commercially available starting materials in 68% overall yield. The functional group compatibility that enables the free indole N-H bond to be tolerated obviates complex protection/deprotection steps, proving a much simpler and more efficient synthesis of Chondriamides A and C compared with previously established synthesis 47,53 .
The decarboxylative desaturation of this substrate was accompanied by the formation of a large amount of intramolecular C −H radical cyclization product (39) 29 . Surprisingly, changing Cy-JohnPhos to a less bulky but more electron-rich trialkyl phosphine, di(1-adamantyl)-n-butylphosphine (cataCXium ® A), resulted in an overwhelming selectivity for cyclization over elimination. Neither decarboxylative cyclization nor decarboxylative desaturation proceeded in the absence of Xantphos. The ligand-dependent selectivity supports a mechanism involving ligand dissociation because less bulky and electron-rich monodentate ligand may strongly coordinate to palladium in hybrid alkyl Pd(I) radical species, thereby preventing phosphine dissociation/alkyl rebinding to allow β-H elimination to proceed. The other evidence to support a mechanism involving phosphine dissociation to allow β-H elimination is that although the choice of monodentate phosphine significantly affected the yield of elimination product, but it has no obvious effect in selectivity of β-H elimination when tertiary carboxylate possessing distinct β-Hs were tested (Supplementary Table 5). The inefficacy of different monodentate phosphines to affect selectivity of β-H elimination suggests a mechanism involving dissociation of monodentate phosphine before β-H elimination to take place.
Discussion
UV-Vis spectrum unambiguously confirmed palladium complex is the only light absorption species in the reaction system (Fig. 6a, see Supplementary Figure 80 for details). Stern-Volmer quenching experiment of Pd(PPh 3 ) 4 with redox-active ester showed redox-active ester effectively quenches photoexcited Pd(0) species, which supports that a photoexcited Pd (0) Figure 81). However, measurement of Stern-Volmer quenching using a Pd(0)-dual ligand system (Xantphos and Cy-Johnphos) was not successful because of very weak emission generated (~620 nm) 33 . The very weak emission of the Pd(0)-dual ligand system under irradiation compared with Pd(PPh 3 ) 4 suggests that ligand dissociation or ligand exchange consumes the absorbed energy from blue LED irradiation.
The generation of hybrid alkyl Pd(I) radical species in the reaction system under irradiation was further supported by radicalclock experiments (Fig. 7) and electron paramagnetic resonance (EPR) studies. As depicted in Fig. 6b, EPR signals with hyperfine coupling constant indicating the formation of a spin adduct of alkyl radical with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was observed 29 (See Supplementary Figure 82 for details). This signal was only observable after irradiation with blue LEDs, which is consistent with our understanding that activation of the redoxactive ester and formation of hybrid alkyl Pd(I) species requires irradiation 28 . Inspired by these results and recent reports by Gevorgyan et al. 30,31,[33][34][35][36] ., we may conclude Pd-alkyl bond under blue LED irradiation exhibits radical property, which resembles to reported radical properties of alkyl-Ni and alkyl-Fe species under thermal conditions 54 . EPR measurements were further used to obtain mechanistic understandings of the dual ligand effect in this transformation. EPR signals of spin adduct formed by trapping alkyl radical with DMPO were also observed when either Pd (Xantphos)Cl 2 alone or PdCl 2 /Cy-JohnPhos was used as catalyst. The EPR results suggest that the dual ligand system is not essential for SET-activation of redox-active ester under irradiation, but may facilitate it (refer to Table 1, entries 1 and 2). The important role of the dual ligand may be related with stabilization of Pd(I) intermediate (e.g. B and C in Fig. 8) before alkyl binding to enable desired catalytic cycle in Fig. 8.
Our mechanistic insights regarding to the irradiation effect and the unusual dual ligand effect on palladium catalysis are provided in Fig. 8. Based on our mechanistic studies and our previous reports [27][28][29] , we considered irradiation induced palladium catalysis involves irradiation-induced single-electron transfer of a dual phosphine-coordinated Pd(0) complex (A) with substrate, and irradiation-induced ligand dissociation/association pathways. As depicted in Fig. 8, in this reaction, we hypothesized that a palladium (0) complex coordinated with both Xantphos and Cy-JohnPhos transfers electron to redox-active ester to induce decarboxylation to form a hybrid alkyl Pd(I) intermediate. The alkyl Pd(I) intermediate possessing two types of phosphine ligand can dissociate one weakly coordinate phosphine ligand under irradiation to allow alkyl binding to undergo β-H elimination. The dissociated monodentate phosphine rebinds to Pd(0) catalyst after releasing olefin product. Accordingly to our observation, while the bidentate phosphine (Xantphos) may be related with photoexcitation, appropriate choice of monodentate phosphine of suitable association and dissociation ability is the key to tune the reactivity of hybrid alkyl Pd(I) intermediate to enable new catalytic transformations.
In summary, we report herein palladium-catalyzed decarboxylative desaturation reaction enabled by a dual ligand system under mild irradiation conditions. The reaction provides a useful method to access various alkenes, including enol ethers, enamides, and peptide enamides, from easily available carboxylates. Synthesis of Chondriamide A and Chondriamide C can be simplified by applying this method. The unusual ligand effect observed herein reveals two phosphine ligands work synergistically through an association/dissociation process under irradiation, and points out opportunity to discover untapped reactivity of irradiation-induced palladium catalysis by exploring dual ligand combinations. | v3-fos-license |
2020-06-02T21:05:51.425Z | 2020-05-28T00:00:00.000 | 219169521 | {
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} | pes2o/s2orc | Citrus aurantium L. Dry Extracts Ameliorate Adipocyte Differentiation of 3T3-L1 Cells Exposed to TNFα by Down-Regulating miR-155 Expression
Citrus aurantium L. dry extracts (CAde) improve adipogenesis in vitro. These effects are dependent from an early modulation of CCAAT/enhancer-binding protein beta (C/Ebpβ) expression and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (CREB) activation. C/Ebpβ and Creb are also targets of miR-155. This study investigated whether CAde regulates miR-155 expression in the early stages of adipogenesis and whether it ameliorates adipocyte differentiation of cells exposed to tumor necrosis factor-alpha (TNFα). Adipogenic stimuli (AS) were performed in 3T3-L1 pre-adipocytes treated with CAde, TNFα, or both. Gene and miRNA expression were determined by quantitative real-time PCR. Adipogenesis was evaluated by Oil-Red O staining. CAde treatment enhanced AS effects during the early adipogenesis phases by further down-regulating miR-155 expression and increasing both C/Ebpβ and Creb mRNA and protein levels. At variance, TNFα inhibited 3T3-L1 adipogenesis and abolished AS effects on miR-155, C/Ebpβ, and Creb expression. However, in cells exposed to TNFα, CAde improved adipocyte differentiation and restored the AS effects on miRNA and gene expression at early time points. In conclusion, this study identified miR-155 down-regulation as part of the mechanism through which CAde enhances adipogenesis of pre-adipocytes in vitro. Furthermore, it provides evidence of CAde efficacy against TNFα negative effects on adipogenesis.
Introduction
Adipocyte differentiation is a highly orchestrated physiological process, which involves a large number of molecular events and whose dysregulation is relevant to human disease by contributing metabolic dysfunction in obesity [1,2]. microRNAs (miRNAs) represent a class of small non-coding RNAs (≈22 nt in length) involved in a variety of cellular processes, whose role is to repress target translation and to induce target mRNA degradation [3]. Oskowitz et al. were the first to detail miRNA expression during human multipotent stromal cell differentiation toward adipogenic lineage and to demonstrate that adipogenic differentiation and lipid accumulation of these cells is disrupted by inhibition of miRNA biogenesis [4]. In the decade that followed, several miRNAs have been implicated in adipocyte fate determination and adipocyte formation from precursor cells; some of them appear to enhance, while some others appear to inhibit adipocyte differentiation [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. For instance, miR-27a and -27b, belonging to the miR-27 gene family, were identified as negative regulators of adipogenesis [7,10,11]. The expression of both is indeed down-regulated during adipogenic differentiation of 3T3-L1 cells, and their over-expression is inhibited in vitro adipocyte formation by reducing the levels of master adipogenic regulators such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) [7]. miR-130 suppresses adipogenesis by directly targeting and inhibiting PPARγ expression [17], while miR-375 enhances adipocyte differentiation via suppression of extracellular signal-regulated kinases (ERK 1/2) signaling [18].
miR-155, initially described as the B-cell integration (bic) cluster gene in chickens [19], is a multifunctional miRNA involved in numerous physiological and pathological processes such as haematopoietic lineage differentiation, immunity, inflammation, cancer, and cardiovascular diseases [20,21]. Its role in the regulation of adipocyte differentiation has also been described [22]. Liu et al. indeed reported that miR-155 expression is down-regulated at the early stage of 3T3-L1 adipogenesis and that its overexpression suppresses adipocyte differentiation by directly targeting the 3 -untranslated regions (3 -UTRs) of C/ebpβ and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (Creb) mRNAs [22]. In addition, it has been identified as the most responsive miRNA to inflammatory stimuli [22,23], and its expression is higher in the adipose tissue of obese compared to normal-weight subjects and correlates with both tumor necrosis factor-alpha (TNFα) expression and body mass index [23]. It follows that the modulation of the miR-155 expression in adipocyte precursor cells may offer new ways to enhance adipose tissue homeostasis during obesity.
Nutraceuticals, which encompass all the substances derived from plants and food sources that provide medical or health benefits, have been described as modulating miRNA expression [24][25][26]. As well as this, nutraceuticals are known to regulate adipocyte cell line activity [27]. In line with this, we have recently demonstrated the nutraceutical properties of CAde, a dry extract preparation obtained from Citrus aurantium L. (CAde) fruit juice, on the regulation of 3T3-L1 cell adipocyte differentiation and function [28]. CAde enhances in vitro terminal adipocyte differentiation of 3T3-L1 pre-adipocytes in terms of increased gene expression of Pparγ, glucose transporter type 4 (Glut4), and fatty acid-binding protein 4 (Fabp4), as well as the functional capacity of 3T3-L1 mature adipocytes in terms of improved insulin-induced glucose uptake [28]. Furthermore, CAde promotes the early differentiation stage as well, by anticipating the 3T3-L1 cell cycle entry and progression during mitotic clonal expansion and by activating CREB and nearly doubling the expression of the transcription factor C/Ebpβ a few hours later in the adipogenic induction [28].
In the present study, the hypothesis was that miR-155 and the regulation of its expression are the missing pieces that link CAde to C/Ebpβ and CREB proteins. Furthermore, we also wondered whether CAde might be effective against micro-environmental insults, which affect adipogenesis and miR-155 expression, such as TNFα.
Citrus aurantium L. Dry Extract (CAde)
Citrus aurantium L. (CAde) fruit juice dry extracts were from [28]. Lyophilized CAde was re-hydrated with distilled H 2 O to a final concentration of 10 mg/mL and used for treatments at the concentration of 100 µg/mL.
Statistical Procedures
Data are given as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Bonferroni correction post-hoc test was used for comparisons with three or more groups. Data were analyzed by using the GraphPad Software (version 6.00 for Windows, La Jolla, CA, USA).
CAde Down-Regulated the Expression of miR-155 and Enhanced C/Ebpβ and Creb Levels during the Early Stage of Adipogenesis in 3T3-L1 Cells
To test the hypothesis that CAde may potentially exert its pro-adipogenic effects in vitro by modulating miR-155 expression, the early stage of fat cell differentiation was investigated in 3T3-L1 pre-adipocytes. In control cells, AS induced at 15 min a 10% reduction of the miR-155 expression, whose levels remained steadily lowered to about 30%-35% at 30 min, 1, 2, and 4 h upon the adipogenic induction ( Figure 1a). In AS + CAde-treated cells, the expression of miR-155 resulted in levels being decreased by 31% already by 15 min, and its levels further declined by 58% at 30 min, 51% at 1 h, 54% at 2 h, and 54% at 4 h upon adipogenic induction compared to control cells at time 0 ( Figure 1a). Interestingly, compared to AS-treated cells, CAde treatment enhanced the effect of AS on miR-155 down-regulation of by 25%-40% at each time point (Figure 1a and Figure S1a). Additionally, we evaluated CAde effect on miR-130a and miR-375, whose role in adipocyte fate determination has already been demonstrated [17,18]. In control 3T3-L1 cells, AS caused a time-dependent down-regulation of the anti-adipogenic miR-130a ( Figure 1b) and up-regulation of the pro-adipogenic miR-375 ( Figure 1c) compared to control cells at time 0. Of note, CAde treatment did not affect the expression of these two miRNAs. Indeed, in AS + CAde-treated cells, the expression levels of both miR-130a ( Figure 1b and Figure S1b) and miR-375 ( Figure 1c and Figure S1c) at each time point were comparable to those levels observed in AS-treated control cells. Altogether, these findings suggest that CAde specifically modulated the expression of miR-155 in the early stage of fat cell differentiation.
Nutrients 2020, 12, x FOR PEER REVIEW 5 of 14 to those levels observed in AS-treated control cells. Altogether, these findings suggest that CAde specifically modulated the expression of miR-155 in the early stage of fat cell differentiation. Statistical significances among groups were tested by one-way ANOVA followed by Bonferroni correction posthoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated 3T3-L1 cells at time 0; # p < 0.05, and ## p < 0.01 vs. control 3T3-L1 cells).
We thus investigated whether CAde may consequently affect the expression of the miR-155 target genes-C/Ebpβ and Creb [21]. In the control cells, AS led to a time-dependent up-regulation of both C/Ebpβ ( Figure 2a) and Creb (Figure 2b) mRNA levels compared to control cells at time 0. It is worth noting that, compared to AS-treated cells, CAde treatment furtherly up-regulated the expression of C/Ebpβ by about 65% and 45% at 2 and 4 h, respectively (Figures 2a and S2a), while the combined stimulation of AS and CAde up-regulated Creb mRNA levels by about 40%, 55%, and 25% at 1, 2, and 4 h, respectively, as compared to treated control cells (Figures 2b and S2b). Coherently with the gene expression data, the protein levels of C/EBPβ isoform liver-enriched activating protein (LAP) resulted as being up-regulated at 2 and 4 h from AS in CAde-treated cells compared to control cells ( Figure 2c). Additionally, the protein levels of CREB were increased, starting from 0.25 h from AS in CAde-treated cells (Figure 2c). Altogether, these findings at the early stage of fat cell differentiation indicated that CAde may exert its function on the adipogenic induction in 3T3-L1 cells, at least in part, by specifically lowering the expression of miR-155 at the early time points and thus up-regulating the mRNA and protein expression of its target genes C/Ebpβ and Creb. Statistical significances among groups were tested by one-way ANOVA followed by Bonferroni correction post-hoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated 3T3-L1 cells at time 0; # p < 0.05, and ## p < 0.01 vs. control 3T3-L1 cells).
We thus investigated whether CAde may consequently affect the expression of the miR-155 target genes-C/Ebpβ and Creb [21]. In the control cells, AS led to a time-dependent up-regulation of both C/Ebpβ ( Figure 2a) and Creb (Figure 2b) mRNA levels compared to control cells at time 0. It is worth noting that, compared to AS-treated cells, CAde treatment furtherly up-regulated the expression of C/Ebpβ by about 65% and 45% at 2 and 4 h, respectively (Figure 2a and Figure S2a), while the combined stimulation of AS and CAde up-regulated Creb mRNA levels by about 40%, 55%, and 25% at 1, 2, and 4 h, respectively, as compared to treated control cells (Figure 2b and Figure S2b). Coherently with the gene expression data, the protein levels of C/EBPβ isoform liver-enriched activating protein (LAP) resulted as being up-regulated at 2 and 4 h from AS in CAde-treated cells compared to control cells ( Figure 2c). Additionally, the protein levels of CREB were increased, starting from 0.25 h from AS in CAde-treated cells (Figure 2c). Altogether, these findings at the early stage of fat cell differentiation indicated that CAde may exert its function on the adipogenic induction in 3T3-L1 cells, at least in part, by specifically lowering the expression of miR-155 at the early time points and thus up-regulating the mRNA and protein expression of its target genes C/Ebpβ and Creb.
CAde Improved Terminal Adipocyte Differentiation of Both 3T3-L1 Cells Over-Expressing miR-155 by Mimic Transfection and 3T3-L1 Cells Exposed to TNFα
Over-expression of miR-155, by a gain of function approach [22] or by pro-inflammatory cytokine induction [22,23], has been reported to impair in vitro adipocyte differentiation of 3T3-L1 cells. We then hypothesized that CAde might preserve adipogenesis of 3T3-L1 cells, where miR-155 was over-expressed by mimic transfection or induced by the pro-inflammatory cytokine TNFα.
miR-155 Gain and Loss of Function Studies
Fat cell differentiation of 3T3-L1 cells was firstly investigated in cells transfected with the miR-155 mimic or the miR-155 inhibitor. The specific over-overexpression of mimic miR-155 reduced the number of 3T3-L1 cells able to differentiate into adipocytes by about 50%, as shown by light microscopy images (Figure 3a) and Oil-Red O lipid accumulation (Figure 3b). In cells treated with CAde, the intracellular lipid accumulation was increased by about 1.4-fold compared to miR-155 overoverexpressing cells (Figure 3a,b). On the other hand, the specific loss of function of miR-155 by hairpin inhibitor transfection increased adipogenesis of 3T3-L1 cells by about 90% ( Figure S3); no further increase of adipogenesis was observed in 3T3-L1 cells transfected with miR-155 hairpin
CAde Improved Terminal Adipocyte Differentiation of Both 3T3-L1 Cells Over-Expressing miR-155 by Mimic Transfection and 3T3-L1 Cells Exposed to TNFα
Over-expression of miR-155, by a gain of function approach [22] or by pro-inflammatory cytokine induction [22,23], has been reported to impair in vitro adipocyte differentiation of 3T3-L1 cells. We then hypothesized that CAde might preserve adipogenesis of 3T3-L1 cells, where miR-155 was over-expressed by mimic transfection or induced by the pro-inflammatory cytokine TNFα.
miR-155 Gain and Loss of Function Studies
Fat cell differentiation of 3T3-L1 cells was firstly investigated in cells transfected with the miR-155 mimic or the miR-155 inhibitor. The specific over-overexpression of mimic miR-155 reduced the number of 3T3-L1 cells able to differentiate into adipocytes by about 50%, as shown by light microscopy images (Figure 3a) and Oil-Red O lipid accumulation (Figure 3b). In cells treated with CAde, the intracellular lipid accumulation was increased by about 1.4-fold compared to miR-155 over-overexpressing cells (Figure 3a,b). On the other hand, the specific loss of function of miR-155 by hairpin inhibitor transfection increased adipogenesis of 3T3-L1 cells by about 90% ( Figure S3); no further increase of adipogenesis was observed in 3T3-L1 cells transfected with miR-155 hairpin inhibitor upon CAde treatment ( Figure S3). These findings indicated that CAde treatment partially prevents the inhibitory effects of miR-155 on adipocyte differentiation of 3T3-L1 cells.
Nutrients 2020, 12, x FOR PEER REVIEW 7 of 14 inhibitor upon CAde treatment ( Figure S3). These findings indicated that CAde treatment partially prevents the inhibitory effects of miR-155 on adipocyte differentiation of 3T3-L1 cells.
Treatment with TNFα
Secondly, we investigated whether CAde may preserve adipogenesis of cells exposed to the pro-inflammatory cytokine TNFα, which exerts profound inhibition of adipocyte differentiation by miR-155 induction [21,22]. As expected, the number of 3T3-L1 pre-adipocytes able to achieve full differentiation was strongly reduced by TNFα treatment, as shown by the light microscopy images (Figure 4a). Consistent with this, TNFα reduced the intracellular lipid accumulation (Figure 4b) by about 60% and the TG deposition by about 70% (Ctrl, 41.5 ± 0.3; TNFα, 11.2 ± 0.3, µg TG · µg DNA −1 ; p < 0.001) compared to control adipocytes. It is worth noting that in cells co-treated with TNFα and CAde, the number of adipocytes was increased compared to TNFα-treated cells (Figure 4a). In addition, upon CAde treatment, the intracellular lipid accumulation (Figure 4b) and TG deposition were increased by 1.5-and about 2.2-fold, respectively, compared to TNFα-treated adipocytes (TNFα + CAde, 26.4 ± 5.8; TNFα, 11.2 ± 0.3, µg TG · µg DNA −1 ; p < 0.05). Altogether, these data suggest that CAde treatment partially protected the adipogenesis of 3T3-L1 cells from the TNFα inhibitory effect.
CAde Prevented TNFα-Induced Dysregulation of miR-155, C/Ebpβ, and Creb Expression during the Early Stage of Adipogenesis in 3T3-L1 Cells
Up-regulation of miR-155 and suppression of C/Ebpβ and Creb expression were identified as one of the mediators of the TNFα-dependent inhibition of adipogenesis [22,23]. We thus evaluated whether CAde may counteract these anti-adipogenic effects of TNFα during the early time points upon adipogenic induction of 3T3-L1 pre-adipocytes. As expected, TNFα already at 15 min from adipogenic induction induced a 30% increase in miR-155 expression, whose level remained elevated at 30 min, 1, 2, and 4 h upon AS stimulation compared to control 3T3-L1 cells at time 0 (Figure 5a). Consistent with this, TNFα treatment also decreased the expression of C/Ebpβ by about 50% at 30 min, 42% at 1 h, 66% at 2 h, and 35% at 4 h upon AS stimulation (Figure 5b) and by Creb by about 52% at 30 min and 62% at 1 h upon AS stimulation (Figure 5c). Interestingly, the co-treatment with TNFα and CAde down-regulated miR-155 expression during the early time points of adipogenic induction to levels comparable to AS-treated control cells (Figures 5a and S4a). At the same time points upon adipogenic induction, an up-regulation of both C/Ebpβ (Figures 5b and S4b) and Creb (Figures 5c and S4c) mRNA expression, to levels comparable to those in AS-treated control cells, was observed in the TNFα + CAde-treated cells. Altogether, these findings suggest that CAde weakened the inhibitory Values are mean ± SEM of determinations from three independent experiments. Statistical significances among groups were tested by one-way ANOVA followed by Bonferroni correction post-hoc test. (* p < 0.05, and ** p < 0.01 vs. Ctrl; # p < 0.05, TNFα + CAde vs. TNFα).
CAde Prevented TNFα-Induced Dysregulation of miR-155, C/Ebpβ, and Creb Expression during the Early Stage of Adipogenesis in 3T3-L1 Cells
Up-regulation of miR-155 and suppression of C/Ebpβ and Creb expression were identified as one of the mediators of the TNFα-dependent inhibition of adipogenesis [22,23]. We thus evaluated whether CAde may counteract these anti-adipogenic effects of TNFα during the early time points upon adipogenic induction of 3T3-L1 pre-adipocytes. As expected, TNFα already at 15 min from adipogenic induction induced a 30% increase in miR-155 expression, whose level remained elevated at 30 min, 1, 2, and 4 h upon AS stimulation compared to control 3T3-L1 cells at time 0 (Figure 5a). Consistent with this, TNFα treatment also decreased the expression of C/Ebpβ by about 50% at 30 min, 42% at 1 h, 66% at 2 h, and 35% at 4 h upon AS stimulation (Figure 5b) and by Creb by about 52% at 30 min and 62% at 1 h upon AS stimulation (Figure 5c). Interestingly, the co-treatment with TNFα and CAde down-regulated miR-155 expression during the early time points of adipogenic induction to levels comparable to AS-treated control cells (Figure 5a and Figure S4a). At the same time points upon adipogenic induction, an up-regulation of both C/Ebpβ (Figure 5b and Figure S4b) and Creb (Figure 5c and Figure S4c) mRNA expression, to levels comparable to those in AS-treated control cells, was observed in the TNFα + CAde-treated cells. Altogether, these findings suggest that CAde weakened the inhibitory effect of TNFα on the adipogenesis of 3T3-L1 cells by restoring the AS effects on miR-155 down-regulation and C/Ebpβ and Creb gene up-regulation at the early differentiation time points. Statistical significances among groups were tested by one-way ANOVA followed by Bonferroni correction post-hoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated 3T3-L1 cells at time 0; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. control 3T3-L1 cells; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. TNFα-treated 3T3-L1 cells).
Discussion
miRNAs have emerged as critical regulators of a variety of biological processes in eukaryotic cells, and the deregulation of their function is associated with an increasing number of human diseases [3]. Furthermore, it has been reported that miRNAs may contribute to the metabolic abnormalities associated with obesity and obesity-related complications by particularly affecting the function of the white adipose tissue (WAT) [35,36]. Indeed, in human WAT, numerous miRNAs are expressed, obesity influences their expression, and their predominant function is to stimulate or inhibit the differentiation of pre-adipocytes into adipocytes, as well as to regulate specific metabolic and endocrine functions, as revealed through loss or gain of function studies on the obesityassociated miRNAs [35][36][37][38][39][40][41]. This makes miRNAs a tangible target for the treatment of adipocyte dysfunction and its related disorders.
Nutraceuticals have been used for decades now in weight management in obese individuals and are described to modulate adipogenesis and to have other positive effects on obesity pathogenesis [27]. Growing evidence sustains the hypothesis that dietary modulation of miRNA expression may explain in part some of the beneficial effects of nutraceuticals on health [24][25][26]. Indeed, an increasing number of studies have reported that several natural food-derived compounds modulate miRNA Values are means ± SEM of three independent experiments. Control value at time 0 was set as 1.00. Statistical significances among groups were tested by one-way ANOVA followed by Bonferroni correction post-hoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated 3T3-L1 cells at time 0; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. control 3T3-L1 cells; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 vs. TNFα-treated 3T3-L1 cells).
Discussion
miRNAs have emerged as critical regulators of a variety of biological processes in eukaryotic cells, and the deregulation of their function is associated with an increasing number of human diseases [3]. Furthermore, it has been reported that miRNAs may contribute to the metabolic abnormalities associated with obesity and obesity-related complications by particularly affecting the function of the white adipose tissue (WAT) [35,36]. Indeed, in human WAT, numerous miRNAs are expressed, obesity influences their expression, and their predominant function is to stimulate or inhibit the differentiation of pre-adipocytes into adipocytes, as well as to regulate specific metabolic and endocrine functions, as revealed through loss or gain of function studies on the obesity-associated miRNAs [35][36][37][38][39][40][41]. This makes miRNAs a tangible target for the treatment of adipocyte dysfunction and its related disorders.
Nutraceuticals have been used for decades now in weight management in obese individuals and are described to modulate adipogenesis and to have other positive effects on obesity pathogenesis [27]. Growing evidence sustains the hypothesis that dietary modulation of miRNA expression may explain in part some of the beneficial effects of nutraceuticals on health [24][25][26]. Indeed, an increasing number of studies have reported that several natural food-derived compounds modulate miRNA expression in different animal cells and tissues [24][25][26]. Extracts from Citrus aurantium L. have been recently used for investigating adipocyte differentiation [28,42,43]. However, quite different results have been obtained, and whether or not extracts from Citrus aurantium L. have pro-or anti-adipogenic properties is still unclear. Kim et al. have indeed reported on the anti-adipogenic effect of preparation from the fruit peel of Citrus aurantium L. in 3T3-L1 cells, where the flavonoids naringin, hesperidin, and nobiletin are predominant [42]. Park et al. have instead demonstrated in 3T3-L1 cells and primary cultured adipocytes the anti-adipogenic and pro-thermogenic actions, respectively, of preparation from the immature dried fruit of Citrus aurantium L., which is abundant in naringin and neohesperidin [43]. We have recently shown that CAde, a dry extract preparation obtained from the fruit juice of Citrus aurantium L., mainly enriched in hesperidin, narirutin, and vicenin-2, increased adipocyte differentiation and function of 3T3-L1 cells [28]. Therefore, reasons for these controversial findings could probably be found on the specificity and the amounts of flavonoids within each of these Citrus aurantium L. extracts, which also depends on their origin.
In the present study, we have further reported new evidence that sustains nutraceutical beneficial effects of CAde on the regulation of miRNA expression and function in vitro in 3T3-L1 pre-adipocytes. In particular, we demonstrated that the treatment of cells with CAde enhanced the down-regulation of the adipogenic suppressor miR-155 [22,23], as early as 15 min upon induction of adipogenesis in 3T3-L1 pre-adipocytes. This results in an up-regulation of the mRNA and protein levels of the miR-155 target genes, C/Ebpβ and Creb [22]. Specifically, CAde treatment further increased the expression of C/Ebpβ mRNA and of the pro-adipogenic C/EBPβ-LAP protein isoform upon 2 h from the adipogenic induction [44]. Additionally, CAde further up-regulated Creb mRNA and protein levels.
It is worth noting that CAde specifically modulated miR-155 expression during the early stage of adipogenesis. Indeed, CAde treatment during the first 4 h post-adipogenic induction with AS did not affect the expression of other miRNAs, such as miR-130a and miR-375, whose role in adipocyte fate determination has been already demonstrated [17,18,41]. miR-155 is biologically relevant to the regulation of adipocyte differentiation, and its perturbation is associated with obesity [22,23]. In addition, miR-155 expression in cells is modulated by nutraceutical compounds [45][46][47]. Indeed, Eseberri et al. have shown that the stilbenoid resveratrol and its metabolites, trans-resveratrol-3-O-glucuronide and trans-resveratrol-4-O-glucuronide, exert their anti-adipogenic effect on 3T3-L1 cells by up-regulating miR-155 expression [45]. On the contrary, others reported the down-regulation of the same miRNA by flavonoid treatment [46,47]. Boesch-Saadatmandi et al. have indeed demonstrated that the flavonoid quercetin and its metabolite isorhamnetin partially neutralize lipopolysaccharide-induced increase of miR-155 in murine RAW264.7 macrophages and state that miR-155 inhibition possibly contributes to the anti-inflammatory properties of both flavonoids [46]. Additionally, Arango et al. have reported that the flavonoid apigenin and a celery-based apigenin-rich diet exert effective anti-inflammatory activity in vivo by reducing expression of miR-155 [47]. As reported above in this section, flavonoids, such as hesperidin, narirutin, and vicenin-2, are very abundant in our dry extract preparation [28] and their presence might be thus responsible for the observed down-regulation of miR-155 expression in CAde-treated cells.
Here, we have also reported that CAde partially preserved adipogenesis of 3T3-L1 cells, where the expression of miR-155 was up-regulated by mimic transfection. CAde did not further enhance adipogenesis of 3T3-L1 cells, where miR-155 activity was abolished by specific hairpin inhibitor. These findings led us to suppose that the treatment with CAde may be effective against any micro-environmental insults, which impair adipocyte differentiation by up-regulation of miR-155.
In accordance with this, we indeed found that the CAde treatment counteracted the detrimental effects of TNFα on adipogenesis. Indeed, terminal adipocyte differentiation of 3T3-L1 cells exposed to TNFα was improved by almost 50% by CAde and was associated with a restoring of the expression of miR-155, C/Ebpβ, and Creb during the early stage of adipogenesis. TNFα is a pleiotropic cytokine that exerts homeostatic and pathogenic bioactivities [48]. High TNFα levels are observed in the WAT during obesity, and they have profound effects on adipocyte metabolism by impairing triglyceride synthesis and storage and inhibiting adipocyte differentiation [22]. Liu et al. have also demonstrated in 3T3-L1 pre-adipocytes that miR-155, whose expression is up-regulated by TNFα as early as 5 min via NFκB-p65 (nuclear factor kappa-light-chain-enhancer of activated B cells) binding to the miR-155 promoter, mediates at least in part the TNFα-induced suppression of adipogenesis by down-regulating early adipogenic transcription factors [22]. These findings, therefore, provide the first piece of evidence for the efficacy of CAde treatment in vitro against micro-environment insults deleterious for the functional capacity of adipose cells. In this scenario, CAde may ameliorate, in the early stage, the differentiation process by blocking NFκB-p65 into the cytosol and thus preventing the NFkB-p65-mediated transcription of miR-155. This hypothesis was indeed sustained by our preliminary data in 3T3-L1 pre-adipocytes short-term treated with TNFα, where the TNFα-induced NFκB-p65 translocation from cytosol to the nucleus was prevented by CAde treatment.
In conclusion, this study demonstrated that miR-155 down-regulation is part of the mechanism through which CAde enhances adipocyte differentiation of pre-adipocytes in vitro. In addition, we herein provide substantial evidence of the efficacy of this nutraceutical compound against micro-environment insults, which are harmful to adipose cell functionality and affect miR-155 expression, such as TNFα, suggesting that the development of CAde-derived compounds may be an effective strategy for the treatment of adipocyte dysfunction and its related disorders. | v3-fos-license |
2019-09-19T09:14:52.415Z | 2019-10-22T00:00:00.000 | 203568420 | {
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} | pes2o/s2orc | A synchrotron-based infrared microspectroscopy study on the cellular response induced by gold nanoparticles combined with X-ray irradiations on F98 and U87-MG glioma cell lines
The inclusion of nanoparticles (NP) in radiotherapy has been shown to increase the damaging effect on tumor cells. However, the mechanisms of action of NP combined with radiotherapy, and the influence of NP parameters and cell type on their radiosensitization capability at molecular and cellular levels still remain unclear. Gold NP (AuNP) have become particularly popular due to their multiple advantages. Within this context, our research work aimed to study the biochemical radiosensitization capacity of F98 and U87-MG glioma cell lines to 1.9 nm AuNP combined with X-ray irradiation. For this purpose, synchrotron-based infrared microspectroscopy (SR-FTIRM) was used as a powerful tool for biochemical composition and treatment response assessment of cells at a single-cell level. SR-FTIRM data, supported by multivariate analysis, revealed clear AuNP-induced changes in the DNA, protein and lipid spectral regions. The AuNP-related biochemical alterations appear prior to the irradiation, which gave us a first indication on the AuNP radiosensitization action. Biochemical modifications induced by the AuNP in the presence of radiotherapy irradiations include enhanced conformational changes in the protein secondary structures, variations in the intensity and position in the phosphodiester bands, and changes in the CH2 and CH3 stretching modes. These changes are better manifested at 24 hours post-irradiation time. SR-FTIRM results showed a clear heterogeneity in the biochemical cell response, probably due to the distinct cell– NP interactions and thus, to different DNA damage and cell death processes.
Introduction
The radiosensitization potential of nanoparticles (NP) has been previously reported in many biological studies (in vitro and in vivo) in the past years (see, for instance ref. [1][2][3][4][5]. Typical radiosensitizers include metallic high-atomic number (Z) materials that can augment the effectiveness of radiotherapy by inducing dose enhancement effects specifically in the tumor site. This leads to an increase in the therapeutic index. The use of NP over the conventional contrast agents resulted in a larger NP accumulation in the tumor due to the enhanced permeability and retention (EPR) effects. 6 In addition, previous studies reported that toxicity is highly reduced when using very small NP (<5 nm). 7 Among all types of NP, gold (Z = 79) nanoparticles (AuNP) are particularly attractive since they are inert, highly biocompatible, relatively easy to synthesize and functionalise, and they have an increased cross section for photoelectric photon absorption (specially for low X-ray energies). 1,4,5,8,9 The latter enhances the secondary electron production (short-range photoelectrons and Auger electrons) and the local dose deposition near the NP. 10 At the same time, these electrons can further increase the generation of reactive oxygen species (ROS) and electron emission from nearby NP. Besides the pure physical effects, the presence of NP itself can also increase the ROS production, and thus, the cellular damage. 4,11 However, the radiosensitization mechanisms at molecular and cellular levels still remain unclear. Results from the previous biological studies indicated that radiosensitization effects might be driven by biological mechanisms induced by the NP rather than by physical (dose enhancement) effects. These mechanisms include oxidative stress, DNA damage induction, cell cycle effects and potential interference with cell communication. [12][13][14][15][16] This, along with the influence of many other parameters like NP size, shape and coating, which at the same time influence the NP distribution inside the cells, as well as the distinct cellular responses, largely complicates research on radiosensitization effects of NP. 4 Fourier transform infrared microspectroscopy (FTIRM) can shed some light on the mechanisms involved in the radiosensitization effects of NP. FTIRM is a non-destructive vibrational technique for the analysis of biological samples. 17 IR active molecular bonds of the main cell biomolecules change the dipole moment as a result of the vibration that occurs when infrared light is absorbed. The mid infrared spectral region is the most important for studying biological specimens since it includes the so-called 'fingerprint region' and the 'lipids region', which provides nucleic acid, protein, lipid, carbohydrate composition and/or conformational changes. This information enables the assessment of cell functionality, cell division, differentiation, growth and metabolism. [17][18][19][20][21] Within this context, the objective of this study is to use synchrotron-based FTIRM (SR-FTIRM) to disentangle radiosensitization effects of gold NP (1.9 nm AuroVist™) 22 in glioma cells. Despite the current treatment options, high grade glioma has poor prognosis in humans and thus, patient outcome could benefit from the enhanced therapeutic index of combined RT-NP modalities. Gaining new insights into the radiosensitization effects of AuNP in glioma cells is essential to progress on the development of NP-based therapeutics. In this work, we present SR-FTIRM data on F98 and U87-MG glioma cell lines to assess distinct cellular responses as a function of the cell type. The highly brilliant infrared source used in SR-FTIRM provided high spatial resolution and high quality information required for the single-cell analysis of our work. To the best of our knowledge, this is the first study that evaluates cell radiosensitization to AuNP by using SR-FTIRM.
Cell culture: F98 and U87-MG
The response of the two glioma cell lines, F98 (rat, ATCC® CRL-2397) and U87-MG (human, ATCC® HTB-14), was studied. The F98 glioma rat cell line simulates the behavior of glioblastoma tumors due to its highly invasive pattern of growth within the brain and low immunogenicity; it is commonly used to evaluate the effectiveness of several therapeutic agents. 23 The U87-MG human tumor model presents a nondiffusely infiltrative growth pattern and it is one of the most widely used cell lines, especially for anti-angiogenic therapy studies. 24 Both cell lines were purchased from LGC Standards (Molsheim, France). They were maintained and passaged in a high glucose (4.5 g L −1 ) Gibco™ DMEM medium (Life Technologies SAS, Courtaboeuf, France) supplemented with 1 mM GlutaMAX™, 1 mM sodium pyruvate, 10% fetal calf serum and 1% penicillin-streptomycin (10 000 U each), at 37°C and 5% CO 2 . On the day prior to the treatments, 10 5 cells per well were seeded in 6-well plates and incubated overnight.
Gold nanoparticles
Gold NP (1.9 nm AuroVist™) were purchased from Nanoprobes (Yaphank, NY, USA). The potential of these NP has been proved in previous biological studies. 1,4,25,26 A stock solution was extemporaneously prepared by mixing the AuNP (40 mg of gold) with 1 mL ultrapure water (Millipore Direct Q8). After rapid dissolution, the solution was filtered through a 0.2 μm centrifugal filter at 15 000g for 8 minutes and kept at 4°C. For cell treatment, the AuNP solution was diluted in fresh supplemented cell medium at a final concentration of 12 μM (500 μg mL −1 ). 25 The cells were then incubated at 37°C and 5% CO 2 for 24 hours. 25 Before the irradiation protocol, the AuNP-containing medium was discarded and replaced by 3 mL of fresh medium.
X-ray irradiation
Kilovoltage irradiations were performed at the Curie Institute (Orsay, France) with an X-ray unit (XRAD 320, Cegelec). The mean energy of this X-ray generator is 90 keV and the dose rate is 1.1 Gy min −1 . Each well plate was irradiated separately at 5, 10 and 20 Gy, respectively, for both cell lines, with and without AuNP. Two non-irradiated wells (0 Gy, without and with AuNP) were kept for controls. Just after irradiation, the medium was replaced by fresh medium.
SR-FTIRM sample preparation
For SR-FTIRM measurements, samples had to undergo a specific preparation protocol, i.e. cell fixation and deposition on infrared transparent calcium fluoride (CaF 2 ) cover glasses. 21,27 The cells were fixed at two post-irradiation times: after the irradiation (T 0h ) to evaluate immediate treatmentinduced effects, and after 24 hours (T 24h ) to assess the cellular response from the treatment.
For both fixation times (T 0h and T 24h ), the medium was removed and kept in order to collect floating cells. Then, 100 μL of 0.05% trypsin-EDTA solution was added into each well and incubated for 5 min. After cell detachment, the medium with floating cells was added back to the trypsinized cells, and 500 μL of fresh supplemented medium was added. The cell suspension was then centrifuged at 1500 rpm for 5 min at 4°C. The cellular pellet was rinsed with PBS, and resuspended in 10% formalin neutral buffered solution (Sigma-Aldrich). After 1 hour at room temperature, the samples were centrifuged at 1500 rpm for 5 min and the pellet was washed 3 times in Millipore water. The final cell suspensions in water were kept at 4°C until cytospinned down onto 0.5 mm thick infrared transparent CaF 2 slides using a Cytospin 4 centrifuge (ThermoFisher; 700 rpm for 5 min). Dried cells were then analyzed by SR-FTIRM.
SR-FTIRM at ALBA Synchrotron
SR-FTIRM experiments were performed at the infrared beamline MIRAS of the ALBA-CELLS synchrotron light source using the Hyperion 3000 microscope coupled to a Vertex 70 spectrometer (Bruker Optik GmbH, Germany). The measurements were performed in the transmission mode of operation of the FTIR microscope. This microscope is operating with a 36× Schwarzschild magnification objective (NA = 0.65) coupled to a 36× magnification condenser. The microscope is furnished with a liquid nitrogen cooled 50 μm mercury cadmium telluride (MCT) detector. Around 70-100 cells were randomly collected from each sample condition. Single point maps of individual cells were collected in the 3800-900 cm −1 mid infrared range at 4 cm −1 spectral resolution with 256 co-added scans per spectrum. All spectra were obtained using a single masking aperture size of 8 μm × 8 μm. Reference or background spectra were collected under the same acquisition parameters of the sample. In particular, measurement in an empty region on the CaF 2 slides (without cells) was repeated every 10 cell measurements, in order to take into account the differences under the ambient conditions along the measurements. The final cell absorbance spectrum was obtained by subtracting the reference spectrum from the raw cell spectra. The subtraction was done directly (automatically) using OPUS 7.5 software (Bruker Optik GmbH, Germany).
SR-FTIRM data analysis
Differences in the spectral features were evaluated by Principal Component Analysis (PCA) using Unscrambler X (CAMO Software AS, Norway). PCA was performed on vector normalized second derivative spectra (Savitzky-Golay algorithm; 3rd polynomial order). Vector normalization and PCA were separately applied in the 3100-2800 and 1800-1000 cm −1 spectral regions.
Raw spectra were corrected following the rubber band method (32 baseline points) using OPUS 7.5 (Bruker Optik GmbH, Germany) in order to assess the area under several spectral bands reported hereafter and represented in Fig. 1.
• Phosphate II: 1146-1004 cm −1 (PhII). The total area under the spectra (except the 2800-1800 cm −1 region) was associated with the total cell biomass (Cell) following previous studies. 28,29 Violin plots showing the probability density of the data for PhI/AII, PhII/ AII, AI/AII and CH 2 /CH 3 ratios were generated, since these ratios have been reported to provide valuable information on cell response after treatments. 28
Microphotography
F98 and U87-MG cells were seeded in a 35 mm polystyrene Petri dishes using the same NP concentration and NP incubation time as for SR-FTIRM measurements. After treatment, the cells were washed in fresh medium and microscopy image acquisition was performed using a 10× Nikon water-immersion DIC objective placed on an Eclipse 80i Nikon microscope (Scop Pro, Marolles-en-Hurepoix, France), which is equipped with a Zyla 5.5 MPX Andor SCMOS cooled camera (Scop Pro, France) and MetaMorph acquisition software (Molecular Devices, Sunnyvale, CA).
Cell viability assay
Metabolic activity was evaluated using the resazurin-resorufin cytotoxicity assay. 32,33 Briefly, after irradiation treatment, the cells were harvested in each well of the 6-well plate by using the usual trypsin-EDTA procedure. The cells were counted and 100 μL of 10 5 cells per mL were seeded in 96-well microplates and incubated for 24 hours at 37°C. After 24 hours, the medium was discarded and replaced by fresh medium containing 30 μg mL −1 of resazurin (from a filtered 1.2 mg mL −1 stock solution in PBS). The cells were incubated for 3 hours at 37°C, and the fluorescence intensity measurement of each well was performed using a Fluoroskan Ascent FL microplate reader (Thermo Fisher Scientific, Illkirch, France) at 530 and 590 nm for excitation and emission, respectively. The percentage of the metabolic activity of the cell populations was evaluated as the ratio of the fluorescence intensity after treatment to the control fluorescence intensity.
Results and discussion
Microphotography images for the qualitative assessment of intracellular NP localization are shown in section 3.1. Then, the results of SR-FTIRM analysis and cell viability assays are presented for both cell lines (F98 and U87-MG) and several irradiation doses (from 0 to 20 Gy) in the presence (+NP) and absence (−NP) of AuNP and two post-irradiation fixation times (T 0h and T 24h ). SR-FTIRM details on the biochemical effects induced by the AuNP without (0 Gy) and with radiation are presented in sections 3.2 and 3.3, respectively. The results of cell viability assays are reported in section 3.4.
Microphotography for intracellular NP localization assessment
Fig . 2 shows the bright field micrographs of F98 and U87-MG cells, respectively, with and without AuNP. While both cell lines show different morphologies (F98 is elongated and U87-MG is flatter and more spread on the cell support), nuclei can be easily distinguished as well as cell boundaries. After AuNP treatment and washing with fresh medium, free NP or NP aggregates can still be clearly seen in the cell surroundings (black dots). An unambiguous punctuated pattern inside the cell cytoplasm can also reproducibly be shown, especially in perinuclear areas. This strongly suggests an internalization process.
Whether this process is active (endocytosis) or passive ( permeation) could not be addressed by this microscopy experiment. However, it has been shown elsewhere and on different cell types that the majority of 1.9 nm particles are endocytosed. 25 While on F98 cells, the size of the dots was quite the same inside and outside of the cells, on U87-MG cells, the size of the black dots seems to indicate that the AuNP are aggregated. This might occur due to the intrinsic differences between the endocytosis processes in both cell lines.
3.2. Biochemical effects induced by the AuNP without irradiation (0 Gy) Fig. 3 and 4 show the PCA scores and the corresponding loading plots on second derivative spectra for both F98 and U87-MG cell lines, respectively. PCA allows the reduction of the dimensionality of features in order to assess the similarities and differences between spectra. In the PCA score plot, each single point represents a cell spectrum. Loading plots express the influence of the variables that are responsible for each principal component as a function of the wavenumber. PCA analysis was performed separately in the fingerprint (1800-1000 cm −1 ) and lipid (3100-2800 cm −1 ) regions.
For the F98 glioma cell line, and prior to the radiotherapy irradiation (0 Gy), the PC1-PC2 plot in the fingerprint region shows a clear separation between NP-treated groups and the control group (−NP), mostly along PC-1, which explains 73% of the variance. PC-1 shows more variance for the NP-treated groups. Fingerprint modifications are already observed at T 0h , which gives us a first indication of the AuNP action. For the U87-MG cell line, there is also some separation along PC-1 in the score plot between the control and NP-treated cells in the fingerprint region mainly at T 0h . The loading plots in the fingerprint region indicate that the largest biochemical changes induced by the NP are associated with protein modifications in the region of 1700-1500 cm −1 for both F98 and U87-MG cell lines. The vector normalized Savitzky-Golay second derivative calculated in the averaged absorbance spectra (Fig. 5) shows NP-induced changes in the protein conformational structures in the amide I and amide II bands, especially in the F98 glioma cell line. These bands are characteristic of peptide bonds of cell peptides and proteins. More precisely, there is a more explicit β-type secondary structure inside the NP-treated cells, [34][35][36] which could be related to an initial apoptotic response of the cells, since previous studies have reported changes in the expression of pre-apoptotic proteins using the same type of NP. 25 The separation of the NP-treated group at T 24h along PC-2 also has some contribution of the asymmetric phosphodiester band of the DNA (1238 cm −1 ), particularly in the case of the F98 cell line. We observe a shift towards the low frequency region for NP-treated cells (see Fig. 5). This shift has been observed in our previous work using gadolinium NP. 21,27 We also observe changes in the distribution of the relative intensities of several DNA spectral bands (PhI/AII, PhII/AII), as shown in Fig. 6 (top). At T 24h , there is a slight decrease in the PhII/AII ratio in the case of U87-MG cells, which might be correlated to cell cycle changes and, particularly, to an increase in the sub G 1 cell population induced by these AuNP. 25 Previous studies have reported similar changes in the DNA region of the infrared spectra due to cell cycle modifications. 36,37 Finally, it is important to note that we clearly observe an increase in the variance of the DNA biochemical content in NP-treated cells with respect to controls (−NP) both at T 0h and T 24h and for both cell lines, as a result of different NP-cell interaction mechanisms.
In the region of 3000-2800 cm −1 , mostly representative of fatty acids encountered in the membranes, there is a separation between the scores of control cells and NP-treated cells in F98 glioma line (Fig. 3). NP-treated cells are correlated with peaks at 2848 cm −1 and 2915 cm −1 , while control cells are correlated with peaks at 2935 cm −1 and 2858 cm −1 . This corresponds to changes in the asymmetric and symmetric vibrations of CH 2 stretching groups. 38 Changes in these peaks were previously related to the changes in the lipid structure and chain length due to oxidative stress and membrane peroxidation. [38][39][40][41] The separation in the PCA scores between −NP and +NP groups is less clear in the case of U87-MG cells (Fig. 4). Fig. 6 (bottom) show a clear increase in the CH 2 /CH 3 ratio distribution for F98 cells (both at T 0h and T 24h ), which might indicate evidences of oxidative stress. 39,40 There are also indications of oxidative stress in the U87-MG cells after NP-treatment (T 0h ), which seems to be reduced at T 24h . The induction of AuNP-related oxidative stress has been previously reported using the same type of NP via increased production of endogenous reactive oxygen species and depletion of intracellular antioxidants. 25,42 Our findings evidence a different biochemical response depending on the tumor cell model, which could be related to a different radiosensitization capability according to NP localization, NP-cell interaction processes or degree of sensitivity to oxidative damage or others. 5,25,26 This could explain the differences in NP internalization relative to the cell type shown in the microscopy pictures (see Fig. 2) and might be related to the subsequent variations in the biochemical effects observed by SR-FTIRM. Fig. 7 and 8 show the PCA scores and the corresponding loading plots on second derivative spectra for both F98 and U87-MG cell lines, respectively, and several configurations: doses (from 5 to 20 Gy), with/without NP (−NP/+NP) and postirradiation times (T 0h /T 24h ). PCA scores were separated as a function of the dose to assess the effect of AuNP for the same irradiation conditions. The two fixation times are compared in the same PCA scores to assess cell response to the treatments as a function of time.
SR-FTIRM study on the radiosensitization effects at several radiation doses
In F98 glioma cells, the protein region of the fingerprint spectral range (1700-1500 cm −1 ) is the main factor responsible for PCA score separation. PC1-PC2 score plots display clearly separated clusters (−NP/NP and T 0h /T 24h ), particularly for 10 Gy and 20 Gy and in the case of T 24h . PC-1 and PC-2 take into account around 70% and 10% of the variance, respectively, for all doses. The loading plots show that wavenumbers around 1628 cm −1 are highly correlated to NPtreated groups, while wavenumbers around 1660 −1 are correlated to irradiated groups without AuNP. This correlation is better manifested at T 24h . While the first is attributed to the β-sheet conformation of the amide I band, the second one is assigned to the α-helix form. 34 This indicates a shift towards lower wavenumbers in the amide for irradiated cells in the presence of AuNP, as can be seen in Fig. 9 (left). Previous studies correlated this shift to an increase in disordered structures due to protein denaturation. 43 In our case, the secondary protein structure changes inside the cells correlated with protein unfolding and/or denaturation might be related to cell death under treatment-induced stress. 36,44 This stress could be enhanced by the increased generation of ROS due to the presence of AuNP, which has been associated with changes in the amide I and II bands. [44][45][46] An amplification in the apoptotic response of irradiated cells in the presence of AuNP is in accordance with our previous FTIRM study using gadolinium NP. 21,27 The protein modifications as a function of the treatment are clearly shown on the vector normalized Savitzky-Golay second derivative plot (Fig. 9, left). In the case of U87-MG cells, the effect is less clear since the PC1-PC2 scores in the fingerprint region show more overlapping between '−NP' and '+NP' groups. For 20 Gy, we observe a slight separation of the NP-treated group for both fixation times. Loading plots mainly correlate this separation with secondary protein structure changes and modifications in the asymmetric phosphodiester bands. These changes, amplified in the presence of NP, are probably related to an enhanced cell death response due to the combination of radiation damage and AuNP-induced biochemical modifications. However, we observed a reduced biochemical response to AuNP with respect to F98 cells in the presence of radiation, since secondary structure protein changes are less clear in the Savitzky-Golay second derivative plot (Fig. 9, right). Fig. 10 shows the distribution of the relative intensities of several spectral band ratios (PhI/AII, PhII/AII, AI/AII and CH 2 / CH 3 ) defined in section 2.6. For the F98 cell line, we observe a decrease in the absorbance of the AI/AII ratio in NP-treated cells. Changes in the amide I and amide II absorbance were previously related to DNA repair processes, which involves many enzymes to repair the damage produced by the ionizing radiation. 47,48 The changes in relative intensity between the areas in amide I and amide II bands have also been correlated with the biochemical changes of cellular proteins following apoptosis, which affects the overall protein folding and localization and thus, the infrared absorption of peptide bonds. 30,31 By looking into the DNA region (1350-1000 cm −1 ) in the vector normalized Savitzky-Golay second derivative plot (Fig. 9), which is assigned to phosphodiester groups mostly found in nucleic acids, we observe several changes in the intensity and position (shifts) of several peaks. This probably implies a number of different variations in DNA organization or local conformation as a result of radiation damage, which is amplified in the presence of AuNP. The investigation of the loadings shows a slight variance emerging from the asymmetric phosphodiester band of the DNA at 1238 cm −1 for NP-treated groups at T 24h . The differential biochemical response to AuNP in the DNA region is more visible in the case of F98 than in the case of U87-MG cells.
For F98 glioma cells, the violin plots of Fig. 10 show a slight reduction in the PhII/AII ratio for NP-treated cells at T 24h , which has been previously correlated with chromatin fragmentation and apoptotic DNA condensation in previous studies. 28,30,49 Previous studies have reported similar variations as a result of radiation damage. 31,44,47,49 Instead, U87-MG cells exhibit a clear increase in the PhI/AII and PhII/AII ratios at T 24h in NP-treated cells. An increase in the phosphodiester bands was already observed in the work of Lipiec and collaborators for PC-3 (human prostate adenocarcinoma) cells following proton irradiation. 47 The increase in the intensity of the DNA backbone was correlated with multiple DNA breakages. 47,50 When comparing NP-treated cells in the lipid region (3100-2800 cm −1 ), the score plots do not display clear separated clusters, despite the fact that the NP-treated groups at T 24h seem to be slightly separated towards positive PC-1 in both cell lines from the other groups (T 0h and T 24h without AuNP). Fig. 10 shows some increase in CH 2 /CH 3 ratio distribution (defined in section 2.6) at T 24h , which is more marked for U87-MG cells. The higher absorbance in the CH 2 asymmetric stretching mode with respect to the CH 3 asymmetric stretching has been reported with cell death processes in previous studies. 28 Indeed, a higher CH 2 /CH 3 ratio has been determined for apoptotic cells by proton nuclear magnetic resonance spectroscopy in previous studies. [51][52][53] Finally, it is important to remark that we observe a higher intra-cellular variation in treated cells, demonstrating the heterogeneity in the biochemical cell response among the same cell population and also among cell lines. This is probably due to different NP-cell interaction processes, and degree of sensitivity to oxidative/radiation damage or others. The radiosensitization potential dependence as a function of the cell type has . Statistical significance in the analysis of the effect of the radiation dose, with or without NP, was determined using a standard two-tailed unpaired t-test. If any, non-significant differences (ns) with p ≥ 0.05 are indicated on the graphs. also been reported in previous studies using the same type of NP. 5,25,26,54 The distinct cellular responses of different cell lines are a key point for the study of radiosensitization effects of NP. Within this context, SR-FTIRM can provide new insights into the molecular and cellular levels for understanding the complex radiosensitization processes involving nanoparticles and radiotherapy, as shown in this work.
Cell viability assay
Fig . 11 shows the results of the resazurin-resorufin proliferation assay performed on treated cells, with or without AuNP, and with or without X-ray irradiations at 24 hours postirradiation time, to correlate with respect to the infrared data at T 24h . F98 glioma cells seem to be more sensitive to AuNP, already in the absence of radiation. Also, in the presence of radiation, U87-MG cells seem to be less sensitive to AuNP compared to F98 cells since the metabolic activity is not significantly different in the presence of AuNP. In F98 glioma cells, we can observe significant changes for the lowest dose (5 Gy), since the F98 metabolic activity is decreased in the presence of AuNP. It is interesting to note that the macroscopical measurement of the metabolic activity through the proliferation assay does not allow the differentiation of the effect of AuNP on both cell lines in all configurations, whereas SR-FTIRM shows more detailed events occurring at the molecular level. In this study, the choice of performing the proliferation test at 24 hours post-irradiation was to be able to correlate with the SR-FTIRM data and early biological events. Larger effects might be expected at larger post-irradiation times, as reported in previous studies using the same type of NP. 25
Conclusions
Despite significant evidence on the radiosensitization effects of AuNP, the exact mechanisms of radiosensitization are not clear. This work constitutes the first study of the radiosensitization effects of 1.9 nm AuNP in the F98 and U87-MG glioma cell lines by synchrotron-based infrared microspectroscopy at the single-cell scale. SR-FTIRM is a powerful tool for monitoring biochemical changes induced by AuNP combined with radiotherapy for assessing different cell responses to the treatments.
Cell-NP interactions differ in both cell lines. Prior to irradiation, AuNP induced spectral variations were observed both in the fingerprint and in the lipid regions. In particular, initial conformational changes in the protein secondary structures (F98, U87-MG), an increase in the CH 2 /CH 3 ratio (F98 cells), and absorbance intensity variations in the phosphodiester vibrations of the DNA (U87-MG) are detected. The biochemical alterations induced by the AuNP on both cell lines give us a first indication on the AuNP action.
PCA analysis reveals clear NP-induced changes when AuNP are combined with radiotherapy. The main spectral variations include a shift toward the low wavenumber in the protein bands (F98), changes in amide I/II relative intensity (F98), and relative amplitude changes in the CH 2 and CH 3 stretching modes (U87-MG), along with several DNA organization or local conformation modifications (F98, U87-MG) due to the presence of AuNP. These changes are better manifested at T 24h and they are probably related to an initial cell death response of the cells, which is amplified in the presence of AuNP. SR-FTIRM results showed the heterogeneity in the biochemical cell response among different cell lines (and among the same cell population), probably due to the different NP-cell interaction processes, degree of sensitivity to oxidative/radiation damage, etc., which implies different radiosensitization capabilities as a function of the cell line.
Despite the fact that SR-FTIRM provides a deeper understanding of the NP-induced molecular and cellular response at a biochemical level, further biological studies are required to assess the role of different ( physical and biochemical) radiosensitization mechanisms as a function of NP and cell type. Comprehensive characterization studies of cellular responses to AuNP are required for the further development of this promising technique towards potential clinical translation.
Conflicts of interest
There are no conflicts to declare. | v3-fos-license |
2021-04-28T14:43:35.608Z | 2021-02-15T00:00:00.000 | 233417662 | {
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} | pes2o/s2orc | Cryopreservation: The secret of modern preservation of brewer’s yeasts – minireview
The aim of the long-term preservation of cells, tissues and organs is to maintain their cellular structures and biological functions for as long as possible. Cryopreservation is a process where biological material is stored and preserved at very low temperatures. However, freezing and thawing processes can cause irreversible cell damage, which is related to formation of ice crystals, osmotic stress, accumulation of reactive forms of oxygen, etc. Therefore the cell viability depends mainly on the freezing rate, the composition of the cryoprotective medium as well as on the thawing rate. Using a suitable cryoprotective medium can increase the viability rate of the yeasts after “revitalization“. Appropriate pre-cultivation before freezing also plays an important role. These facts show that cell freezing and thawing processes must be controlled to avoid cell damage.
Introduction
It is essential that even after a long-term storage, brewer's yeasts maintain their original properties which constitute one of the main requirements for their use in brewing.
The preservation process should not only ensure high viability of the cells, but also the preservation of their physiological as well as technological properties. Various techniques can be used for preservation of yeasts, for example regular re-inoculation on a suitable medium (a liquid or solidified soil), drying methods, freeze-drying (lyophilization), freezing in liquid nitrogen (cryopreservation), vitrification, etc. (Kumar et al., 2013;Day and Stacey, 2007;Kirsop and Doyle, 1991).
This paper is focused mainly on the long-term storage of brewing yeast using the cryopreservation technique. I addition, it also offers a brief overview of other preservation techniques to enable a comparison of their particular advantages and disadvantages.
Traditional techniques for non-freezing yeast preservation
The yeast preservation techniques can be generally divided according to the length of the preservation period into short term and long-term, and according to the applied process into cultivation, drying and freezing. A short-term period is considered to be the preservation of the sample of up to 1 year, while long term is considered as permanent (Nakasone et al., 2004).
Cultivation methods -periodic transfer
The repeated cultivation is a basic short-term preservation technique which maintains yeast growth on agar soils (Nakasone et al., 2004). In the brewing environment, it constitutes a widely employed technique mostly using a slant agar (e.g. malt agar) and a storage temperatures of up to 4 °C (Matoulková and Sigler, 2011). This method is simple and cheap, because specialised equipment is not required. On the other hand, it is rather time consuming and labour intensive since it involves the steps of re-inoculation, regular checking for contamination and desiccation (Nakasone et al., 2004). That is why this method is recommended primarily for collections with a small number of cultures which are used constantly and for short periods of time i.e. less than 1 year (Matoulková and Sigler, 2011).
It is possible to prolong the intervals between re-inoculations by using sterile mineral oil. The medium used to overlay yeast cultures is mineral oil, and thus the yeast metabolism is slowed down (Suga et al., 2000). The oil level in preservation vessels (e.g. tubes, vials, etc.) must be periodically checked, and oil should be added, if necessary. To revive an active oil-free culture, it is suggested to re-cultivate the harvested colony several times (Nakasone et al., 2004).
The use of the re-inoculation method has its limits because the yeast viability cannot be maintained for more than 5-6 months. Moreover, the multiple re-inoculation can lead to significant changes in viability, morphology, physiology and genetic stability. Respiratory-deficient mutants, changes in cell morphology as well as varieties with different flocculation properties can appear (Kirsop and Doyle, 1991;Kirsop, 1974). Furthermore, frequent handling of cultures increases the risk of contamination (Boulton and Quain, 2001).
Drying methods
Likewise, drying methods have established their strong position in the brewing industry due to the fact that they are simple, fast and cost-effective. The literature describes a number of techniques ranging from preparation of active dried yeasts (ADY) to protocols incorporating yeasts into a gel structure (Gelinas, 2019;Nyanga et al., 2012;Jekins et al., 2011;Cyr et al., 2007;Nakasone et al., 2004). However, drying methods can influence the yeast viability undesirably. Several studies point out that especially lager strains of Saccharomyces pastorianus are extremely sensitive to drying process; subsequently abnormal flocculation, haze formation and a less stable foam structure can arise (Jekins et al., 2011;Cyr et al., 2007;Russell and Stewart, 1981).
Fluidised bed drying has become a widespread and gentle technique for preparation of ADY for brewing. Moreover, spray drying should be mentioned as well, although it is not commonly used in brewing because it produces low viability cultures.
Desiccation using anhydrous silica gel is considered to be a medium-term and cheap drying method. Silica gel is a porous form of silica (SiO 2 ) with a pore diameter of 0.1 to 10 μm. This technique is based on mixing a yeast suspension with a precursor of gel, thus the process of gelatination starts. During gelatination water evaporates and gel dries. This results in incorporation of yeasts into gel structure (Uo et al., 2004). Revival of cultures involves scattering a few silica gel crystals on an agar plate. The viability of cultures stored in this way depends on the yeast strain and the medium on which it was cultivated before the preservation process began. The advantage of silica gel is that it prevents all fungal growth and metabolism. (Nakasone et al., 2004).
It should be added that also lyophilisation (freeze-drying method) can be included in this chapters, nevertheless we preferred to assign it to the next chapter dealing with freezing methods.
Freezing methods
Freezing methods are versatile and widely applicable in a long-term preservation of brewer's yeasts, however, these methods require specialized and expensive equipment. The easiest variant of freezing methods is to place suitably prepared cultures in mechanical freezers in a temperature range between -20 and -80 °C, although the viability decreases with the time (Bond, 2007). Electric freezers enable the yeast preservation even at lower temperatures between -100 and -150 °C. Nevertheless, a frequently utilized, rather inexpensive and simply accessible protocol is a combination of 25% (v/v) glycerol as a cryoprotectant and freezing temperatures from -60 to -80 °C (Bond, 2007;Nakasone et al., 2004).
It is relevant to add that the above-mentioned freezing technique is sometimes referred to as cryopreservation in the scientific literature (Cabrera et al., 2020;Grimalt-Alemany et al., 2020;Wing et al., 2020;Homolka, 2013). In this paper we will define the freezing techniques of deep freezing and the cryopreservation as a cell storage in liquid or vapour nitrogen.
Lyophilization/freeze-drying technique
Lyophilization is based on the sublimation of water from a sample at a low temperature and pressure. The process involves three basic steps: freezing and two high vacuum drying phases (Alonso, 2016; Morgan et al., 2006). Although the loss of the cell viability is quite high during the lyophilisation process, it is minimal during the subsequent storage of the lyophilised yeasts, which remains viable for more than 30 years (Day and Stacey, 2007). The freeze-drying technique is thus generally regarded as a gentle and suitable process of yeast preservation even from the point of view of further manipulation and transport. As about 90% of water is removed, the lyophilised cultures are very light. Although they should be kept at -18 °C to maintain stability, the cold chain can be interrupted for a short time, e.g. during transport (Foerst and Santivarangkna, 2015). Despite its numerous advantages, it is also necessary to point out that lyophilization is energy and cost demanding (Foerst and Santivarangkna, 2015).
Cryopreservation -storage using nitrogen
Cryopreservation is a method of a long-term storage of viable organisms in a frozen state. To be exact, we should understand this method as a storage of (micro)organisms at the temperature below -139 °C, when the cells occur in the state of anabiosis, that means a reversible suspension of life processes (Kirsop and Doyle, 1991). In other words, the rates of biophysical processes are too slow to affect cell vital properties at -139 °C, i.e. the temperature in which not even ice crystals grow (Nakasone et al., 2004). Various liquified gases are used to reach this extremely low temperature, for example nitrogen, argon or xenon. Nevertheless, liquid or vapour phase nitrogen, with its working temperature range between -160 °C to -196 °C, is used almost exclusively due to safety and financial reasons (Matoulková and Sigler, 2011;Bond, 2007).
We may assume that the rates of mutation in cultured yeasts approximately correspond to those of cell division and metabolic activity. Therefore the cryopreservation prevents increased genetic variability of stored cultures owing to the complete halt of cell division as well as the total arrest of metabolism. In addition, the method is timesaving, reducing labour needs compared to the handling of living cells. It prevents culture contamination and increases assurance of the long-term availability of cultures. Of course, there are several drawbacks such as the high cost of the equipment and the fact that liquid nitrogen must be checked every 2 days and refilled if necessary; further a sufficient space for refrigeration units is required (Bond, 2007;Nakasone et al., 2004).
Let's briefly examine the effects of freezing of living cells. The cells are exposed to 2 basic stressors, namely osmotic and thermal stress during both processes of freezing and thawing. The cell suspension can be considered as an aqueous solution of yeast cells and various solutes coming from the cultivation medium. When such an aqueous solution begins to cool below the freezing point, the water in the external environment will freeze the first, because it is much less concentrated than the cytoplasm inside the yeasts. The frozen water starts to be released from the cell suspension in the form of ice crystals, whereby the substances dissolved in the remaining non-frozen fraction of the solution are concentrated. The osmotic pressure of the environment increases and the cells respond by expelling water from the intracellular environment. Thus the yeasts are dehydrated and wrinkle their shape (Hubálek, 1996;Morris et al., 1998).
Since yeasts are relatively sensitive to the above described osmotic and thermal stresses, the addition of cryoprotective substances to the freezing medium is often used in order to prevent damage of the cells as much as possible. In general, these substances should be readily soluble in water and non-toxic or very slightly toxic. Cryoprotectants differ in their ability to penetrate through the cell membrane. On the basis of this key ability, they show different protective effects: i) penetrating cryoprotectants -such as glycerol or dimethyl sulfoxide (DMSO), which easily pass through the cell membrane and ensure intracellular as well as extracellular protection; ii) non-penetrating cryoprotectants -such as various sugars (e.g. sucrose, lactose, glucose), alcohols (e.g. mannitol, sorbitol), amino acids, proteins or a number of other substances with different structure and molecular weight (e.g. dextran, polyvinyl-pyrrolidone, and hydroxyethyl starch), which protect the cells extracellularly (Nakasone et al., 2004;Hubálek, 2003;Hubálek, 1996).
The principle of the cryoprotectant effect is to increase the total concentration of all solutes in the system and thus reduce the ice formation (Bond, 2007).
The cryoprotective principle of glycerol as an intracellular cryoprotective agent lies in the forming of strong hydrogen bonds with water molecules through hydroxyl groups. These bonds prevent excessive cell dehydration, intracellular ice crystal formation as well as reduction of salt toxicity by decreasing the amount of frozen water. They also increase the plasticity of the cell wall and bind intracellular water, which increases the cell's resistance to hyperosmotic stress (Hubálek, 2003).
As we mentioned above, the cells are submerged in liquid nitrogen (Figure 1) usually at the temperature range from -160 to -196 °C, at which all biochemical and crystallization processes cease. Freezing, or more precisely the transition of cells from the optimal ("room") temperature to the very low temperature of e.g. -196 °C (= boiling point of nitrogen at sea level), is a controlled process, depending on the technical equipment, by means of which the efficiency of cryopreservation can be influ-enced. The alternative to storing cultures directly submerged in liquid nitrogen is to place them in special refrigeration boxes filled with nitrogen vapours (Figure 1) with the temperature of -140 °C (Kirsop and Doyle, 1991).
The rate of freezing largely determines the degree of cell dehydration and the formation of ice crystals together with their size and location. During slow freezing (< 10 °C/min), the cells have enough time to equalize the osmotic pressure of the medium and therefore only extracellular ice crystals are formed. On the contrary, during rapid freezing (cca 102 °C/min), the cells do not have enough time to expel water to the external environment, thus intracellular as well as extracellular ice crystals are formed. In case of very fast freezing (> 103 °C/min), the cells do not lose water at all and therefore contain a high amount of very small ice crystals (Dumont et al. 2004;Dumont et al., 2003;Hubálek, 1996). In the two latter cases, there is a risk of cell damage during revitalization because the growing ice crystals inside the cells can damage cytoplasmic membrane, the cell organelles and the cell wall of the yeasts (Momose et al., 2010;Seki et al., 2009). An improperly chosen freezing method can also lead to cell damage at the level of nuclear or mitochondrial DNA (Stamenova et al., 2008;Stoycheva et al., 2007).
The most effective approach is a combination of slow freezing with fast recovery. Figure 2 illustrates how cryosamples of brewer's yeasts are pulled up from the cryogenic Dewar vessel filled with liquid nitrogen. The optimum revitalization rate is 200 °C/min. This roughly corresponds to immersion of a frozen culture, which has been placed in a polypropylene straw, into a water bath at 37 °C (Hubálek, 1996). The literature reports and practice often implements a non-programmable conventional method, which is based on pre-freezing of cultures in ultra-low temperature freezers at -80 °C and their subsequent immersion into liquid nitrogen (Yang et al., 2010). The disadvantage of this procedure is that the rate of freezing cannot be controlled, and thus there is a risk that intracellular crystals will form in the cells.
The resistance of yeast cells to thermal and osmotic stress can be increased by a suitably selected cultivation method used immediately before an immersion into liquid nitrogen. Aeration, i.e. shaking, during cultivation as well as the presence and the concentration of a cryoprotective component added into the incubation medium usually affect yeast resistance (Polezhaeva et al., 2014;Suga et al., 2000). The choice of the cryoprotective component together with a corresponding methodological procedure represent a significant aspect. For example, glycerol as the most common cryoprotective substance enters into yeast cells relatively slowly. When glycerol is used, it is recommended to leave the cells in a medium supplemented with this cryoprotectant at a room temperature for about 2 hours prior to freezing. This phase is called the "equilibration phase" and it gives glycerol the time necessary to enter into the cells (Hubálek, 2003). The storage of the cell suspension in a cryoprotective medium is provided in suitable vessels (cryotubes, cryovials) or in thin cryotubes, called straws, which are then placed in cryotubes. The volume of one straw is between 50-80 microlitres of the cell suspension. It means that a large enough number of straws is necessary to store each strain. Freezing is performed in a chamber with a programmable and controlled rate of freezing. An open straw with the cell suspension is placed aside for each frozen batch so that the cooling process can be monitored and the program adjusted. A record of progress is kept for each frozen batch. The freezing itself occurs when the cryotubes with straws are placed in a freezing chamber into which nitrogen vapours are fed in a controlled manner. As soon as a required temperature is reached (e.g. -160 °C), the cooling process is terminated. Finally, the frozen cryotubes are placed in racks and containers in a Dewar vessel and submersed in liquid nitrogen (Matoulková and Sigler, 2011).
Conclusion
The general purpose of the long-term deposition of cells is to keep them viable for as long as possible. Moreover, in the case of technologically important microorganims, it is necessary to ensure the stability of their physiological and technological properties. The cryopreservation, as a storage in liquid nitrogen, is an effective way to preserve many (micro)organisms including brewer's yeasts. However, the process requires correctly set conditions that enable the long-term storage.
Acknowledgments
The article was supported by the Ministry of Agriculture of the Czech Republic, within Institutional Support MZE-RO1920. | v3-fos-license |
2019-04-06T13:08:13.780Z | 2012-12-18T00:00:00.000 | 96697591 | {
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} | pes2o/s2orc | Direct measurement of the oceanic carbon monoxide flux by eddy correlation
. This report presents results from a field trial of ship-based air–sea flux measurements of carbon monoxide (CO) by direct eddy correlation with an infrared-laser trace gas analyzer. The analyzer utilizes Off-Axis Integrated-Cavity-Output Spectroscopy (OA-ICOS) to achieve high se-lectivity for CO, rapid response ( ∼ 2 Hz) and low noise. Over a two-day sea trial, peak daytime seawater CO concentrations were ∼ 1.5 nM and wind speeds were consistently 10– 12 m s − 1 . A clear diel cycle in CO flux with an early afternoon maximum was observed. An analysis of flux error suggests the effects of non-stationarity are important, and air–sea CO flux measurements are best performed in regions remote from continental pollution sources.
Introduction
Carbon monoxide (CO) is produced in the ocean surface mixed layer by photolysis of chromophoric dissolved organic matter (CDOM) (Wilson et al., 1970;Lamontagne et al., 1971;Zuo and Jones, 1995). Daytime photolytic production and continual consumption by microbes Seiler, 1980, 1982) leads to a pronounced diel cycle in surface seawater CO concentration, with a pre-dawn minimum and an early afternoon maximum (Lamontagne et al., 1971;Johnson and Bates, 1996;Stubbins et al., 2006;Zafiriou et al., 2008). Considerable variability is possible in both the rate of production (Valentine and Zepp, 1993;Zuo and Jones, 1995) and rate of consumption (Jones, 1991;Jones and Amador, 1993;Johnson and Bates, 1996). In two detailed studies of the water column CO budget, ventilation to the atmosphere was less significant than loss to microbial consumption (Bates et al., 1995;Zafiriou et al., 2003).
Although marine emissions represent a minor fraction of the global CO budget (Bates et al., 1995;Stubbins et al., 2006), the ocean may be a significant source of CO to the remote marine boundary layer in the Southern Hemisphere (Erickson and Taylor, 1992). In addition, CO is recognized as a useful tracer for studies of sea surface mixed layer processes because it couples to biological, photochemical, and physical mixing dynamics (Najjar and Erickson III, 1995;Zafiriou et al., 2008).
Due to the lack of a suitable direct flux measurement, previous studies of oceanic CO emissions utilize empirical gas exchange formulations (e.g., Wanninkhof, 1992;Nightingale et al., 2000). CO solubility in seawater is quite low. Empirical models of air-sea gas exchange typically focus on gases of similarly low solubility (e.g., Rn or He/SF 6 dual tracer methods), so these formulations may also provide a reasonable representation of CO transfer. An eddy correlation flux measurement allows a practical test of this assumption. In a more fundamental sense, direct measurement of the CO flux facilitates development of physical gas transfer algorithms specifying the solubility dependence of the gas exchange coefficient (e.g., Fairall et al., 2011, and references therein).
In this submission we present results from a short field trial of a new method for direct measurement of the oceanic CO flux by eddy correlation. To our knowledge, this is the first reported CO flux measurement from a ship. The data set presented here is brief. Our intent in submitting this article is to (1) present evidence that air-sea flux measurements of CO are feasible with current state-of-the-art instrumentation, and (2) examine flux error related to departure from stationary conditions (see Businger, 1986, for a discussion of stationarity and flux measurement).
Experiment
An LGR model 907-0014 N 2 O/CO analyzer (Los Gatos Research, Inc.) was used in this trial. This instrument employs a continuous narrow-band infrared laser source for Off-Axis Integrated-Cavity-Output Spectroscopy (OA-ICOS) absorption measurements of N 2 O, CO and H 2 O (O'Keefe et al., 1999;Baer et al., 2002). In this test, we use a 200-tube Nafion air drier (Perma Pure PD-200T-24-SS) to reduce the dew point to < −10 • C, yielding essentially dry-air concentration values. The analyzer data rate is 10 Hz, but, as we note below, in this configuration at a sample flow of ∼ 10 std L min −1 frequency response is ∼ 1-2 Hz. Air was subsampled from a 20 m long high-flow Teflon inlet (3/8 ID) drawing air at ∼ 80 std L min −1 . The air inlet, sonic anemometer (Gill Model R2) and a six-channel motion sensor (Systron-Donner MotionPak) were mounted at the top of a 10 m meteorological tower on the bow of the University of Hawaii research vessel Kilo Moana. The motion sensor was located 0.6 m below the sonic volume. A short pulse of nitrogen injected at the inlet tip each hour facilitated precise synchronization of wind and CO measurements. Procedures for correcting wind data for ship motion interference have been described previously (Edson et al., 1998;Blomquist et al., 2010).
The mean ten-minute CO variance spectrum (Fig. 1) shows a "pink" background noise (∼ 1/f n ) in addition to the −5/3 dependency due to turbulent dissipation. The integrated noise variance over the flux bandpass (0.00167 to 2 Hz) is 0.015 ppb 2 (σ co = 0.12 ppb). An instrument artifact signal is evident at 3 Hz, but this is beyond the flux frequency response and has been filtered from the data. A comparison of shipboard spectra with laboratory data (not shown) shows negligible analyzer sensitivity to ship motion.
Fluxes are derived from ten-minute segments of CO and vertical wind velocity data with 50 % overlap of successive segments (i.e., 11 ten-minute segments per hour). The linear trend is subtracted from each segment and a Hamming window function applied to limit leakage of low frequency variance unrelated to surface flux. The slope from the linear trend in CO is retained as a measure of ∂CO/∂t for each segment. Flux is computed as the area of the cospectrum.
This gas inlet has been used extensively for dimethyl sulfide (DMS) flux measurements and the transfer characteristics studied in detail (Blomquist et al., 2010). At flow rates ≥ 80 L min −1 , frequency attenuation in the main sample line is not significant; the half-power frequency is ∼ 10 Hz. The Nafion air dryer produces the greatest signal attenuation, lowering the half-power frequency to ∼ 1-2 Hz. The frequency response of the CO analyzer in this configuration is quoted by the manufacturer at 1-2 Hz as well. For DMS, we Fig. 1. Mean bin-averaged CO variance spectrum for ∼ 400 tenminute data segments at sea. Valid data segments were selected using criteria described in Sect. 3. The peak at 3 Hz is an analyzer artifact.
have consistently found a flux attenuation correction of 4-5 % at wind speeds comparable to this project (e.g., Yang et al., 2011). We therefore apply the same correction to CO flux. In the future a more thorough examination of flux losses is warranted. However, bias associated with imprecision in this correction is unlikely to alter the conclusions of this report. Ten-minute flux results are selected to eliminate periods unsuitable for eddy correlation measurements. Data acquisition was continuous, but roughly one-third of the ten-minute flux values over the two-day period were discarded on the basis of selection criteria. Selection limits relating to sampling conditions are: relative wind direction within ± 60 • of the bow, standard deviation in relative wind direction < 10 • , and heading change < 25 • in ten minutes. In addition, it was necessary to impose stationarity criteria limiting the magnitude of ∂CO/∂t and horizontal turbulent flux, as described in Sect. 4.
Results
The CO flux trial was conducted on a routine cruise to the Hawaii Ocean Time-series (HOT) station ALOHA, located at 22 • 45 N, 158 • W (cruise HOT-238, 18-22 December 2011). Over two days of sampling on-station, wind speed was consistently 10-12 m s −1 , and relative wind direction remained within ± 20 • of the bow. Seawater CO concentration at 5 m depth ( Fig. 2) was measured from selected daytime CTD (conductivity, temperature, depth) casts using the method of Xie et al. (2002). An afternoon maximum of ∼ 1.5 nM is evident. Nighttime samples were not analyzed but were most likely ≤ 0.5 nM based on data from prior cruises, implying a mean daily concentration of < 1 nM. Seawater CO measurements at station ALOHA on prior cruises since 2008 (also Fig. 2) show a diel pattern typical for blue water regions in the Pacific and Atlantic (e.g., Johnson and Bates, 1996; Stubbins et al., 2006;Zafiriou et al., 2008): an early afternoon maximum of 2-3 nM and mean daily concentration of ∼ 1 nM. In contrast, peak seawater CO concentrations in Fig. 2 for this trial were about half the typical value, providing a stringent test of the flux measurement method. Figure 3 is a time series of hourly CO flux for the cruise, computed as the mean of all ten-minute flux measurements each hour meeting selection criteria. Figure 4 shows tenminute results bin-averaged to hour-of-day (local time). The diel cycle in CO flux from Fig. 4 closely mirrors the cycle in seawater concentration in Fig. 2, with a pre-dawn minimum and early afternoon maximum.
For a seawater CO concentration of 1.5 nM at the peak in the flux diel cycle (02:00-03:00 p.m. LT -local time), the CO gas transfer coefficient computed from Eq. (1) at the reference Schmidt number 660 (k 660 ) is 41 cm h −1 ; where F co is flux, α co is dimensionless solubility, P co is the interfacial concentration gradient, (C sw /α − C air ), and Sc co is the Schmidt number in seawater at ambient temperature. For these conditions, at U 10 ∼ 11 m s −1 , the Wanninkhof (1992) (1997) yields k 660 = 40 cm h −1 (α, Sc and k 660 computed using the Rscripts of Johnson, 2010). Clearly, the observed k 660 is close to the expected value based on several of these models, but with limited data it is difficult to draw too much from this agreement. More extensive sampling of the seawater CO concentration is necessary to critically assess the observed transfer coefficient. Rowe et al. (2011) have analyzed sensor resolution requirements for eddy correlation measurements. For the conditions of this study and the noise level of the CO analyzer, their analysis predicts a 60 % random error for hourly average CO flux measurements at a seawater concentration of 0.5 nM. In this test, however, scatter in hourly CO flux from midnight to 06:00 a.m. LT (when seawater CO is ∼ 0.5 nM) suggests a larger error: 0.00063 ± 0.00118 ppb m s −1 or 187 % as the relative standard deviation. The expected CO variance from surface flux may be predicted from similarity theory as a function of friction velocity (u * ) and flux magnitude (|w co |) (Fairall et al., 2000;Blomquist et al., 2010). Assuming neutral stability, the relationship takes the form σ co,sim = 3 |w co |/u * .
Sources of flux uncertainty
(2) As a point of comparison, data for similarity-predicted and observed variance in DMS and CO concentration are shown in Table 1. The observed DMS standard deviation (σ obs ) from a cruise in the Sargasso Sea is quite close to the similarity estimate (σ sim ). This is reasonable, as the sea surface is the sole source of DMS, and its atmospheric lifetime is sufficiently short (2-3 days) to limit the influence of advection from distant sources. However, σ obs for CO is seen to be four times greater than the similarity-predicted value, further suggesting additional CO variance from non-surface-flux sources. Conditions relating to non-stationarity are an obvious source of additional error. Small gradients in CO concentration can yield variance from horizontal turbulent flux which greatly exceeds variance from surface flux, even for typically clean conditions in the remote marine boundary layer at station ALOHA, where the relative standard deviation in ten-minute mean CO concentration was just 2 % over two days on-station. Figure 5 illustrates the relationship between components of the horizontal turbulent flux and ∂CO/∂t. A positive correlation exists between the alongwind component of horizontal flux (u c ) and ∂CO/∂t, indicating advection of the CO gradient past the ship. For numerous samples, horizontal turbulent flux is many times larger than the expected magnitude of CO surface flux. . Fig. 6. Mean cospectra illustrating vertical (w co , red) and horizontal (|u co |, blue) CO turbulent flux for ∼ 35 selected ten-minute afternoon flux segments (01:00-04:00 p.m. LT). Horizontal flux is computed as the mean of absolute values. After eliminating periods of excessive non-stationarity, the magnitude of horizontal turbulent flux is still several times greater than the vertical flux.
The following additional criteria were therefore applied to eliminate ten-minute segments with excessive gradient influence: |∂CO/∂t| < 2.7 ppb h −1 and |u c | < 0.026 ppb m s −1 (shown as a bounding box in Fig. 5). Even for samples which meet these criteria, the magnitude of |u c | is large compared to w c . Figure 6 presents cospectra for w c and |u c |, representing mean fluxes for selected early afternoon ten-minute segments (35 segments, 01:00-04:00 p.m. LT). The w c cospectrum is positive but noisy due to limited sample size and the effects of residual CO variance from other sources. The integrated area of the |u c | cospectrum is quite large, with a significant component at low frequencies mirrored in the CO variance spectrum (Fig. 1). The mean absolute horizontal turbulent flux from all ten-minute segments meeting selection criteria ( |u c | all ) is 0.008 ppb m s −1 ( u c all = −0.0034 ± 0.0091 ppb m s −1 ), or eight times greater than the mean vertical flux ( w c all ) of 0.001 ppb m s −1 .
The scalar variance budget production term associated with CO turbulent flux in a mean gradient is (Stull, 1988) −2 u i co where u i and x i specify the full turbulent wind field. From similarity theory, an estimate for the vertical CO gradient is which is ∼ 3.5 × 10 −4 ppb m −1 (0.35 ppb km −1 ) for mean conditions of this test (i.e., mean flux = 0.001 ppb m s −1 ; u * = 0.4 m s −1 ; measurement height, z = 18 m; and the von Karman constant κ = 0.4). The terms (Eq. 3) for horizontal and vertical flux components are therefore comparable when ∂CO/∂x ∼ 4.3 × 10 −5 ppb m −1 (4.3 ppb per 100 km). This is a very low threshold gradient for a species with a mean background concentration of 60-150 ppb. Assuming a CO gradient aligned with mean wind at u = 10 m s −1 , this corresponds to |∂CO/∂t| = 1.5 ppb h −1 , or approximately half the 2.7 ppb h −1 selection limit. The stationarity criteria therefore limit the variance from horizontal turbulent flux to approximately twice the variance from mean surface flux. In this test, this is sufficient to eliminate the majority of outlier observations.
Flux detection limit
To investigate the flux detection limit, we examine theoretical error as a function of air-sea concentration gradient, P co , and wind speed, u. Flux error may be specified as a function of variance in both vertical wind (w) and scalar (CO) measurements, where CO variance is composed of an atmospheric vertical turbulent flux component (σ 2 co a ) and an "other noise variance" component (σ 2 co n , from analyzer white noise, etc.), and where T is sampling time in seconds (after Fairall et al., 2000).
The two terms in Eq. (5) are assumed to be independent, with characteristic integral time scales (τ ). From nighttime flux measurements under conditions where P co is very low (and therefore σ 2 co a ∼ 0), we can solve Eq. (5) for the "other noise" term, yielding σ 2 co n τ co n = 0.00041. Using similarity relationships to represent σ 2 co a , σ w and τ w co , and employing empirical functions for the stability dependence of these parameters (see Blomquist et al., 2010) yields an expression for error as a function of u and u * , which can be further extended to the following relationship for P co (u) (Eq. 6) by assuming an arbitrary error condition (e.g., δF /F = 1, or 100 % error) and substitution of the standard flux formulation: F co = αk(u) P co .
Here, f i (z/L) are functions defining stability dependence (unity for neutral conditions assumed here), u r is mean relative wind speed (equivalent to u if the ship is not moving), and k(u) is the gas transfer coefficient function. For CO, k(u) may be estimated from a cubic wind speed dependence (e.g., Edson et al., 2011). An estimate for u * (u) may be obtained by assuming a roughness length typical of the open ocean (∼ 0.0003 m), or by a fit to observed u * , or from a fit to bulk flux model derived u * (e.g., COARE 3.0). Note that in Eq. (6), F co is dependent on P co and u, so an iterative solution is necessary. Alternately, Eq. (6) may be further rearranged and solved for P co following substitution for F co . The detection limit criterion P co (u) computed from Eq. (6) is shown in Fig. 7. This curve should be a theoretical limit under neutral stationary conditions (the similarity functions assume stationarity), but in fact it may be an upper limit, as σ 2 co a is not exactly zero in the empirical determination of the "other error" term above. We also note that, other things being equal, as SST (sea surface temperature) decreases so does P co , and therefore flux error increases.
Early afternoon conditions for this field trial ( P co = 1700 ppb, u = 11 m s −1 ) lie a factor of three above the curve in Fig. 7, implying an expected error of 33 % for hourly mean flux at the peak in the diel cycle. This is approximately the observed error from Fig. 4 (22-37 % relative error at 01:00-03:00 p.m. LT). Results in Fig. 4 represent the average of all 10-min flux measurements for each hour which meet selection criteria, and in this case the afternoon values include only a little more than one hour of total sampling time, which is approximately the sampling time assumed in Fig. 7. The detection limit presented in Fig. 7 supports the conclusion that oceanic CO flux measurements are feasible under favorable conditions with appropriate selection criteria.
Conclusions
The analytical performance of a commercially available infrared OA-ICOS trace gas analyzer is sufficient for shipbased flux measurements of CO at moderate-to-high wind speeds when seawater concentration is > 1 nM. A clear diel cycle in CO flux, mirroring the cycle in seawater concentration, was observed over two days at a research site near Oahu in the oligotrophic North Pacific subtropical gyre. CO flux measurements by eddy correlation demonstrated here are a potentially important development for studies of biogeochemical and physical dynamics in the ocean's surface mixed layer. Additionally, CO is an important low solubility end-member in the spectrum of gases involved in airsea exchange. As such, it should exhibit significant bubblemediated gas exchange enhancement at moderate-to-high wind speeds, providing an interesting and important test of physical gas transfer theory.
The effects of non-stationarity are clearly significant for CO. The moderately long atmospheric lifetime of CO (∼ 50 days), combined with vigorous natural and anthropogenic sources, yields a high, variable background atmospheric concentration of ∼ 100 ppb in the Northern Hemisphere and half that value in the Southern Hemisphere. CO variance from horizontal turbulent diffusion of atmospheric gradients as small as 1-2 % of the mean concentration per 100 km reduces precision of the eddy correlation flux measurement. Removing 10-min flux values which exhibit excessive horizontal flux or ∂CO/∂t (Fig. 5) is critical to reducing error to near the theoretical limit and resolving the diel cycle in CO flux. These conclusions may also apply to flux measurements of other long-lived gases with high mean background concentrations.
In practice, ideal stationarity is never realized in the field and the question "How much non-stationarity is too much?" is an interesting one. The answer probably depends on the nature of the scalar and the scientific question to be addressed. Criteria are often somewhat subjective. In this case, flux data in Fig. 5 show a clear central cluster surrounded by a widely scattered cloud of outliers. Limits were chosen to include the densest portion of the central cluster. Furthermore, it is apparent from Fig. 5 that ∂CO/∂t shows the greatest range as an indicator of non-stationary conditions, but it is not always sufficient. Some samples exhibit significant horizontal turbulent flux and low ∂CO/∂t. It therefore seems wise to examine stationarity from a variety of perspectives. These considerations may impose stringent location selection criteria for future CO air-sea flux studies. On the basis of this test, conditions at station ALOHA appear suitable and many locations in the Southern Hemisphere should be equally acceptable. | v3-fos-license |
2019-04-04T13:09:31.749Z | 2015-10-20T00:00:00.000 | 94051623 | {
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} | pes2o/s2orc | Quantification of acidic sites of nanoscopic hydroxylated magnesium fluorides by FTIR and 15N MAS NMR spectroscopy
Lewis and Brønsted sites were quantified in a series of weak acidic hydroxylated magnesium fluorides by Fourier transform infrared spectroscopy (FTIR) and solid state nuclear magnetic resonance spectroscopy (NMR) with pyridine as probe molecule. Molar extinction coefficients, which are necessary for quantitative FTIR measurements, were calculated by an easy approach. It utilizes the fact that both signals, used for the quantification by FTIR, are caused by the same deformation vibration mode of pyridine. Comparison of quantitative FTIR experiments and quantification by NMR shows that concentrations of acidic sites determined by FTIR spectroscopy have to be interpreted with caution. Furthermore, it is shown that the transfer of molar extinction coefficients from one catalyst to another may lead to wrong results. Molar extinction coefficients and concentrations of acidic sites determined by FTIR spectroscopy are affected by grinding and probably the particle size of the sample. High temperature during FTIR experiments has further impact on the quantification results.
Introduction
Metal uorides and hydroxide uorides are interesting acidic materials because they are able to catalyze various reactions such as dehydration reactions, 1,2 cyclization 3 or halogen exchange reactions. 4Their catalytic activity is related to Lewis and Brønsted acid sites on their surfaces.For instance, the reaction mechanism of the carbohydrate dehydration reaction shows a correlation to the acidic surface properties of hydroxylated magnesium uoride catalysts.Furthermore, the acidic properties of these samples can be altered by modifying surface OH groups with uorosulfonic species. 2 Distinction and quantication of acidic sites, especially of Lewis and Brønsted sites, is therefore an important task in the characterization of acidic catalysts.
The application of the Lambert-Beer law requires reliable values for molar extinction coefficients.Such coefficients have been mostly determined by comparing the pyridine adsorption at various catalysts 12,14 and are transferred from one catalyst to another.Thereby, it is assumed that molar extinction coefficients are intrinsic to the probe molecule.Hence, that they are independent from the type of catalyst, the acidic strength of the adsorption site and the coverage degree of the surface.Selli and Forni 14 list molar extinction coefficients which were determined by various authors.In contrast to the assumption that molar extinction coefficients are intrinsic to the probe molecule (pyridine), molar extinction coefficients reported by Selli and Forni 14 show a broad distribution and differ between 0.078 and 3.03 cm mmol À1 for Brønsted sites and between 0.269 and 3.26 cm mmol À1 for Lewis sites.Hence, the determination of molar extinction coefficients by comparing pyridine adsorption at various catalysts and transfer of molar extinction coefficients from one sample to another is doubtful and these coefficients may depend on several factors. 23Selli and Forni 14 discuss that the distribution of molar extinction coefficients is due to different experimental conditions used for the determination of these coefficients.Hence, it would be desirable to determine molar extinction coefficients for each sample individually.7][18] However, this combination of FTIR and micro-gravimetry requires specialized equipment 18 or sample mass and IR signal areas cannot be determined simultaneously.
The present study reports on an easy method for the calculation of molar extinction coefficients of pyridine at Lewis and Brønsted sites for each sample individually.This method assumes that molar extinction coefficients for Lewis and Brønsted sites are similar in size because the signals used for the quantication of Lewis and Brønsted sites both arise from the same ring deformation mode of pyridine n 19b .
5][26] Furthermore, concentrations of acidic sites determined by FTIR are compared with quantitative measurements by 15 N MAS NMR.Pyridine was used as probe molecule in all quantication experiments to ensure that the results are comparable.
Preparation of the samples
Samples were prepared under argon atmosphere using Schlenk techniques.Magnesium (Aldrich, 99.98%) (7.8 g, 325 mmol) was dissolved in 400 mL methanol (dried over Mg) at room temperature overnight.Aer Mg was completely dissolved, the corresponding amount of hydrouoric acid (Mg : F ratio 1 : 2) was added at room temperature.The mixtures were vigorously stirred and reacted to form highly viscous transparent sols.Four different hydroxylated magnesium uoride catalysts were prepared, denoted as M-40, M-57, M-71 and M-87.The number refers to the HF wt% of the hydrouoric acid, which was added to the magnesium methoxide precursor solution.The concentration of the hydrouoric acid was checked by titration.They were aged at room temperature overnight and dried under vacuum (10 À2 mbar) at a heating rate of 1 K min À1 until 100 C and kept at this temperature for 2 h.
NMR experiments
Solid state NMR experiments were performed on a Bruker Avance 600 spectrometer (14.1 T).All experiments were carried out at room temperature using a 7 mm magic angle sample spinning (MAS) probe for solid state NMR experiments.Proton decoupling was carried out with a 15 two pulse phase modulation (TPPM) sequence. 27Data analysis was performed with the soware TopSpin 2.1 (and 3.0).DMFIT was used for line ts. 28 15 MAS NMR spectra were recorded using the EASY method 29 for removing acoustic ringing at a Larmor frequency of 60.8 MHz. 15 N chemical shis (d) are reported relative to CH 3 NO 2 with internal NH 4 Cl as secondary standard (d ¼ À341 ppm).30 1 H- 15 N CPMAS (cross-polarization with magic angle sample spinning) experiments are needed for the determination of the T 1 correction factors of the time optimized 15 N MAS NMR spectra using the Torchia method.31 Quantitative spectra are obtained with pulse repetition delay of at least one T 1 . Sil areas are corrected according to their T 1 value and concentrations of acidic sites are calculated with respect to the signal area of the added NH 4 Cl.Details are described elsewhere.20 For the NMR measurements, 600 mg of sample were weighted in a Schlenk ask, followed by an annealing step at 200 C under vacuum for 2 h to remove physisorbed water. Thn, excess of 15 N-pyridine (30 mL; 367 mmol) were added and the powder was stirred for 30 min at 150 C to ensure homogeneous pyridine distribution.Aer that, the sample was evacuated for 1 h at 150 C. Rotors for magic angle spinning (MAS) NMR experiments were carefully lled in the glovebox.
FTIR experiments
FTIR spectra were taken on a Nicolet iS10 FTIR spectrometer of Thermo Fisher Scientic Inc. with a dTGS (deuterated triglycine sulfate) detector.Data analysis was performed with the spectrometer soware Omnic 8.1.Presented spectra are difference spectra, i.e., the spectrum recorded before adsorption of pyridine was subtracted from spectra taken with pyridine adsorption.
For FTIR experiments, about 10-30 mg of a sample was grounded for one minute in a vibrating mill, if not described differently, and was pressed with a pressure of 0.5 t in a selfsupporting disc (radius 0.65 mm) in air.The disc was placed in a quartz cell equipped with KBr windows.Before starting adsorption and FTIR analysis, samples were heat-treated at 200 C in vacuum (10 À5 to 10 À6 mbar) for 2 h.Addition of known amounts of gaseous probe molecule pyridine in the cell was possible via a known volume connected to the quartz cell.By lling this known volume with pyridine at known pressure, controlled by a pressure gauge, the amount of introduced pyridine could be calculated according to the ideal gas law.
Aer the stepwise adsorption of pyridine, samples were saturated with pyridine at a pressure of 5 mbar for 10 min and weakly adsorbed pyridine molecules were desorbed at room temperature or 150 C in vacuum (10 À5 to 10 À6 mbar) for 30 min.sites (BPy).Lewis and Brønsted sites can be identied through the signals of coordinated pyridine and protonated pyridine, pyridinium ions.Table 1 shows the wave numbers of the four vibration bands which are used for their identication.
The signals at about 1450 cm À1 (LPy) and 1540 cm À1 (BPy) are both due to the n 19b ring deformation mode of pyridine which is affected differently by the interactions of pyridine with the adsorption sites.][14][15][16][17][18] According to the Lambert-Beer law, the concentration c(Y) [mmol cm À3 ] of an acidic site Y, i.e. the concentration of pyridine molecules adsorbed at such sites, can be calculated from the signal area A Y [cm À1 ] of a related signal.
Thereby, d [cm] is the thickness of the self-supporting disc and 3 Y [cm mmol À1 ] is the molar extinction coefficient of the pyridine signal at the acidic sites Y.
For the comparison of various catalysts, it is advantageous to compare the number of acidic sites n(Y) [mmol] per catalyst mass or per surface area.The number of acidic sites n is obtained by the combination of the acidic site concentration c and the disc thickness d.This combination results in number of acidic sites per area.A signal can only be obtained in the area where the IR beam interacts with the sample.Hence, it is reasonable to include the area of the IR beam in the calculation.The area of the IR beam is constant during the whole FTIR experiment and is incorporated into the molar extinction coefficient.Accordingly, the Lambert-Beer law is modi-ed to: Molar extinction coefficients are according to this equation of the dimension cm À1 mmol À1 and can be determined by stepwise adsorption of pyridine at the catalyst.It is assumed that molar extinction coefficients are independent from the coverage degree and do not change during the adsorption.Hence, during the stepwise adsorption of pyridine the signal areas of the signals at about 1540 cm À1 and 1450 cm À1 increase linearly and are further plotted versus the amount of introduced pyridine molecules.
The total amount of introduced pyridine n is in the rst adsorption steps the sum of pyridine molecules at Lewis and Brønsted sites.In combination with eqn (2) results: Derivative of eqn (3) with respect to the amount of pyridine molecules n results in: dl Py /dn and dA BPy /dn are the slopes of the signal areas versus the amount of introduced pyridine molecules in the rst adsorption steps determined in the experiments.However, eqn (4) can only be solved if only one kind of acidic sites is present in a sample.In such a case the slope of the acidic site which does not occur is zero.Hence, one summand of eqn ( 4) is zero and the molar extinction coefficient of the occurring site can be determined.Otherwise, if Lewis and Brønsted sites are present, eqn (4) cannot be easily solved, as there are two unknown variables.Therefore, in samples in which LPy and BPy occur, an additional condition for 3 0 LPy and 3 0 BPy has to be found to solve eqn (4).
Two possibilities for the calculation of 3 0 LPy and 3 0 BPy have been described in the literature.One possibility is to compare the slopes dA LPy /dn and dA BPy /dn obtained for various catalysts.The molar extinction coefficients are then calculated from the various slopes under the assumption that the extinction coef-cients are the same for each catalyst. 12,14However, Selli and Forni 14 showed that a broad distribution of molar extinction coefficients can be found in the literature, and Rosenberg et al. even found different molar extinction coefficients for series of similar catalysts. 16,177][18] However, this combination requires specialized equipment 18 or sample mass and IR signal areas cannot be determined simultaneously.Therefore, another approach is chosen to determine molar extinction coefficients in this study.
Both signals at about 1540 cm À1 and 1450 cm À1 , which are used for the quantication of acidic sites, are due to the n 19b ring deformation mode of protonated pyridine at Brønsted sites and coordinated pyridine at Lewis sites.Furthermore, molar extinction coefficients for Lewis and Brønsted sites listed by Selli and Forni 14 or calculated in the group of Anderson 13,16,17 are in the same order of magnitude, whereby in most cases the molar extinction coefficient for Lewis sites is up to three times larger than the molar extinction coefficient for Brønsted sites.
The concentrations of acidic sites per catalyst mass are calculated by dividing the amount of acidic sites by the mass of the investigated self-supporting disc.
Results
Hydroxylated magnesium uorides are biacidic catalysts.Hence, besides acidic Lewis sites these catalysts also exhibit acidic Brønsted sites.The Brønsted acidic character of these catalysts is surprising because MgOH groups are usually of basic character.The partial acid character of hydroxyl groups in hydroxylated magnesium uorides is probably caused by the mixed coordination of magnesium by uorine and hydroxyl groups at the particle surfaces, as shown schematically in Scheme 1. [24][25][26] Besides FTIR spectroscopy, 15 N MAS NMR spectroscopy can be used to distinguish and quantify acidic Lewis and Brønsted sites.Hence, it was used as reference method for the quantitative FTIR experiments in the investigated series of catalysts.
The most common method for the quantication of acidic sites NH 3 -TPD is not used in this study as these samples are sensitive to temperature. 24As example, ESI Fig. 1 † shows the X-ray pattern of a hydroxylated magnesium uoride sample before and aer it was calcinated at 300 C. The decrease in the peak width shows that at 300 C the crystallite size increase and probably some of the acidic sites are destroyed by surface rearrangement.
15 N MAS NMR spectroscopy
Fig. 2 shows the 15 N MAS NMR spectra of the four hydroxylated magnesium uoride samples aer adsorption of pyridine.The spectra show four signals.The narrow signal at À341 ppm is assigned to ammonium chloride which was added as internal standard for the quantication.The other three signals are assigned to pyridine in different adsorption states.All samples show a signal at À102 ppm with a broad sideband pattern typical for pyridine molecules coordinated at acidic Lewis sites LPy.This signal overlaps with the signal of pyridine molecules adsorbed via hydrogen bridges HPy at À89 ppm.A signal for protonated pyridine at Brønsted sites BPy at À175 ppm is only observed in three of the samples and not in M-40.None of the 15 N MAS NMR spectra show a signal of bulk pyridine at about À64 ppm. 32,33This means that all pyridine molecules are adsorbed at the catalyst surfaces.The T 1 values of all signals are determined using the Torchia method 31 and signal areas of signals in the 15 N MAS NMR spectra are corrected accordingly.The concentrations of each adsorption state are determined by comparing the corrected signal areas of the individual sites with the signal area of the added internal standard ammonium chloride. 3,20Concentrations of pyridine molecules adsorbed via hydrogen bridges were not determined as their concentrations differ depending on the sample preparation. 19able 2 lists the concentrations of Lewis and Brønsted sites in the four samples.The concentrations of acidic Lewis sites are nearly equal in all samples, whereas the concentration of acidic Brønsted sites changes.It decreases from M-57 to M-87, while M-40 shows no Brønsted sites.The reason for the decrease in the concentration of Brønsted sites is that less hydroxyl groups are present in samples synthesized with highly concentrated hydrouoric acid than in samples synthesized with diluted hydrouoric acid.Hence, the number of hydroxyl groups/Brønsted sites decreases with increasing concentration of the hydrouoric acid used for the synthesis. 25The reason that M-40 shows no Brønsted sites is probably that the hydroxyl groups in this sample are too weak to protonate pyridine.It is assumed that the acid strength of MgOH groups decrease with the increasing concentration of hydroxyl groups at the surface.Hence, hydroxyl groups at the surface of M-40 may already exhibit neutral or even basic character.
FTIR spectroscopy
FTIR spectroscopy can distinguish between Lewis and Brønsted sites.However, determination of molar extinction coefficients and therefore quantication of acidic sites is oen challenging.As has been pointed out, 14,23 experimental conditions can affect the quantication by FTIR.Hence, molar extinction coefficients and concentrations of acidic sites were calculated under various sample preparation and adsorption conditions.Especially, grounding of the samples was considered because we found that ungrounded samples can be easier pressed in selfsupporting discs.
4.2.1.Ungrounded samples.The rst samples were pressed in self-supporting discs without further sample preparation, just as they were synthesized.The investigated hydroxylated magnesium uorides are nanocrystalline what can be shown by XRD (Fig. 2 in ESI †) but can form larger agglomerates in the synthesis. 34ig. 3 shows exemplary the stepwise adsorption spectra of pyridine on the hydroxylated magnesium uoride sample M-57.The FTIR spectra of the stepwise pyridine adsorption on the other three samples can be found in the ESI (Fig. 3 sites are mostly inaccessible for pyridine molecules during adsorption.Additionally, Fig. 3 (and ESI Fig. 3-5 †) shows the plots of the signal areas of the signals at about 1545 and 1446 cm À1 versus the amount of pyridine introduced.These plots show the expected behavior for the signal area of the Lewis sites versus introduced pyridine molecules: a rst linear increase of the signal area with increasing pyridine concentration and attening of the curve aer all accessible acidic sites are saturated with pyridine.The Brønsted sites in sample M-57 are not fully saturated during the pyridine adsorption, this is an indication that the Brønsted sites are also difficult to access for pyridine in M-57 like in the other samples.
Molar extinction coefficients are calculated as described in Chapter 3 from the slopes of the signal areas versus the adsorbed amount of pyridine.With the molar extinction coefficients the concentrations of Lewis and Brønsted sites are calculated aer all acid sites have been saturated with pyridine and weakly adsorbed pyridine has been desorbed.
Each sample was investigated up to three times by stepwise adsorption of pyridine.Table 3 lists the calculated molar extinction coefficients and determined concentrations of acidic sites.The molar extinction coefficients for Brønsted sites and concentrations of Brønsted sites were only determined for M-57 because these sites appear only aer pyridine desorption at higher temperature in sample M-71 and M-87.
The molar extinction coefficients, shown in Table 3, exhibit a broad distribution.Even for the same sample, extinction coefficients differ up to a factor of 12.The concentrations of acidic sites, however, are in the same order of magnitude for each sample but show an error of up to 40% and are mostly smaller as detected by 15 N MAS NMR.Table 4 lists molar extinction coefficients and concentrations of acidic sites for the samples which were grounded before they were pressed in self-supporting discs.The molar extinction coefficients are bigger and show a much narrower distribution (maximum factor of 1.4 in a single sample) in the grounded samples as in the ungrounded samples.However, they still differ between the catalysts up to a factor of two.
The calculated concentrations of acidic sites also change in the grounded samples.In sample M-40, M-71 and M-87 concentrations of acidic sites are now in the same order of magnitude as determined by 15 N MAS NMR.Expect for sample M-57, pyridine seems to reach all Lewis acidic sites during adsorption in the grounded samples.However, no Brønsted sites were detected for sample M-71 and M-87.The error of the quantication is smaller but still in the order of 30%.linearly with increasing pyridine concentration in the beginning of adsorption and aer the acidic sites are saturated the curve levels off.However, in the plot of sample M-71 and M-87, the signal areas of pyridine at Brønsted sites increase until the end of the stepwise adsorption.Again, this is an indication for the difficult accessibility of Brønsted sites in these samples.Table 5 lists the molar extinction coefficients and concentrations of acidic sites calculated for the series of grounded samples and pyridine adsorption at 150 C. Molar extinction coefficients for Lewis sites are in the same order of magnitude as for the grounded samples and adsorption at 25 C.However, they still differ between the samples, especially for sample M-57.The molar extinction coefficients for Brønsted sites, in contrast, are twice as large at higher adsorption temperature.
The concentrations of acidic sites at 150 C are smaller as calculated at 25 C. Especially for the samples M-40, M-57 and M-71, the concentration of Lewis sites are only about half as large at 150 C compared to the concentrations at 25 C.The reason will be discussed in detail in the next chapters.
Discussion
Quantitative FTIR investigations by stepwise adsorption of pyridine in a series of hydroxylated magnesium uorides show that the grinding of the samples and the temperature, at which pyridine is adsorbed have a huge impact on the calculated molar extinction coefficients and concentrations of acidic sites.
Comparison of molar extinction coefficients
First of all, molar extinction coefficients determined under various conditions are discussed.Molar extinction coefficients determined for grounded samples are larger in comparison to extinction coefficients of ungrounded samples.The reason may be the presence of large particles/agglomerates of hydroxylated magnesium uoride in the ungrounded samples.Chalmers 35 reported that the signal intensity of an IR signal depends on the particle sizes in the sample and increases with decreasing particle size.The effect of grinding on the signal intensity becomes most visible in sample M-71.The signal intensities of the pure samples before the adsorption of pyridine (see Fig. 14 in ESI † on the le side) increase aer grinding of the sample.Hence, grinding seems to lead to smaller particles/ agglomerates.Surprisingly, the signal intensities of pyridine (Fig. 14 in ESI † on the right, note that the spectra are shown in the same order as on the le side) show the same trend.Not only the signal intensities of the catalysts are affected by their particle size but also the signal intensities of pyridine adsorbed on their surface.
Molar extinction coefficients are calculated from the signal areas of the rst adsorption steps and therefore are also affected by the particle sizes in the sample.Hence, molar extinction coefficients increase in the same way as the signal area of adsorbed pyridine with decreasing particle size.It has been reported very recently that particle sizes have an effect on molar extinction coefficients of adsorbed molecules. 36However, Jento et al. 36 found that molar extinction coefficients of adsorbed alkanes increase with higher scattering of a sample, respectively larger particles.This is in contrast to the presented observations that molar extinction coefficients of pyridine increase with decreasing scattering/particle size of a sample.
Variation of sample weights can be ruled out as reason for the difference in the signal areas, respectively the molar extinction coefficients (differ up to a factor of 12), because the maximum variation in sample weight was of a factor of three.
Furthermore, molar extinction coefficients show a much lower distribution between them aer grinding.Probably grinding of samples leads to smaller particles and smaller distribution in particles size, because agglomerates in the sample are broken up.Hence, signal intensities of pyridine signals are higher in grounded samples which lead to larger molar extinction coefficients that can be determined more reproducible.
Brønsted sites are only detected for the majority of the samples at an adsorption temperature of 150 C. First of all it is very interesting to note that molar extinction coefficients of Lewis sites do not change regardless if Brønsted sites are detected in the sample or not.This shows that the method presented in Chapter 3 is suitable for the calculation of molar extinction coefficients.However, molar extinction coefficients of Brønsted sites of sample M-57 are about twice as large at 150 C as coefficients determined at 25 C (M-57 is the only sample where Brønsted sites are observed at 25 C).One reason may be that higher kinetic energy of hydroxyl groups and pyridine at 150 C lead to a higher protolysis as at 25 C and, therefore, to the observed increase in the molar extinction coefficients.
Comparison of concentrations of acidic sites
Concentrations of acidic sites determined by 15 N MAS NMR and FTIR under various conditions are shown in Fig. 4 for Lewis sites and Fig. 5 for Brønsted sites.
It has been pointed out that molar extinction coefficients in FTIR spectroscopy exhibit a broad distribution in ungrounded samples and differ even for the same sample up to a factor of 12. Surprisingly, concentrations of acidic sites are in the same order of magnitude.The reason for different distributions in concentrations and extinction coefficients in FTIR spectroscopy is that the position of the IR beam, where it penetrates the selfsupporting disc, is the same in the entire experiment.The scattering of the IR beam and the detected signal intensities depend on the particle sizes with which the IR beam interacts. 35osition of the IR beam and, thus, scattering of the IR beam is the same during the entire experiment.Therefore, the signal intensities of the pyridine signal are of the same order of magnitude during the adsorption of pyridine and aer all acidic sites are saturated with pyridine.Hence, the effect of the particle sizes on the signal intensity is cancelled out in the division of the signal area by the molar extinction coefficient in the calculation of the amount of acidic sites (eqn (5)).Therefore, concentrations of acidic sites can be determined reproducibly although molar extinction coefficients differ.Concentrations of acidic Lewis sites are larger in grounded samples in comparison to ungrounded samples.To ensure that the grinding does not create new surfaces and therefore new acidic sites, the surface area was measured by nitrogen sorption before and aer grinding of one sample (see Table 1 in the ESI †).It can be seen that the surface area does not increase signicantly aer grinding of the sample.Hence, no new acidic sites are created due to the grinding.Nevertheless, concentrations of acidic sites determined by FTIR are affected by grinding of the samples.In two samples M-40 and M-71, three times more acidic Lewis sites are detected by FTIR aer grinding.The reason may be that bigger particles/agglomerates in ungrounded samples are held together by the interaction of basic hydroxyl groups or uoride with acidic Lewis sites.Grinding of the samples probably breaks up these agglomerates, such that these acidic Lewis sites are accessible to pyridine at 25 C.
Furthermore, it has been seen that FTIR signals of pyridine at Brønsted sites occur in some samples only aer treatment at 150 C. Hence, FTIR experiments were performed at pyridine adsorption temperature of 150 C. FTIR adsorption experiments at 150 C show two differences compared to the same experiments at 25 C. Firstly, Brønsted sites can be detected in three of the samples at 150 C, whereas at 25 C only M-57 shows signals for pyridine at Brønsted sites.The reason may be that Brønsted sites are difficult to access even in grounded samples and hence the increased protolysis or pyridine mobility at 150 C is necessary to protonate pyridine.Secondly, concentrations of acidic sites are lower in all samples at higher temperature.The reason for the lower concentrations may the fact that during desorption (150 C and high vacuum 10 À5 to 10 À6 mbar) of weakly adsorbed pyridine molecules pyridine also partly desorbs from the weak acidic sites of the investigated samples.Furthermore, adsorption of pyridine at an acidic site is an exothermal process and therefore less favorable at higher temperatures and weak sites.Hence, the equilibrium constants of the reactions between pyridine and the acidic Lewis and Brønsted sites may be small at 150 C.
Comparison of quantication by NMR (sample preparation: excess of 15 N-pyridine was added and distributed in the sample for 30 min at 150 C. Aer that, the sample was evacuated (10 À2 mbar) for 1 h at 150 C) with the quantitative experiments by FTIR (see Fig. 4 and 5) shows that the total concentration of acidic sites cannot be measured properly with any of the used experimental conditions in the FTIR experiments (either in grounded or ungrounded samples and pyridine adsorbed via gas phase at 25 C or 150 C).
Concentrations of Lewis sites could be determined properly, for the majority of the catalysts, only in grounded samples at 25 C by FTIR.However, under these conditions Brønsted sites were only detected in one catalyst by FTIR while NMR detect in three of the catalysts (M-57, M-71 and M-87) Brønsted sites.That in these three catalysts Brønsted sites exist is supported by FTIR experiments at 150 C.
Moreover, the determined concentrations of acidic sites of sample M-57 by 15 N MAS NMR are higher than in all FTIR investigations, regardless which experimental condition was used.Quantication by NMR shows that this catalyst exhibits the highest amount of acidic Brønsted sites.Maybe interaction of pyridine molecules with hydroxyl groups disturb the background signals between 1750 and 1300 cm À1 (see Fig. 15 in ESI †), which lead to errors in the FTIR difference spectra.
Conclusion
A series of weak acidic hydroxylated magnesium uorides was investigated by quantitative FTIR carried out under various experimental conditions and quantitative solid state 15 N MAS NMR.Both methods use pyridine as probe molecule, so that the determined concentrations of acidic sites can be compared.For the quantication by FTIR spectroscopy, it is crucial to determine molar extinction coefficients for Lewis and Brønsted sites.An easy method was presented which allow the calculation of molar extinction coefficient from a single sample.Determination of molar extinction coefficients from a single sample is important because this investigation shows that molar extinction coefficients differ in the investigated series of samples and even between various measurements of the same sample.Hence, molar extinction coefficients determined for one sample cannot be transferred to another sample.The variance in molar extinction coefficients can be explained by different particle sizes in the samples which inuence the signal intensity the FTIR spectra.Accordingly, molar extinction coefficients can be calculated more reproducible if the samples are nely grounded to ensure a small distribution of particle sizes.
Furthermore, it was found that acidic sites are partially not accessible for pyridine adsorbed via gas phase at 25 C in ungrounded samples.One reason may be that large particles/ agglomerates are held together by the interaction of basic hydroxyl groups or uoride with acidic Lewis sites.Grinding of the samples breaks up these agglomerates, such that all Lewis sites were accessible to pyridine.However, even in nely grounded samples, Brønsted sites were only detected at high adsorption temperatures of pyridine in most of the samples.The reason is that Brønsted sites are difficult to access even in grounded samples and increased protolysis and/or higher pyridine mobility at higher temperature are necessary for the protonation of pyridine.
Hence, experimental conditions have to be chosen carefully for quantitative FTIR experiments and results should be compared with other quantication methods to ensure that all acidic sites were detected.
Solid state 15 N NMR is a much more reliable method for the quantication of acid sites as both kind of acidic sites could be reliable detected and quantied.
Fig. 1 shows a plot for eqn ( 4 ) of possible values for 3 0and 3 0 3 0and 3 0 3 0and 3 0 3 0and 3 0
LPyBPy with dA LPy /dn ¼ 1 and dA BPy /dn ¼ 0.5.As we know that LPy BPy have to be in the same order of size, it is further assumed that the correct pair of values for LPy BPy is the point of eqn (4) nearest to the origin of the coordinate system.The closest pair of values (LPy BPy ) to the origin of the coordinate system is calculated by searching the minimal value for the sum of 3 LPy 02 and 3 BPy 02 under the conditions that eqn (4) is fullled and both values are positive.Differences in the molar extinction coefficients due to the nature of the solid or the acid strength of the adsorption sites between various samples have already an inuence on the slope of the signal areas measured in the stepwise adsorption of
3 0and 3 0
pyridine.Hence, these factors inuence eqn (4) and are therefore considered in the calculation of molar extinction coefficients.Calculated LPy BPy are used to determine the amount of acidic sites.For this purpose, all acidic sites are saturated with pyridine and weakly adsorbed molecules, physisorbed pyridine or bound via hydrogen bridges, are desorbed from the catalyst.According to eqn (2) the amount of acidic sites can be calculated from the areas of the signals at 1540 cm À1 or 1450 cm À1 and their molar extinction coefficients.
Fig. 2
Fig. 2 15 N MAS NMR spectra of the four hydroxylated magnesium fluorides after adsorption of pyridine.Additionally, the line fits of sample M-87 for pyridine at Lewis sites, Brønsted sites (solid lines) and pyridine molecules adsorbed via hydrogen bridges (dotted line) are shown.Spectra were obtained under comparable experimental conditions.MAS spinning frequency was 6.5 kHz except for M-71 (6 kHz).The signal intensity of sample M-40 is divided by two because of faster T 1 relaxation.*MAS spinning sidebands.
-5 †).All spectra show signals for pyridine molecules coordinated at Lewis sites at about 1606, 1578, 1492, and 1445 cm À1 .Surprisingly, only sample M-57 shows signals of pyridine molecules protonated at Brønsted sites at 1645 (very weak or not detected at all), 1578, 1545, and 1493 cm À1 during adsorption of pyridine.Brønsted sites were also detected by 15 N MAS NMR in sample M-75 and M-87.In these samples, the FTIR signals of pyridine molecules at Brønsted sites only appear aer desorption of pyridine at 150 C.These results suggest that Brønsted
Fig. 3
Fig. 3 FTIR spectra after stepwise pyridine adsorption at ungrounded M-57 and the integrated intensity of n 19b band of coordinated and protonated pyridine at about 1446 and 1545 cm À1 .Also shown are the integrated intensities of n 19b band after saturation with pyridine (open symbol).
4. 2 . 2 .
Grounded samples.Molar extinction coefficients and concentrations of acidic sites change if samples were nely grounded before they were pressed in self-supporting discs.Spectra of the stepwise pyridine adsorption and the plots of signal area versus the amount of adsorbed pyridine are shown in the ESI (Fig.6-9 †).As for the ungrounded samples, only in sample M-57 signals of Brønsted sites can be detected but the Brønsted sites are not saturated during the stepwise adsorption of pyridine.
4 . 2 . 3 .
Adsorption at 150 C. Finally, pyridine was adsorbed at grounded samples at 150 C. The FTIR spectra of the stepwise adsorption for these samples and the plotted signal areas versus the amount of pyridine are shown in the ESI (Fig. 10-13 †).The spectra show that Brønsted sites are detected at an adsorption temperature of 150 C in the samples M-57, M-71 and M-87.Plots of signal areas versus adsorbed pyridine show in all samples the expected adsorption behavior for Lewis sites, and in sample M-57 also for Brønsted sites; the signal area increase
Fig. 4
Fig. 4 Concentration of Lewis sites determined by 15 N MAS NMR and FTIR under various conditions.Error bars shown are determined by measuring several samples (NMR).Lines indicate the average values of several FTIR measurements.
Fig. 5
Fig. 5 Concentration of Brønsted sites determined by 15 N MAS NMR and FTIR under various conditions.Error bars shown are determined by measuring several samples (NMR).
Table 1
FTIR-bands [in cm À1] of adsorbed pyridine between 1700 and 1400 cm À1 .LPy: pyridine coordinated at Lewis sites; BPy: pyridine protonated at Brønsted sites This journal is © The Royal Society of Chemistry 2015
Table 2
15ncentrations of acidic sites determined by15N MAS NMR spectroscopy for the four hydroxylated magnesium fluoride samples.The errors were determined by multiple measurements of the samples and various simulations of the spectra (NMR)
Table 3
Calculated molar extinction coefficients of coordinated (Lewis) and protonated pyridine molecules (Brønsted) and the concentration of Lewis and Brønsted sites in the ungrounded hydroxylated magnesium fluoride samples.Each catalyst has been investigated several times
Table 4 Calculated
molar extinction coefficients of coordinated (Lewis) and protonated pyridine molecules (Brønsted) and the concentration of Lewis and Brønsted sites in the grounded hydroxylated magnesium fluoride samples.Each catalyst has been investigated several times
Table 5
Calculated molar extinction coefficients of coordinated (Lewis) and protonated pyridine molecules (Brønsted) and the concentration of Lewis and Brønsted sites in the grounded hydroxylated magnesium fluoride samples at an adsorption temperature of 150 C | v3-fos-license |
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} | pes2o/s2orc | Nonlinear rheological characteristics of single species bacterial biofilms.
Bacterial biofilms in natural and artificial environments perform a wide array of beneficial or detrimental functions and exhibit resistance to physical as well as chemical perturbations. In dynamic environments, where periodic or aperiodic flows over surfaces are involved, biofilms can be subjected to large shear forces. The ability to withstand these forces, which is often attributed to the resilience of the extracellular matrix. This attribute of the extracellular matrix is referred to as viscoelasticity and is a result of self-assembly and cross-linking of multiple polymeric components that are secreted by the microbes. We aim to understand the viscoelastic characteristic of biofilms subjected to large shear forces by performing Large Amplitude Oscillatory Shear (LAOS) experiments on four species of bacterial biofilms: Bacillus subtilis, Comamonas denitrificans, Pseudomonas fluorescens and Pseudomonas aeruginosa. We find that nonlinear viscoelastic measures such as intracycle strain stiffening and intracycle shear thickening for each of the tested species, exhibit subtle or distinct differences in the plot of strain amplitude versus frequency (Pipkin diagram). The biofilms also exhibit variability in the onset of nonlinear behaviour and energy dissipation characteristics, which could be a result of heterogeneity of the extracellular matrix constituents of the different biofilms. The results provide insight into the nonlinear rheological behaviour of biofilms as they are subjected to large strains or strain rates; a situation that is commonly encountered in nature, but rarely investigated.
INTRODUCTION
Bacterial biofilms occur in diverse environments, such as aquifers 1 , rivers 2,3 , hydrothermal springs 4 , within sewer pipelines 5 , in bioremediation plants 6 and various other places. Within these environments the biofilms can be subjected to extreme temperatures 4,7 , variation in physical forces 8 , changes in chemical concentrations 9 , changes in salinity 10 and pH 11 . These extreme conditions impact a biofilm's lifecycle, and yet, they are able to thrive by colonising a variety of surfaces. The ability of biofilms to withstand the above-mentioned dynamic environments is often attributed to the extracellular matrix (ECM), 12,13 which is commonly described as a network of polymers, including extracellular polysaccharides (EPS), extracellular DNA (eDNA), proteins and various other components 14 . The ECM holds the polymeric constituents and bacterial cells together, thereby conferring the biofilm its rigidity and viscosity. In addition, the ECM is also known to perform a wide array of additional functions: such as acting as a reservoir of metabolites and signalling molecules 15 , offering decreased permeability to invaders or chemicals 16 and as a promoter of virulence of microbes 17 . Despite its multifunctional nature, the ECM is often dubbed as the 'dark matter' owing to the limited information about its composition and the organisation of the polymers that constitute it 14 . Since the polymeric composition and its organisation within the ECM dictates the rheological behaviour of the biofilms (in addition to other functionalities); it becomes important to investigate the matrix viscoelasticity [17][18][19] . This could allow one to understand the role of biopolymers not only in conferring structure to the biofilm, but also, its biological and environmental functionalities.
Biofilms exhibit contrasting mechanical behaviour during their lifecycle: in early stages of growth in liquid cultures, cells become connected through the ECM and behave like viscoelastic liquid 20 . While, after growth on surfaces they can exhibit rheological characteristics similar to that of viscoelastic solids or liquids, which is dependent on a variety of environmental and physical factors.
To study such complex rheological responses, a wide array of mechanical and spectroscopic techniques have been developed at length scales ranging from nanometers to a few millimetres [21][22][23] . Examples of such techniques include particle tracking rheology [24][25][26] , diffusing wave spectroscopy 27 , Brillouin-Raman microscopy [28][29][30] and optical tweezing 31 . These techniques have been employed to quantify the structural heterogeneity within biofilm matrices at the scales of few microns, that arise due to changes in chemical environment 32 or changes arising from modification of the genetic characteristics 18 . Rheometers are the most commonly used instrument for investigating viscoelasticity of biofilms and provides bulk characteristics (at millimeter scale) by performing dynamic oscillatory measurements 22 . Tests including amplitude/ frequency sweep or creep and relaxation [33][34][35][36] are the most commonly employed techniques. These measurement schemes have allowed investigators to shed light on: the functionality of eDNA in controlling the relaxation times 37 , the effect of chemical treatments on the viscoelastic moduli 38,39 and the polysaccharide production mediated alteration in the mechanical toughness of biofilms 19 and various other effects. Interfacial rheometry [40][41][42] , extensional measurements 43 , micro-cantilever 44 and bioreactors interfaced with rheometers 45 have allowed the growth of biofilm and pellicles under dynamic conditions; thereby allowing one to study the effects of dynamic conditions on the biofilm structure and variation in rheological properties. In most of these situations the strain applied to the biofilm is within the linear viscoelastic regime, as a result the biofilm structure remains intact. However, 1 in realistic situations like in rivers, on ship hulls and in bioreactors the shear forces and rates near solid walls can be extremely high; which can induce large deformations within the biofilm network, thereby disrupting the structure of the underlying polymeric network. Such large strain or strain rates can result in local stiffening or softening, or appearance of significantly different material characteristics, which are rarely explored in case of biofilms 42 .
Large amplitude oscillatory shear (LAOS) is a type of dynamic oscillatory test performed on a strain-controlled rheometer, which involves measuring material response (stress) to increasing values of imposed oscillatory strain 46,47 . With the increasing strain amplitudes, the material response transitions from exhibiting a sinusoidal (linear) stress waveform to a non-sinusoidal (nonlinear) stress waveform 48 . By decomposing the nonlinear stress waveforms based on symmetry arguments 49 and using Chebyshev polynomial analysis 50 one can analyse the contribution of higher order harmonics to gain invaluable insights. Material descriptors like intracycle strain stiffening (or softening) and intracycle shear thickening (or thinning) indices can reveal a unique fingerprint of the material 50 . Owing to higher sensitivity, LAOS also finds application in detection of architecture and branching characteristics of polymer melts [51][52][53] . In the past decade, LAOS has become a canonical technique for quantifying the rheological characteristics of polymers, soft solids, gels, emulsions and various other complex fluids and materials 48,54 . The techniques have also been successfully applied to understand the intracycle strain dependent hardening/softening or thickening/ thinning of biological materials, examples of which include: mucus of the gastropod 55 , hagfish slime 56 , fibrobalst cells 57 , fibrin/collagen gels 58 , vocal fold tissues 59 , pluronic/hyaluronic acid 60,61 , Xanthan gum 62 and blood 63 . For biofilms, LAOS provides us with the ability to determine the nonlinear material response of biofilms when subjected to large shear. In addition, one might also be able to perform genetic manipulation to control polymer production in biofilms and study how the interactions of the polymers play a role in shaping the rigidity and viscosity or other biological functionalities of biofilms.
In this paper we employ a combination of small and large amplitude oscillatory shear (SAOS and LAOS) tests to characterise the rheology of four species of bacterial biofilms: Bacillus subtilis (BS), Comamonas denitrificans (CD), Pseudomonas fluorescens (PF) and Pseudomonas aeruginosa (PA). Biofilms from these microbes tend to spread out radially into circle shaped colonies as seen in Fig. 1 (a-d) when a droplet of bacteria culture deposited on an agar plate. Whereas if the microbes are smeared uniformly on the agar plates, they form a continuous mat like biofilm. Scrapings of these mat like biofilms exhibit contrasting differences in cellular packing under a confocal microscope, which can be seen in Fig. 1 (e-h). Small amplitude oscillatory tests reveal that the linear viscoelastic moduli and yield stresses of the single species biofilms vary by orders of magnitude. Furthermore, by performing LAOS experiments and using Chebyshev polynomial analysis we are able to capture the subtle as well as distinct changes in the intracycle strain stiffening and shear thickening characteristics of the biofilms in the Pipkin diagram. The biofilms also exhibit differences in transition to nonlinear viscoelastic behaviour and energy dissipation characteristics, indicating that polymeric composition can play a role in dictating such nonlinear behaviours. Together, these results show that LAOS can be a useful tool in characterising the nonlinear rheological behaviour in biofilms of different species.
RESULTS
The following sections describes our findings on the linear and nonlinear rheological characteristics of the different species of biofilms. We first perform the linear viscoelastic tests to quantify the differences in the single species biofilms.
Linear viscoelastic moduli varies across single species biofilms Figure 2 (a) shows the frequency sweep results for the different species of biofilms. Pseudomonas fluorescens biofilms exhibit the lowest elastic moduli amongst the tested species while Pseudomonas aeruginosa exhibited the minimum viscous modulus. The elastic (G 0 ) and viscous moduli (G″) of P. fluorescens measures 385Pa and 75Pa at 1 Hz, while the measurements for P. aeruginosa was measured to be 655Pa and 52Pa at 1 Hz, respectively. In contrast, for Comamonas denitrificans the elastic (G 0 ) and viscous moduli (G″) measured 35770 Pa and 7400 Pa at 1 Hz, which are higher by a factor of~10 2 when compared to P. fluorescens. The moduli for Bacillus subtilis are in the intermediary range; G 0~2 250 Pa and G″~172 Pa at 1 Hz. For all the species of biofilms the moduli exhibits a plateau up to 10 Hz. Beyond 10 Hz the inertia of the instrument dominates and therefore the data have been discarded. Figure 2 (b) shows the results from oscillatory amplitude sweep for the four different species of biofilms that were carried out at 1 Hz for strain amplitudes ranging from 0.025-400%. It is evident from the plot that the linear viscoelastic regime, which is denoted by the plateau of G 0 in the amplitude sweep plot varies for the different biofilms. P fluorescens exhibits linear behaviour for strain amplitudes (γ < 10%), P. aeruginosa for (γ < 4%), B. subtilis for (γ < 5%) and C. denitrificans for (γ < 0.5%). Beyond the linear viscoelastic regime the elastic moduli continuously decreases for increasing strain amplitudes and exhibits a consistent decay, indicating a global strain softening behaviour up to a strain amplitude of 500%. The viscous modulus for B. subtilis, C. denitrificans and P. aeruginosa show distinct humps at γ = 30%, γ = 0.7% and γ = 25%, which are indicative of Type-III weak strain overshoot. Such behaviour is commonly seen in soft glassy materials and can arise due to structural rearrangements arising within the biofilm 51 . A closer look at the viscous modulus component of the amplitude sweep for CD shows a two step yielding signature at 0.7% and 200% strain, similar to that observed in colloidal systems [64][65][66] , which hypothesises successive bond and cage breaking at two separate strain amplitudes. However, such phenomena for biofilms are yet to be quantified and physically understood in terms of the underlying structure and the matrix constituents. The single species biofilms also show distinct crossover points for elastic and viscous modulus in the amplitude sweep plot. The corresponding yield stresses (σ Y ) for BS, CD, PF and PA are found to be 248 Pa, 822 Pa, 57 Pa and 100 Pa, respectively.
Intracyle strain stiffening/softening characteristics shows subtle differences amongst biofilm of different species We subsequently turn our attention to the behaviour of the bacterial biofilms when they are subjected to large strain amplitudes. The raw waveforms, which are recorded by the rheometer as numerical values of stress versus time and strain versus time, can also be parametrically represented as stress versus strain plots. These plots are commonly known as elastic Lissajous Bowditch (LB) plots and provide a geometrical description of the state of the material. In the linear viscoelastic regime, the LB plots takes the shape of a prolate ellipse; but the shape becomes progressively distorted into parallelogram like shapes as the material is subjected to higher values of strain amplitudes and the behaviour of the biofilm transitions to the nonlinear regime. Figure 3 (a-d) shows the elastic Lissajous-bowditch plots for each of the tested strains of single species biofilms at 2 Hz for strain amplitudes ranging from 1-100%. One can observe from Fig. 3 (a-d) that elastic LB plot for each species depicts a distinct shape and size for a given strain amplitude. The maximum total stress seen in the LB plots for CD biofilms is~500 Pa, which is about five times higher as compared to PF or PA biofilms~100 Pa; while for BS the maximum value of the stress is~180 Pa. Occurrence of parallelogram like shapes are indicative of plastic flow in the biofilms and changes in the slope of major axis of the parallelogram refers to the presence of strong nonlinearities in the material.
A more quantitative description of the LB plots can be obtained by calculating the third (e 3 ) and the first (e 1 ) order Chebyshev polynomial coefficients using MITlaos software. The third order coefficient of Chebyshev polynomial aids in a physical interpretation, a positive or negative value denotes intracycle strain hardening or softening, respectively; and gives us a measure of elastic nonlinearities in the material. Stiffening can be understood as sudden increase in stress due to increasing values of applied strain, while softening represents a decrease in the value of stress due to increase in the applied strain. Depending on the polymeric composition of the matrix and the interactions between the constituent polymers, stiffening or softening can occur at different points in the Pipkin space. Figure 3 (e-h) shows a plot of the ratio of e 3 ∕e 1 at various points in the Pipkin space for the different species biofilms (for standard deviation of e 3 ∕e 1 see Supplementary Fig. 1). We find that PF biofilms shows strain stiffening characteristics at 0.5 Hz and 1 Hz, with the maximum values of e 3 ∕e 1 occurring in between strain amplitudes 200-500% and 100-200%, respectively. Beyond strain amplitude values of 200% at 1 Hz, the PF biofilms soften. BS biofilms show similar strain stiffening characteristics when compared to PF biofilms at both 0.5 Hz and 1 Hz. Strain stiffening for BS biofilms occur at a slightly smaller strain amplitude (75-400% at 0.5 Hz and 50-100% at 1 Hz) and the softening also happens only slightly earlier compared to biofilms of PF. CD shows strain stiffening at 0.5 Hz and 1 Hz for all values of strain amplitudes and doesn't show any softening behaviour at this particular frequencies. The peak values of strain stiffening for CD occurs at 2 Hz and 4 Hz, at strain amplitudes of 200% and 50%, respectively, followed by softening and recovery. Similar to CD biofilms the peak values occur for PF biofilms at 75% and 25% strain amplitude at 2 Hz and 4 Hz. In contrast, BS biofilms only show slight evidence of strain stiffening at 2 and 4 Hz. PA biofilms exhibit persistent strain stiffening which starts at 25% strain amplitude at 0.5 Hz and lasts up to 50%. Similarly, at frequencies of 1 Hz and 2 Hz, strain stiffening initiates at 10% strain amplitude and persists upto 100% and 25% respectively. The softening beyond these values of strain amplitudes is indicative of breakdown of the underlying structure of the PA biofilms after which it softens rapidly. At a frequency of 4 Hz, the PA biofilms soften at strain amplitude of 25% and subsequently shows recovery of the structure as the value e 3 ∕e 1 increases. The stiffening behaviour of PF and PA biofilms which belong to the same genus, differ in certain aspects: Firstly, the onset of stiffening is much quicker for PA biofilms which is evidenced by pipkin diagrams 3 (b3-b4). Secondly the softening or breakdown of structure is more extensive in PA biofilms and it takes longer to recover when compared to PF biofilms.
We also present the results of strain stiffening index (S) for the different biofilms in Fig. 3 (i-l), which is defined by equation (4) (for standard deviation of S see Supplementary Fig. 2). A value of S > 0 indicates intracycle strain stiffening behaviour while S < 0 represents strain softening behaviour. In the Pipkin space, all four species of biofilms show evidence of strain stiffening at 0.5 Hz for strain amplitudes <500%. As the frequency of oscillation increased, the transition from stiffening to softening occurs at progressively smaller strain amplitudes for all the biofilms. The largest value of strain stiffening index at 0.5 Hz occurs for BS biofilms (S~2), while for CD, PF and PA biofilms, the indices show a much lower value. At 2 Hz, CD, PF and PA biofilms shows persistent stiffening until strain amplitudes of 200%, 100% and 25%, respectively. The stiffening indices reach a maximum value of S~1, S~2 and S~1, respectively, for CD, PF and PA biofilms. BS biofilms at 2 Hz mildly strain stiffen at intermediate strain amplitudes upto 25% and 50% and subsequently exhibit strain softening. At 4 Hz, for BS, PF and PA biofilms the strain stiffening index is minimal for all values of strain amplitudes, while CD biofilms are found to exhibit the maximum value of the stiffening index at strain amplitude of 50%.
Intracyle shear thickening/thinning characteristics shows distinct differences amongst biofilm of different species To investigate the intracycle viscous nonlinearities occurring in the biofilms we turn our focus to the viscous Lissajous Bowditch plots, which show the variation of stress (σ) with strain rate ( _ γ). Akin to elastic LB plots, the viscous LB plots can be constructed by parametrically eliminating time, from the time series signals of stress and strain rates. The viscous LB plots describe the viscous response of the material through a graphical representation. Figure 4 (a-d) shows the viscous LB plots for the different species of biofilms at an oscillatory frequency of 2 Hz and for strain amplitudes ranging from 1 to 100%. At small values of strain amplitudes, the LB plots takes a circular shape representing the expected behaviour in the linear viscoelastic regime. However, as the biofilm is subjected to increasing values of strain amplitudes, the circle gets distorted to different shapes indicating the presence of nonlinearities in the material. For CD biofilms the viscous LB plots resemble sigmoids ( Fig. 4 (b)), indicating the existence of strain stiffening or softening behaviour. For both PF and PA biofilms, the material exhibits large nonlinearities at certain intermediate and high values of strain amplitudes and one also observes the appearance of self-intersecting loops (Fig. 4 (c-d)). These secondary loops are indicative of a phenomena known as stress overshoot, where reversible structural breakdown of the material occurs 67 .
While the viscous LB plots aids in qualitative description of the material, a more quantitative description of the nonlinearities can be gained by plotting the ratio of third order to first order Chebyshev coefficients (ν 3 ∕ν 1 ) in the Pipkin space. A positive value of ν 3 indicates intracycle shear thickening while a negative value indicates shear thinning. Thickening can be understood as sudden increase in stress as function of increasing strain rate, while thinning represents a decrease in the value of stress due to an increase in the applied rate. It is evident from Fig. 4 (e-h), that each of the tested biofilms shows a varying degree of viscous nonlinearities in the Pipkin space (for standard deviation of ν 3 ∕ν 1 see Supplementary Fig. 1). For all the tested frequencies, BS biofilms exhibit slight intracycle thickening at amplitudes below 25%, beyond which they show thinning behaviour. The maximum thickening for BS biofilms occurs at 0.5 Hz and 4 Hz for strain amplitudes of 3-5% and 5-10%, respectively. While, the minimum values of thickening for BS biofilms is seen at 0.5 and 1 Hz, for strain amplitudes ranging from 400 to 500%. For PF biofilms, shear thinning occurs at 0.5, 1 and 2 Hz, for strain amplitudes ranging from 200 to 500%. PF biofilms also show mild strain stiffening for strain amplitudes ranging from 3 to 10% at frequencies below 4 Hz, thereby showing a behaviour similar to BS biofilms. However, the maximum thickening for PF occurs at 4 Hz in between strain amplitudes of 10-100% and the biofilm doesn't show any thinning behaviour at the given frequency. This is quite different to the behaviour of BS biofilms at 4 Hz for which thinning starts for strain values >25%. PA biofilms exhibit a behaviour similar to that of PF biofilms at both 0.5 Hz and 4 Hz. For strain amplitudes >100% at 4 Hz magnitudes of ratio of thickening indices is larger when compared to biofilms of PF. At 2 Hz and for strain amplitudes ranging from 5 to 25% the magnitude of thickening indices are higher when compared to that of PF (or BS). CD biofilms exhibit the most contrasting behaviour amongst the tested species, showing only a slight value of shear thickening across the Pipkin space. The peak of the thickening behaviour for CD occurs at 0.5 Hz, in between strain amplitudes of 0.8-8%. The minimum value of thickening for CD occurs at 2 and 4 Hz, in between strain amplitudes of 50-200%.
We also present the plot of thickening index (T) in Fig. 4 (i-l) for the four species of biofilms that we had investigated (for standard deviation of T see Supplementary Fig. 2). A value of T > 0 indicates intracycle shear thickening behaviour, while T < 0 represents shear thinning behaviour. At 4 Hz, thickening is the dominant behaviour for both BS, PF and PA biofilms, though it occurs at different strain amplitudes for the different species. For BS biofilms, thickening index shows a negative value for stain amplitude of 75%, in contrast PA and PF show persistently positive values of T for strain amplitudes >5% at 4 Hz. The maximum value of the thickening index (amongst all species) T~0.5 is found to occur for PA biofilms at 4 Hz. CD exhibits a contrasting behaviour compared to the other species as it shows a consistent thinning behaviour at 4 Hz. Thinning behaviour is also predominant at other frequencies and for strain amplitudes >10%. The minima of T for CD biofilms occurs close to 100% strain after which an increase in the values of T is seen for all the frequencies. The minimum value of T~− 0.75 for CD occurs at 4 Hz and strain amplitude of 100%, while the maximum value of T~0.15 occurs at 0.5 Hz at 1% strain amplitude. At large values of strain amplitudes >100% and for all the tested frequencies, CD biofilms exhibit recovery of the network structure (demonstrated by increase of T after a minimum), which is sparingly observed for the biofilms of any other species. The negative values of T at specific strain amplitudes, could also be correlated to the two step yielding behaviour. The dip in T might be representative of the characteristic bond breaking behaviour seen in colloidal systems [64][65][66] and could explain the presence of a two step hump as seen in the amplitude sweep ( Fig. 2 (b)). All of these measures capture the variation in the intracycle viscous nonlinearities that occur in the different species.
C. denitrificans biofilms exhibit highest energy dissipation To gain insight into the energy dissipation characteristics of the biofilms, we numerically integrated the area enclosed by the elastic Lissajous plots. For a perfectly elastic material, the plot of stress vs. strain typically yields a straight line; indicating zero energy dissipation and a complete recovery of the material to its original state. However, for complex materials like biofilms and other soft solids the energy dissipated can be nonzero, indicating an incomplete recovery of material. Figure 5 (a) and (b) shows the Figures e, f, g, h show the heat maps of the normalized value of ν 3 with respect to ν 1 , which indicates the distinct regions in the Pipkin space where shear thickening/ thinning occur (n ≥ 5, for standard deviation please see Supplementary Fig. 1). Figures i, j, k, l show the value of the shear thickening index T at different frequencies for the different species of biofilms that were tested (n ≥ 5, for standard deviation please see Supplementary Fig. 2). energy dissipated by the various species of biofilms at 1 Hz and 4 Hz, respectively. Within the linear regime, the dissipated energy for all the species that were tested show quadratic dependence on the strain amplitude γ o , which confirms the analytic solution E d ¼ πγ 2 o G 00 1 50,68 . Amongst the different species the variation in energy dissipation is rather large; with PA showing the least amount of dissipation. BS is found to dissipate slightly more energy than PF, whereas dissipation by CD is an order of magnitude higher as compared to BS. The larger amount of dissipation indicates that CD is more prone to having irreversible structural changes as compared to any of the other biofilms. Amongst the same genus of Pseudomonads, aeruginosa exhibits comparatively better elastic recovery as compared to fluorescens, which points to the existence of biomolecules aiding in elastic recovery. Beyond the linear regime and at 1 Hz, E d $ γ 1 o ; however across all values of strain amplitudes, CD consistently dissipates higher energy by at least half an order of magnitude when compared to all the other biofilms. At 4 Hz and for γ 0 > 10%, E d scales as γ 3 o ; which possibly indicates failure of the material, allowing it to dissipate energy rapidly after the point of failure. Similar effects of material failure, on energy dissipation characteristics has been discussed in context of optical coatings by Chen et al. 69 .
Onset of nonlinearity defined by Medium amplitude oscillatory shear region differs amongst species
In the linear regime, there is minimal distortion of the stress signal; as a result the first order harmonic signals dominate and contribution from higher order signals are minimal. As the strain amplitude on the biofilm sample is increased, the stress waveforms distort and contributions from higher order harmonics become significant. Increase in intensity of higher order harmonics (I n ) provides one with an understanding of the nature of nonlinearity developing within the material. Normalized values of higher order harmonics (I n ∕I 1 ) can be obtained from the files that are output by MITlaos and contains information on fourier transform spectrum of stress. One way of exploring the onset of nonlinearity in the different species of biofilm is by comparing the intensity of third to first order harmonic (I 3 ∕I 1 ) as a function of the strain amplitude, at a constant frequency of 1 Hz. Similar tests undertaken on typical model polymers exhibit a scaling of o . This particular region of quadratic scaling is known as Medium Amplitude Oscillatory Shear (MAOS) and denotes the transition from linear to nonlinear behaviour of the sample under consideration. Figures 6 (a), (b), (c) and (d) show the scaling relation, which is indicative of the onset of nonlinearity for the different species of biofilms. B. subtilis shows a scaling of 1.8 for strain amplitudes ranging between 6 and 12%, while C. denitrificans shows a scaling of ∝ 1.5 for strain amplitude ranging between 0.55 and 0.7%, P. fluorescens exhibits a scaling of ∝ 1.8 for strain amplitude from 8 to 13% and P. aeruginosa exhibits a scaling of ∝ 1.6 for strain amplitude from 0.9 to 4%. By comparing the MAOS regions of different microbial species, once comes to the conclusion that C. denitrificans starts exhibiting nonlinearity at very small strain amplitudes; but the rate of growth of nonlinearity is smaller when compared to any of the other species. For model polymeric systems, like linear polymers a scaling exponent of~2 is usually observed; while a slope of~1.8 has been observed for branched polymer systems 51 have found power law dependent scaling characteristics in MAOS region have only been performed for model and synthetic polymer systems.
DISCUSSION
We have investigated the rheological characteristics of four different species of bacterial biofilms in the linear viscoelastic and nonlinear regime. In the linear regime, biofilms show substantial differences in viscoelastic moduli and yield stresses that are orders of magnitude different from each other. The amplitude sweeps for most of the species exhibit a type III hump for the viscous modulus with increasing strain amplitude, which is reminiscent of the structural rearrangement that occur in soft colloidal systems. The frequency sweeps exhibit a weak power law pointing towards similarities between biofilms and colloidal gels. By subjecting the biofilms to increasing values of strain amplitudes at different frequencies we are able to capture the elastic and viscous nonlinearities occurring in the biofilms using the Pipkin diagram. The elastic nonlinearities show subtle variations in the phase space of frequency and strain amplitude (i.e. the Pipkin diagram), while the viscous nonlinearities show contrasting differences dependent on the species. The biofilms also exhibit large differences in the elastic energy dissipation characteristics indicating a varying amount of mechanical resilience of the extracellular matrix. By studying the slope of the third to first harmonic, we find that the onset of nonlinearity occurs much earlier in C. denitrificans when compared to the other species. The nonlinear parameter (I 3 ∕I 1 ) grows at a much higher rate for B. subtilis and P. fluorescens indicating a larger degree of nonlinearity of the ECM for these two species (albeit their linear viscoelastic moduli is significantly lower when compared to C. denitrificans).
Such contrast in the rheological measures of biofilms might be a result of a combination of factors. The first factor might be the way cells order and pack themselves in biofilms. As seen in Fig. 1 (b2) the packing in CD biofilms is quite disordered and the disorder in packing extends to the out of focal plane. Whereas in PF the cells lie mostly within the focal plane but there is lot of spatial disorder. PA shows very sparse packing of cells, which can be seen in Fig. 1(b4) and cellular disorientation spans out of the focal plane, even greater than that observed for CD. The second factor is the extracellular polymers that constitute the ECM which are known to form links between cells and between the cells and the matrix providing the biofilm its rigidity and structure. Proteins like RbmA are known to control cell-cell connections and therefore determine the ordering of cells within V. cholerae a biofilm 71 . However, similar knowledge of the polymeric components of the matrix and their roles for the species that we have tested are still lacking. For example in B. subtilis, the matrix comprises of a large molecular weight exo-polysaccharide (EPS), whose exact composition remains yet undiscovered 72 . While, the major protein components are TasA which causes the rugosity of the biofilm colonies and BslA that controls the hydrophobicity. Minor protein component TapA is known to control the assembly of TasA fibres. However, the role of individual polymers in determining the rheology of the overall matrix in B. subtilis still continue to be investigated 73 . The matrix components of biofilms of C. denitrificans still remain unknown and have only been quantified in terms of mass fraction of proteins, nucleic acids and polysaccharides 74 . Rheological tests on this particular species of biofilms have not been performed to the best of our knowledge. In P. fluorescens, the biofilms consist of an acetylated form of cellulose fibre, together with lipo-polysachharides, fibrils and attachment factors like PNAG (poly-N-acetylglucosamine); which provide structural integrity to the wrinkly spreader biofilms 75,76 . Functional amyloids produced by the fap operon have been found to modulate the hydrophobicity of P. fluorescens biofilms and also confer mechanical strength 77 . Our knowledge of the matrix constituents of P aeruginosa is substantially advanced, as compared to other microbes owing to decades of research on the model organism. It is well known that the polysaccharide Pel acts as a scaffold for the biofilm and helps in maintaining the intercellular interactions 78 , while the polysaccharide component Psl initiates biofilm formation by modulating cell-cell and cellsurface attachment 79,80 . An over-expression of the polysachharide alginate results in a mucoid biofilm 19 . The protein CdrA has been found to control the cellular packing of cells and provide protection against proteolysis by interacting with Psl 81 . More recently the matrix protein LecB has been found to bind to Psl and helps in retention of cells and polysaccharides within the PA matrix 82 . This highlights the diversity of polymers in the various biofilms, however their exact contribution to rheology of biofilms and especially large strain behaviour needs to be carefully investigated.
In our experiments we scrape a uniform mat of biofilm and then use the pooled scrapings for rheological measurements. One of the major concerns is that the process of scraping can introduce defects or disrupt the structure of a continuous material, thereby affecting the rheological measurements. This is an plausible situation if the cultivated biofilms are very weak and especially for submerged biofilms. Since our biofilms are cultivated at the agarwater-air interface, they are more stiffer than their submerged counterparts. While scraping with a glass slide (imposing large strains), we find that the torn part of the biofilm mat retains its cohesion and tears off cleanly at the edges. This possibly indicates that the material still retains its continuous properties even after application of large strain (please see Supplementary Fig. 3, Supplementary note 1). Multiple instances of such scrapings with continuous material properties are pooled together to create a composite biofilm test sample, which is expected to exhibit rheological characteristics similar to the bulk material. In addition, biofilms are hypothesised to be thixotropic materials which means that their material structure and rheological properties can recover, even after the application of large strains. Such behaviour was recently confirmed by Yan et al. in V. cholerae biofilms 83 , where even after two cycles of amplitude sweep of up to 1000% strain; the linear viscoelastic measures were found to be similar to each other. Our experiments also show a similarity of elastic or viscous LB plots when the CD biofilms are subjected to LAOS sequences of up to 500% strain amplitude (please see Supplementary Fig. 4, Supplementary note 2).
While our study uses biofilm grown on agar plates which is an artificial system, in reality biofilms within natural environments are subjected to shear forces, varying amounts of hydration (water content), temperature and various other factors. Many of these factors including a variety of tools have confounded rheological measurements of biofilms in the past 84 , as describing the rheological state of a biofilm (akin to colloids) remains a challenging proposition. Part of the problem can be attributed to the active nature of biofilms. For example, as the biofilms start growing on surfaces, they secrete extracellular polymers and the concentration of the polymers depends on the water content of the system. However, as a biofilm's growth progresses over time (given a fixed volume of water), the concentration of the polymeric substances would increase, which could result in transition from a dilute to a concentrated polymeric system, resulting in a different rheological state. Another complexity arises from the fact that extracellular polymers of biofilms are a mixture of multiple polymers; which could interact amongst themselves. In addition, shear forces play an implicit role in shaping the structure of biofilms, the constituent polymeric molecules in the matrix can stretch, reorienting the cells thereby causing local ordering 85,86 . Beyond a certain limit of shear forces, the structures can rupture forming clusters and the clusters can hydro-dynamically interact with each other. On a rheometer, such interactions can show up as two step yielding signatures, which we have observed in the case of C. denitrificans [64][65][66] . To summarise, in order to better characterise biofilm rheology a better description of the rheological state of the biofilm, which includes a description of constituent macromolecules and their interactions would be essential.
With advances in molecular microbiology, investigations involving the impact of biomolecules/biopolymers have led to interesting insights into a variety of biofilm functionalities. However, a similar knowledge of the molecular constituents and their effects on biofilm rheology is still limited and only spans a few species [17][18][19] . Biochemists in particular can isolate and help decipher the role of small molecules that cause short-and longrange interactions amongst the matrix constituents and the cells, which may cause stiffening/thickening of the matrix or various other effects. Alternatively, microbiologists can also engineer mutants that lack ability to express a particular biomolecule and investigate the absence of polymeric component on the rheological behaviour of the biofilm matrix, using LAOS. This could allow one to shed light on the role of the particular component in biofilm rheology. While we have described LAOS as a tool to mechanically explore biofilm rheology, it is by using tools from biochemistry/molecular microbiology; that the real potential of LAOS in deciphering the interactions of biopolymers can be realised. Advanced tools like confocal rheoscope 87 , small angle neutron scattering 88 , together with LAOS can help us visualise the cellular structure/orientation of cells and greatly enhance our knowledge of matrix viscoelasticity of biofilms.
Overall, our results provide insights into the nonlinearities occurring in biofilms at large shear forces, a situation that could be more prevalent than perceived. It also lays the groundwork for future investigations that could possibly use genetic manipulation to dissect the role of matrix polymers and their interactions, in altering matrix viscoelastic characteristics. By combining the above-mentioned array of interdisciplinary tools, we hope to continue to gain novel insights into the material characteristics of biofilms and their functionalities, in the near future.
Bacteria and biofilm growth conditions
Pseudomonas fluorescens is a gram-negative, multi-flagellate, obligate aerobe that was isolated from prefilter tanks in Sweden and can also be found acting as a biocontrol agent on the plant roots. Bacillus subtilis is a soil and gut dwelling, multi-flagellate, gram-positive bacterium and is a model system for studying various characteristics of biofilms. Comamonas denitrificans is a uniflagellate, gram-negative bacterium that plays a role in the denitrification process and is one of the dominant species found in the Birtley Wastewater Treatment plant (Northumbrian Water). Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in humans and is also commonly found in lungs of cystic fibrosis patients. Single species biofilms of Bacillus subtilis (B. subtilis 168), Pseudomonas fluorescens (DSMZ-50090) Comamonas denitrificans (DSMZ-17887) and Pseudomonas aeruginosa (DSMZ-22644) were grown overnight in LB Miller (Molecular Dimensions MD12-103, 25g/L), Nutrient (Sigma-Aldrich N7519, 8g/L), Tryptic Soy (Sigma-Aldrich 22092, 30g/L) and Nutrient (Sigma-Aldrich N7519, 8g/L) broth respectively. For imaging of radially spreading biofilms, a 2 μL droplet of bacteria culture is deposited on the respective agar plates and were stored at room temperature for six days. For all rheology experiments, a 150 μL droplet of the overnight culture was pipetted onto the respective 1.5% agar plates, respectively, and was smeared aseptically over the agar surface using a L shaped spreader. The inoculated bacteria were allowed to develop thick mat like continuous biofilms on the agar surfaces for 72 h (at room temperature) and were then harvested for experiments.
Rheometry
For rheological experiments, mat like biofilms were gently scraped off from the agar plates using a glass slide and the aggregate is placed on the stage of a Malvern Kinexus Pro + rheometer. During scraping minimal pressure was applied to the agar surface to avoid chunks of agar detaching and contaminating the biofilm sample. The non-abrasive side of a Silicon carbide grinding paper (from Struers online store, Grit#2000) was attached to double sided sticky tape (3M 9084, Double Sided Paper Tape) and 20 mm holes were punched through. These circular adhesive backed waterproof grit papers were placed at the end of the rotating head and the bottom plate of the rheometer to reduce instances of slip between the plate and the biofilm sample. Zero gap configuration was established with the grit papers attached to both plates and then the biofilm sample was loaded onto the bottom stage. A constant normal force of 0.1N was applied and the sample was allowed to reach a steady state for at least 60s, which ensured uniform contact between the top plate and the biofilm sample. If required, any excess sample was trimmed to reduce instances of overfill. A solvent trap system was used to keep the samples hydrated for the duration of the measurements. For LAOS studies, the rheometer was operated in a strain-controlled mode and a 20 mm parallel plate geometry was used for our tests. A constant gap height was maintained for each run, which can vary between 0.5 and 1 mm due to the differences in biofilm volume harvested (The volume harvested per agar plate varies substantially for each of the tested species). After ensuring a constant gap height and a steady state normal force of 0.1N the biofilm sample was subjected to increasing values of oscillatory strain γ (0.3%, 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 25%, 50%, 75%, 100%, 200%, 400% and 500%) at a given frequency ω (0.5, 1, 2, 4 Hz). The raw values of angular displacement and torque were output from the Rspace software as a CSV file. The CSV files were read using MATLAB following which the waveforms were checked for stability and truncated to five cycles. The truncated waveforms were analysed using MITlaos software to get the LAOS measures. Standardised frequency and amplitude sweeps were performed on the sample to understand the material characteristics and map out the linear viscoelastic regime. All SAOS as well as LAOS experiments were repeated a minimum of five times. Experiments required for determining the slope of I 3 ∕I 1 vs. strain amplitude were performed once, since we had to sample finer values of strain amplitudes. To calculate the energy dissipation from the Lissajous bowditch plots, we used numerical integration in MATLAB. The endpoints of the averaged LB plots from MITlaos were joined to ensure that the curve is closed before performing the integration. The elastic Lissajous Bowditch plots can self-intersect, which can cause ambiguity in determination of area under the curve. To avoid ambiguities due to self-intersection, we use MATLAB code 'intersections' to check for points of intersection in the elastic LB plots. If self intersections are detected, the areas are segmented and total area is determined by summation of areas of each of the segments (Please see Supplementary Fig. 5, Supplementary note 3).
Confocal microscopy
Following the growth of mat like biofilms on agar plates, 500 μL of 10 μM Syto 63 (S11345 from Thermo-fisher scientific) solution in Tris buffer was pipetted on the agar plate. The dye was allowed to stain the biofilms for 30 min. The biofilms were then scraped on the gene-frames (AB0576 from Thermo-fisher scientific) attached to a glass slide and sealed with a coverslip on the top. Images of the cellular structure were acquired with a Leica SP8 confocal microscope at a distance of 10-15 μm from the bottom glass coverslip using a ×100, 1.4 numerical aperture objective lens.
Analysis of recorded waveforms using Chebyshev polynomial analysis
As the rheometer is operated in strain-controlled mode, the oscillatory strain imposed on the sample can be described as γ = γ o sin(ωt). The response of the material is assumed to be sinusoidal and can be written as σ = σ o sin(ωt + δ). Where, γ o is the strain amplitude, σ o is the magnitude of stress, ω is the oscillatory frequency of rheometer, δ is the phase angle between the input (strain) and output (stress) waveforms and t denotes time. Using the equations described in Ferry et al. 89 and assuming that stress waveforms are sinusoidal (for various strain amplitudes), one can decompose the total stress into elastic and viscous components and calculate the elastic (G' ) and viscous modulus (G″) of the material.
However, at large strain amplitudes the material enters the nonlinear regime and the stress waveform is not a simple sinusoid anymore. As a result, higher order harmonics need to be considered in order capture the true meaning of the waveform. The non-sinusoidal stress waveform can therefore be written in terms of Fourier expansion as: σðt; ω; γ 0 Þ ¼ γ o X n ¼ 1;3;:::: G 0 n ðω; γ o ÞsinðnωtÞ þ G 00 n ðω; γ o ÞcosðnωtÞ (1) where n represents the higher order harmonics. Only odd harmonics are considered because stress is assumed to bear odd symmetry with respect to shear strain or strain rate 49 . Ewoldt et al. 50 proposed the use of orthogonal Chebyshev polynomials of first kind to approximate the nonlinear waveforms, as the higher order Chebyshev coefficients have physical meanings. Therefore Eq. (1) can be rewritten in terms of Chebyshev polynomials as: σðt; ω; γ 0 Þ ¼ γ o X n ¼ 1;3;:::: e n ðω; γ o ÞT n ðxÞ þ ν n ðω; γ o ÞT n ðyÞ where, e n , ν n are the n-th order elastic and viscous Chebyshev coefficents, T n denotes the Chebyshev polynomial of first kind of n-th order, x = γ∕γ o = sin(ωt) and y ¼ _ γ=γ o ¼ cosðωtÞ. Furthermore, by using the recursion identities of Chebyshev polynomials T n cos(ωt) = cos(nωt) and sin(ωt) = cos (π∕2 − ωt), which yields T n sinðωtÞ ¼ ðÀ1Þ nÀ1 2 sinðnωtÞ, one can directly express the Chebyshev coefficients in terms of the n-th order moduli: 2 ; ν n ¼ G 00 n ω ; n 2 1; 3; ::::: For n = 1, one can recover e 1 ¼ G 0 1 and ν 1 ¼ G 00 1 ω . Traditionally, G 0 1 $ G 0 is known as the elastic modulus, G 00 1 ¼ ν 1 ω $ G 00 is known as the viscous modulus and ν 1 is the viscosity. The higher order coefficients can be related their respective moduli and in particular, the third order elastic (e 3 ) and viscous (ν 3 ) Chebyshev coefficients represent a physical meaning. A positive value, i.e. e 3 > 0 and ν 3 > 0 represents intracyle strain hardening and shear thickening, respectively; whereas negative values e 3 < 0 and ν 3 < 0 represents strain softening and shear thinning. These measures represent the a quantitative way of describing the elastic and viscous nonlinearities occurring in the material. One can also calculate the dimensionless strain stiffening index (S) and shear thickening index (T), which can be defined in terms of the higher order Chebyshev coefficients as follows: S ¼ 4e 3 :::: e 1 þ e 3 þ ::::: ; T ¼ 4e 3 ::: ν 1 þ ν 3 þ ::::: A more detailed description of the derivations of the above-mentioned equations and the physical interpretation can be found in the articles by Ewoldt et al. 50,90,91 . Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.
DATA AVAILABILITY
All the raw data that were gathered can be found at the link: https://doi.org/10.6084/ m9.figshare.11917890.
CODE AVAILABILITY
All the codes used to pre-process the raw data can be found at the link: https://doi. org/10.6084/m9.figshare.11917890. MITlaos software 91 used to analyze the recorded waveforms can be requested from Professor Randy Ewoldt by emailing mitlaos@mit. edu. | v3-fos-license |
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} | pes2o/s2orc | Effects of cooking and fermentation on the chemical composition, functional, and antinutritional properties of kariya (Hildergardia barteri) seeds
Abstract The effects of natural fermentation and cooking on kariya seeds functional properties, chemical composition, and antinutritional properties were evaluated. Result showed a reduction in antinutritional properties and improvement in protein content which were observed to increase with cooking (at 100°C) and fermentation period (24–96 hr). Functional analyses showed an increase in foaming and emulsion properties, while water absorption capacity and swelling power were observed to likewise increase with an increasing temperature between 60°C and 90°C. There was also an improvement in foaming properties with increase in salt (NaCl) concentration, while emulsifying property decreases with an increase in salt (NaCl) concentration. Based on the result of the findings of this study, it can be stated that the cooking and fermentation processes employed in this study can enhance the domestic and industrial utilization of these seeds.
| INTRODUCTION
Quite a number of oil seeds have been characterized but the vast majorities have not been evaluated adequately (Oderinde & Ajayi, 1998). This is also mostly valid for vegetation and crops cultivated in Nigeria which has one of the most widespread floras in Africa (Oderinde & Ajayi, 1998). In the last decade, there has been increased interest in research focusing on underutilized oil seeds for possible food product development and use (Obasi & Okolie, 1993). In Nigeria and other states in Africa, there has been increased utilization of these underutilized seeds by locals for domestic and industrial purposes. Kariya (Hildegardia barteri) falls into this group of underutilized species of plants.
Kariya (Hildegardia barteri) is predominantly an ornamental tree in West Africa that grows in Nigeria and other countries in Africa. It is called Krobo Christmas tree (Irvine, 1961), grown only for its bright beautiful flowers which bud during the dry season. The flowers of this plant which develop into one-seeded pods are borne on leafless branches around the months of December to March annually. Each leaf of this plant is approximately 50 mm in length (Hildergadia Notes, 2009). When dried after attaining maturity, the pods drop and mostly dispose of as refuse. But in some parts of West Africa, the kernel is extracted from the pods and processed by roasting it like peanuts, with peanut flavor for human consumption (Inglett, Cavins, & Spencer, 1973). In some other instances, these kernels are used as condiments in the preparation of food by locals. In a report by Ogunsina, Olaoye, Adegbenjo, and Babawale (2011), kariya kernels were reported to contain 6.5%, 2.8%, 37.5%, and 17.5% of crude fiber, ash, crude fat, and protein. Research efforts on kariya have been limited to the physical properties of the seeds (Adebayo, Ogunsina, & Gbadamosi, 2013;Inglett et al., 1973;Ogunsina et al., 2011). Adebayo et al. (2013) have likewise made some interesting findings on the physicochemical and | 1107 FAWALE Et AL. functional properties of Kariya flours. Gbadamosi and Famuwagun (2016) reported that the antinutrient content in Kariya seed isolates was reduced significantly when fermented. In another study by the same investigator, it was observed that when protein hydrolysate was processed from Kariya seeds and fermented, functional, antioxidant and proximate properties were improved as a result. In another related study by Abiose (2015a, 2015b), the functional properties like foaming and emulsifying properties, water absorption capacity, and gelling capability were enhanced when kariya seeds were processed into its protein concentrate and isolate.
Furthermore, kariya seeds have found restricted application as food and food ingredient due to its antinutrient composition. In order to increase the utilization of underutilize seeds, Gbadamosi, Abiose, and Aluko (2012) suggested processing of the whole flour of these seeds into defatted flours and examining its suitability as functional ingredients and food supplements. It was also said that the ultimate success of utilizing any plant protein as food ingredients mainly depends on its functional and nutritional properties (Gbadamosi et al., 2012).
Fermented plant proteins are a vital nutritional source to the populace of the Oriental and African continents. Fermentation has over the years been demonstrated to improve the biological availability and enrichment of food substrates such as protein, essential amino acids, and vitamins, while also resulting in the elimination of antinutrients (McGovern et al., 2004). Fermented foods improve the digestibility, flavor, aroma, health-promoting benefits, and availability of bio-nutrients (Jeyaram et al., 2009). It has been reported that fermentation improves the nutritional, shelf-life structural properties as well as reduction of antinutritional content (Fowomola & Akindahunsi, 2008). Despite the importance of these seeds, there is still a dearth of information on the functionality of kariya seed flour. This study therefore aimed to investigate the functional, chemical composition, and antinutritional properties of cooked and fermented kariya flour with a view to improving its utilization as food ingredients.
| Sample collection
Kariya (Hildegardia barteri) seeds for this research were harvested from various locations in University, Ile-Ife, Nigeria. The outer shell of the seeds was manually decorticated and air-dried in a cabinet dryer at 45°C, stored in tight and moisture-free plastic containers for use in further analysis. All chemicals used were obtained from Sigma Chemicals, (St. Louis, MO) and were of analytical grade.
| Preparation of fermented samples
Fermentation of H. bateri seeds was carried out by the method described by Fowomola and Akindahunsi (2008). The decorticated dried seeds were divided into four portions. The first part was kept as control (unfermented seeds), the second portion was cooked (100°C) but not fermented, and the third portion was fermented but not cooked.
The fourth portion of the kernel was cooked and transferred into a calabash pot, lined uniformly with banana leaves (up to 5 layers) and allowed to undergo natural fermentation at ambient temperature for 5 days inside the incubator (30°C) after which the fermentation process was terminated by oven drying at 70°C. This was followed by milling to obtain flour which was afterward subjected to cold extraction process using acetone. Extraction was done on a magnetic stirrer (Cenco, Breda, Netherland) for 4 hr, while a 1:5 w/v flour-to-solvent ratio was adopted after which slurry gotten was filtered and residue re-extracted as described previously. De-solventization of the resulting flour was carried out by drying in a fume hood and ground into fine flour particles using a Kenwood grinder. Homogenous defatted flour samples were obtained using sieving through a 200-mm sieve mesh and stored in air tight containers at 4°C until used for analysis.
Likewise, some dried whole seeds were not defatted and kept under similar conditions as described previously.
| Proximate composition
The proximate compositions of the fermented and cooked samples were determined according to AOAC methods (2000). Ash, protein, and fat content were among factors determined. The analysis was done in triplicate while data were reported as means ± standard deviation.
| Determination of physicochemical and functional properties
The method described by Beuchat (1977) was used for the determination of the oil absorption capacity of the samples. One gram of sample weight was centrifuged at 4,000g for 30 min using an MSE-Harrier centrifuge. Swelling capacity was determined by the method described by Takashi and Seib (1998). This was carried out at room temperature and at 60°C and 90°C and expressed as milligram per gram. Water absorption capacity (WAC) was determined at room temperature and at a higher temperature between 60°C and 90°C using the technique described by Adepeju, Gbadamosi, Adeniran, and Omobuwajo (2011). Two gram of sample was used for this process and centrifugation was performed at 4,000g for 30 min using an MSE-Harrier centrifuge (Sydenham, London, UK). Water absorption was expressed as a percentage increase in the weight of the sample.
| Protein digestibility determination
The method described by Chavan, Mckenzie, and Shahidi (2001) as modified by Gbadamosi et al. (2012) was used in the determination of protein digestibility. Sample weight of 250 mg was suspended in 15 ml of HCL of 0.1 mol/L having 1.5 mg of pepsin. This was followed by moderate shaking for a duration of 1 hr. The resultant suspension was neutralized with NaOH of 0.5 mol/L concentration and treated with 4.0 mg pancreatin in 7.5 ml of phosphate buffer (0.2 mol/L, pH 8.0). The resulting mixture was shaken at room temperature for 24 hr, filtered using What No. 1 filter paper. Residues recovered were washed with distilled water and air-dried. This was used for protein determination using the method earlier described by AOAC (2000).
Protein digestibility was calculated using the following equation: where, I is protein content of sample before digestion and F is protein content of sample after digestion.
| Determination of tannins
The concentration of tannin in tested samples was determined using the modified vanillin-hydrochloric acid (MV -HCl) method of Price, Van Scoyoc, and Butler (1974).
Various concentrations (0.0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml) of the catechin standard solution was pipetted into clean dried test tubes in duplicate. To one set was added 5.0 ml of freshly prepared vanillin -HCl reagent prepared by mixing equal volume of 4% (w/v) vanillin/MeOH and 16% (v/v) HCl/MeOH, and to the second set was added 5.0 ml of 4% (v/v) HCl/methanol to serve as blank. The solutions were left for 20 min before the absorbance was taken at 500 nm.
The absorbance of the blank was subtracted from that of the standards. The difference was used to plot a standard graph of absorbance against concentration.
where, x is value obtained from standard catechin graph.
| Determination of oxalate
Oxalate was determined using titrimetric method by Falade, Dare, Bello, Osuntogun, and Adewusi (2004). Two grams of the sample was weighed in triplicate into conical flasks and extracted with a 190 ml distilled water and 10 ml 6 mol/L HCl. The suspension was placed in boiling water for 2 hr and filtered and made up to 250 ml with water in a volumetric flask. To 50 ml aliquot was added 10 ml of 6 mol/L HCl and filtered and the precipitate washed with hot water. The filtrate and the wash water was combined and titrated against conc. NH 4 OH until the salmon pink color of the methyl red indicator changed to faint yellow. The solution was heated to 90°C and 10 ml 5% (w/v) CaCl 2 solution was added to precipitate the oxalate overnight. The precipitate was washed free of calcium with distilled water and then washed into 100 ml conical flask with 10 ml hot 25% (v/v) H 2 SO 4 and then with 15 ml distilled water.
The final solution was heated to 90°C and titrated against a standard 0.05 mol/L KMnO 4 until a faint purple solution persisted for 30 s.
The oxalate was calculated as the sodium oxalate equivalent.
| Determination of saponin
The spectrophotometric method of Brunner (1984) was used for saponin analysis. One gram of finely ground sample was weighed into 250-ml beaker and 100 ml of isobutyl alcohol was added. The mixture was shaken for 2 hr to ensure uniform mixing. Thereafter, the mixture was filtered through a Whatman No. 1 filter paper into a 100-ml beaker and 20 ml of 40% saturated solution of magnesium carbonate was added and the mixture made up to 250 ml. The mixture obtained with saturated MgCO 3 was again filtered through a Whatman No. 1 filter paper to obtain a clear colorless solution. One milliliter of the colorless solution was pipetted into a 50 ml volumetric flask and 2 ml of 5% FeCl 3 solution was added and made up to mark with distilled water. It was allowed to stand for 30 min for blood red color to develop. Saponin stock solution was prepared and 1-10 ppm standard saponin solutions were prepared from saponin stock solution. The standard solution was treated similarly with 5% of FeCl 3 solution as done for 1 ml of sample above. A dilution of 1-10 was made from the prepared solution. The absorbances of the samples as well as that of the standard solution were read after color development in a 752S Spectrum lab UV, VIS Spectrophotometer at a wavelength of 380 nm.
| Effect of pH and NaCl concentration on emulsifying activity index and emulsion stability index
The effect of pH on emulsifying activity index (EAI) and emulsion stability was determined by the method described by Pearce and Kinsella (1978) with slight modifications. About 500 mg of the samples was dispersed in 100 ml of distilled water at different NaCl concentrations (0.0, 0.5 and 1.0 mol/L). The pH of the protein solution was then adjusted separately to pH 2-10 with either 1 N HCl or 1 N NaOH and the solution was mixed with 50 ml of pure Gino oil and homogenized using a blender (SN2200 Qlink, Beijing, China) set at high speed for 60 s. Approximately, 50 ml aliquot of the emulsion was transferred from the bottom of the blender after homogenization and mixed with 5 ml of 0.1% sodium dodecyl sulfate solution. The absorbance of the diluted solution was then measured at 500 nm using spectrophotometer (722-2000 Spectronic 20D, England).
The absorbance obtained was used to calculate the EAI as shown below; where, A is absorbance at 0 min after homogenization.
To determine the emulsion stability, the emulsions were allowed to stand for 10 min at room temperature and the ESI was determined as 1ml of 0.05 mol/L KMn0 4 = 2mg sodium oxalate equivalent/g of sample.
Saponins = Absorbance of sample × dil.factor × gradient of standard graph sample weight × 10,000 (mg/g) Emulsifying activity index(m 2 ∕g) = 2 × 2.303 × A 0.25 × protein weight (g) described below and expressed based on the absorbance at 0, 10 min, and the time difference as shown in the formula; 2.8 | Effect of pH and NaCl concentration on foaming capacity and stability A modified method described by Chavan et al. (2001) was used for the determination of the foaming capacity (FC) and foaming stability (FS) of the samples as influenced by salt concentration and pH. To 100 ml of NaCl solution at concentrations of 0.0, 0.5, and 1.0 was added 2 g of the samples. The pH of the protein solution was separately modified to pH 2, 4, 6, 8, and 10 with either 1 mol/L NaOH or 1 mol/L HCl until the set pH was achieved. The resultant solution was standardized for 2 min using a blender (SN2200 Qlink, Beijing, China) set at high speed.
This was then transferred into a 250 ml measuring cylinder and volume increase to the original volume of solution in measuring cylinder noted.
Percentage volume increase to that of the original volume of protein solution in the measuring cylinder was calculated and expressed as foaming capacity or whippability. Foam stability was expressed as a percentage of the volume of foam remaining in the measuring cylinder to that of the original volume after 30 min of quiescent period.
| Statistical analysis
All the analyses were conducted in triplicate and subjected to statistical analysis using analysis of variance (ANOVA). Means were separated using Duncan's multiple range test. Table 1 shows the chemical composition of the fermented and cooked kariya seed. Moisture content was observed to range between 9.67% and 16.52% with cooked unfermented kariya (CUK) flour having the lowest moisture content and cooked fermented kariya (CFK) exhibiting the highest moisture content. The moisture content was observed to increase with fermentation days. The moisture content achieved in this study were within the range of values stated for pigeon pea flour (10.20%-13.24%) and tiger nut flour samples (9.93%-10.42%) by Adebowale and Maliki (2011) and Adejuyitan, Otunla, Akande, Bolarinwa, and Oladokun(2009) but higher than the values reported by (Osungbade, Olasunkanmi, & Oladipupo, 2016) for sandbox (Hura crepitans) seeds, respectively.
| Chemical composition of cooked and fermented kariya seeds
Increased duration of fermentation period was observed to increase (p ≤ .05) the crude protein content of kariya flour. The crude protein of kariya flour samples increased (p ≤ .05) with fermentation period. The protein content ranged between 33.91% and 44.57% with the raw unfermented sample (RUK) having the lowest protein value, while the cooked fermented kariya (CFK) had the highest protein content. The increase in protein during fermentation observed in this study agrees with previous values (21.8%-23.9% and 24.08%-52.10%) reported for pigeon pea (Cajanus cajan) and sand box seed flour (Adebowale & Maliki, 2011;Osungbade et al., 2016). The increase in protein confers a nutritional benefit on the fermented kariya seed flour. The increase in protein value with fermentation time observed in this study could be ascribed to the net synthesis of protein by fermenting seeds (Uwagbute, Iroegbu, & Eke, 2000). This agrees with other scientific findings that processing techniques such as fermentation improve the nutritional quality of the food products, particularly in terms of protein content (Enujiugha, 2003;Fasasi, Eleyinmi, & Oyarekua, 2009). Table 1, crude fat content ranges between 8.16% and 9.12% and were significantly different (p ≤ .05) among nonfermented and fermented samples. However, a significant difference (p ≤ .05) between all fermented samples was not observed. Increased lipolytic enzyme activities during the fermentation process might be responsible for the decrease in fat content observed in these set of samples.
As shown in
Lipolytic enzymes are known to hydrolyze fat components into glycerol and fatty acid.
| Effect of cooking and fermentation on oil absorption capacity
The effects of fermentation on the functional properties of kariya seeds flour are shown in
| Effect of cooking and fermentation on protein digestibility (IVPD)
Protein digestibility of all samples is shown in Table 2. Cooking and fermentation causes significant (p ≤ .05) improvement in IVPD in all samples. The increase was in the range of 63.71% to 85.51% in all samples (RFK, CUK, RUK, CFK, CFK1, CFK2, and CFK3). The cooking process increased the rate at which the antinutrients were leached out and destruction of protease inhibitors. The improvement in protein digestibility caused by fermentation could be attributed to partial degradation of complex proteins to more simple and stable products which could be a possible reason for the improvement of the protein digestibility of fermented samples. Hassan and El Tinay (1995) attributes this effect to the degradation of polyphenols, tannins, and phytic acid by microbial enzymes. Also, elimination of phytic acid during the degradation process by microbial enzymes during fermentation results in the improvement of protein digestibility in fermented millet (Khetarpaul, 1991).
| Effect of temperature on water absorption capacity and swelling capacity
The result of swelling capacity as influenced by temperature is shown in Figure 1. The swelling capacity values of RFK, CUK, RUK, and CFK between 30°C and 90°C ranged between 1.34 and 1.93 ml/g, 1.12 and 1.35 ml/g, 1.19 and 1.56 ml/g, and 0.90 and 1.25 ml/g, respectively, with lowest swelling capacity occurring at 60°C and highest at 90°C. The swelling capacity was observed to increase with an increase in temperature of all samples (Figure 1b). Swelling capacity is a function of processing conditions, material nature, and treatment type.
| Effect of pH and salt (NaCl) concentrations on emulsifying activity index (EAI) and emulsion stability index (ESI) of cooked and fermented kariya seeds
The EAI and ESI at neutral pH of the samples ranged between 8.75 m 2 /g and 29.79 m 2 /g and 59.85% and 167.43%, respectively ( EAI value for RFK and as seen in CUK and CFK might be due to protein denaturation caused by the fermentation and the cooking process which exposed more hydrophobic groups. Denaturation has been revealed to increase the emulsifying properties of proteins, due to improved hydrophobic surface and flexibility (Dickinson & Hong, 1994;Moure, Sineirob, Domíngueza, & Parajó, 2006). The values attained in this work are within the range of values obtained for conophor defatted flour (CDF), conophor protein concentrate (CPC), isoelectric protein isolate (CII), and neutral protein isolate (CNI) (40.70, 27.15, 24.42, 20.17 m 2 /g) as reported by Gbadamosi et al. (2012). Raw, cooked, or 14.57%), respectively, were establish around the isoelectric pH established earlier, while the highest EAI and ESI of RFK (44.40 m 2 /g and 113.59%), CUK (33.97 m 2 /g and 76.98%), RUK (40.77 m 2 /g and 84.57%), and CFK (43.13 m 2 /g and 66.25%), respectively were observed at pH 10 and the differences were significant (p < .05) as shown in Figure 2. According to Adiamo et al. (2015aAdiamo et al. ( , 2015b, most food proteins are poorly hydrated, lack electrostatic repulsive forces, sparingly soluble, and are generally poor emulsifiers at the isoelectric pH. The EAI of the samples was observed to increase after the isoelectric pH point with increased salt concentration when salt concentration was increased from 0.5 to 1.0 mol/L. At high salt concentrations, salts compete with protein for water resulting in low extractability of protein due to the salting out effect at this concentration (Osungbade et al., 2016) which is a possible explanation for the decrease in EAI of samples under study. Salting out occurs at high salt concentrations when salts compete with the protein for water (Adiamo et al., 2015a(Adiamo et al., , 2015b (Figures 3-5).
At 0.5 mol/L NaCl, there was a reduction in EAI and ESI values which showed a minimal increase as the salt concentration was increased to 1.0 mol/L. This observation agrees with an earlier report by Osman, Amro, Mohamed-Ahmed, and Babiker (2005) in which it was observed that at 0.0 mol/L NaCl, the EAI and ESI of chickpea were higher but afterward decreased at 0.2 mol/L NaCl although no marginal reduction occurred at 0.6 mol/L NaCl. This trend has been affirmed by previous research on the effect of salt concentration on conophor proteins in a study by Gbadamosi et al. (2012). This investigator reported that at high salt concentration, EAI values were low and attributed this to the increase in repulsive forces which is as a result of the poor interfacial properties of the interfacial film of protein molecules. At high salt concentration, negatively charged chloride ions are induced to interact with positively charged proteins, thereby resulting in a reduction in electrostatic repulsion and enhance hydrophobic interaction. This results in a higher tendency for the protein to form insoluble aggregates which result in decreasing solubility and emulsifying activity. This observation by this investigator is also in agreement with the previous report on pea and hyacinths bean as reported by Ahmed, Babiker, Mohamed Ahmed, Eltayeb, and Faridullah (2012).
| Effect of pH and salt (NaCl) concentrations on the foaming capacity and stability of cooked and fermented kariya seeds
The FC and FS at neutral pH of the kariya flour samples ranged between 14.29% and 33.33% and 9.52% and 15.79%, respectively ( Table 2). The FC of RUK (33.33%) was higher than those of RFK Increase in foam capacity of the samples as salt concentration increases may be due to the fact that salt diminishes protein films rigidity surface viscosity while causing an increase in their spreading rate, thereby resulting in the weakening interpeptide interactions and consequently increase foam volumes for certain proteins (Oshodi, 1992).
The result obtained in this study compared favorably with the reports of Berhanu and Amare (2013), on defatted soy bean which was found to exhibit increased foam capacity as the salt concentration increased.
The foam stability of the samples was also found to increase as the salt concentration increases. This result obtained is in line with what was reported by Osman et al. (2005) and by Mahajan, Bhardwaj, and Dua (1999) who found out that as the concentration of NaCl increased, the foam stability of chickpea flour also increased significantly. From this report, it can be deduced that kariya seed flour foaming capacity and stability improves with increase in salt concentration. Arogundade, Akinfenwa, and Salawu (2004)) previously reported that improved foaming capacity and stability of seed flour in the presence of NaCl will improve its functional properties and invariably its application in food processing such as frosting, whipped toppings, and cake mixes where foaming is of vital prominence.
| Effect of cooking and fermentation on the antinutritional content of kariya flour
The effects of cooking and fermentation on the antinutrient levels of kariya flour samples are shown in Table 3. All the flour samples showed a reduction in oxalate (8.13-6.50 mg/100 g), tannins (1.63-1.20 mg/100 g), and saponins (0.18-013 mg/100 g), respectively.
The values of antinutrient of all samples with respect to the tested antinutrient were significant differences (p < .05). The result showed that the concentrations of each tested antinutrient reduced significantly (p < .05) with an increase in the duration of fermentation days.
The result obtained in this study compares favorably with the work of Fowomola and Akindahunsi (2008) who reported that an increase in fermentation days caused a significant reduction in the antinutrient (oxalate, tannin, saponin, alkaloid, phytate, and cyanide) contents present in Hura crepitans seed. The observed progressive reductions of antinutritional factors as the fermentation progresses is in line with the assertion that the antinutrient levels were generally reduced due to the microflora activities and secreted polyphenol oxidase (Achinewhu & Isichei, 1990). The activities of polyphenol oxidase secreted by fermentative micro flora on tannin might have caused a reduction in tannin content during the processing of the seed flour (Reddy & Pierson, 1994). Also, the cooking process as observed in samples CFK, CFK1, CFK2, and CFK3 resulted in significant reduction in the antinutrients of samples coupled with the fermentation process. Provided that food is adequately cooked to certain temperature and time duration, the toxic effect of antinutrients such as tannin, oxalate, or phytate will be significantly reduced to a permissible level which is nonharmful to health (Enechi & Odonwodu, 2003). Similar observation was reported by Ikemefuna, Obizoba, and Atii (1991) where the combination of cooking and fermentation was reported to drastically lessen the antinutritional factors in sorghum (Guinesia) seeds drastically to a safe level. The values of the antinutrients observed in this study were lesser than the values (tannin: 18.61-5.8 mg/100 g; oxalates: 23.7-3.6 mg/100 g; and saponins: 8.5-1.4 mg/100 g) reported by Fowomola and Akindahunsi (2008) that cooking coupled with increase in fermentation days result in significant reduction in the antinutrients content of Hura crepitans seed. The results agreed with the reports of Ibukun and Anyasi (2012) where a reduction was observed in the oxalate, tannin, and saponin levels of fermented sesame seeds (Sesanum indicum) (2.57-0.36, 0.019-0.008, and 2.68-1.01 mg/g extract, respectively); musk melon seeds (Cucumis melo) (2.1-0.27, 0.007-0.004, and 5.1-2.8 mg/g extract, respectively) and; white melon seeds (Cucumeropsis mannii) (1.35-0.14, 0.008-0.005, and 3.5-1.9 mg/g extract), respectively. Therefore, a combination of cooking and fermentation will result in significant reduction in the antinutrients and improvement of the nutritional quality of kariya seeds.
| CONCLUSION
Cooking and fermentation of H. barteri seeds significantly enhance their protein contents and digestibility. The antinutritional content was also observed to be decreased to safe and acceptable levels after subjecting to fermentation and cooking at conditions stated in this study. Likewise, the processing method employed in this study improves the functional properties of kariya seed flour and is readily available for use as food ingredient in food formulation and supplementation.
T A B L E 3 Antinutritional properties of cooked and fermented flour (RFK, CUK, RUK, and CFK) | v3-fos-license |
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} | pes2o/s2orc | Purification of a n Acyl-CoA Hydrolase from Rat Intestinal Microsomes A CANDIDATE ACYL-ENZYME INTERMEDIATE IN GLYCEROLIPJD ACYLATION*
We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol sug- gesting the presence of a cysteine residue(s) at or near the active site. The enzyme carbon acyl-CoA not utilize diacylglyc-erols as potential and at concentrations be-low 100 or
Purification of an Acyl-CoA Hydrolase from Rat Intestinal Microsomes
A CANDIDATE ACYL-ENZYME INTERMEDIATE IN GLYCEROLIPJD ACYLATION* (Received for publication, April 12, 1993, andin revised form, July 22, 1993) Richard Lehner We have purified to apparent homogeneity an acyl-CoA hydrolase activity from rat intestinal villus cell microsomes by heparin and anion exchange and affinity chromatography. The purified 54-kDa acyl-CoA hydrolase along with several microsomal proteins form a covalent acyl-protein bond upon incubation with an activated fatty acid (acyl-CoA). The acyl moiety of the acylated acyl-CoA hydrolase is stable to denaturation and extraction with organic solvents, but is displaced by neutral hydroxylamine or mercaptoethanol, indicating a labile high energy (thio)ester linkage. The enzyme activity is inhibited by thiol-directed reagents and activated by the presence of dithiothreitol suggesting the presence of a cysteine residue(s) at or near the active site.
Common serine-esterase inhibitors (NaF, phenylmethylsulfonyl fluoride) and activators (Mg2+, Ca2+) had no effect on the hydrolase activity. The enzyme hydrolyzed (transferred to water) 14-20 carbon acyl-CoA with similar efficiencies and did not utilize glycerophospholipids or mono-and diacylglycerols as potential acyl donors/acceptors. Phospholipids and mono-and diradylglycerols at concentrations below 100 pM or polyclonal antibodies raised against the purified hydrolase did not inhibit the enzyme activity. However, the acyl-CoA hydrolase activity could be immunoprecipitated from solubilized microsomes or purified enzyme preparations with corresponding decrease of the hydrolase activity in the supernatant of the immunoprecipitate. Immunoblotting studies show cross-reactivity with a protein of an identical molecular mass in other rat or human tissues. It is concluded that the microsomal acyl-CoA hydrolase deserves consideration as a candidate acyl-enzyme intermediate in glycerolipid synthesis when associated with appropriate acyltransferases.
Long chain fatty acyl-CoAs are synthesized from endogenous and exogenous fatty acid pools by acyl-CoA ligase in the endoplasmic reticulum, which also contains several acyl-CoAdependent enzymes catalyzing the transfer of the acyl moiety from acyl-CoA to suitable acceptors (acylglycerols, cholesterol, and proteins). A long chain acyl-CoA hydrolase activity was observed in particulate and soluble fractions of various cells (1)(2)(3)(4)(5)(6), and several isozymes with distinct electrophoretic mobilities have been at least partially purified from rat liver (1,2) and brain (5) and from bovine (3) and rabbit (6) hearts. The metabolic role of these enzymes has remained uncertain as their function as intermediates in glycerolipid biosynthesis has been compromised by their hydrolytic activity toward glyceryl esters.
We report purification of an acyl-CoA hydrolase activity from rat intestinal mucosa, which does not attack glyceryl esters and could serve as an intermediate in glycerolipid synthesis. The intestinal enzyme also possesses physicochemical properties different from those of the liver enzyme as judged by substrate specificities and inhibition studies. Direct attempts to demonstrate that the intestinal microsomal acyl-CoA hydrolase acts by forming a covalent high energy thioester acyl-protein as an intermediate during acylglycerol synthesis are in progress.
Preparation of Microsomal Membranes-Male rats (Wistar, Charles River Canada Inc., La Salk, Que.) weighing 250-300 g were fed ad libitum with standard diet. They were anaesthetized with .
Purification and
Characterization of Acyl-CoA Hydrolase 24727 diethyl ether and exsanguinated via their abdominal aortae. The upper two thirds of the small intestine were removed and rinsed with 0.9% NaCl and 2 mM Hepes (pH 7.1), and the mucosal scrapings were obtained as described by Hoffman and Kuksis (9). Homogenization and low speed centrifugation procedures were adapted from a method by Pind and Kuksis (10). The scrapings from three or four rats were suspended in approximately 200 ml of 300 mM mannitol, 5 mM EDTA, and 5 mM Hepes (pH 7.1) and homogenized in a Waring blender, set at low speed (power setting of 50 on a Powerstat) for 30 s. The homogenate was then gently filtered through a single layer of gauze (Nu Gauze, Johnson and Johnson Inc., Toronto, Ont.), followed by a double layer of I l l -p m pore size polyethylene mesh (Spectromesh PE, Spectrum Medical Industries Ltd., Los Angeles, CA), to remove mucus and fat particles. The homogenate was centrifuged at 1,500 X g for 5 min. Supernatant was centrifuged at 25,000 X g for 10 min, and the postmitochondrial supernatant was further centrifuged at 106,000 X g for 60 min to pellet microsomal membranes. Microsomes were washed by homogenization in 5 ml of 50 mM potassium phosphate (pH 7.4) containing 0.5 M KCl, 1 mM EDTA, 2 mM dithiothreitol, and 0.1% CHAPS with seven strokes of a motor-driven Potter-Elvehjem homogenizer, followed by centrifugation at 106,000 X g for 60 min. All steps were carried out at 4 "C. Washed microsomes were suspended in 50 mM potassium phosphate (pH 7.4), 1 mM EDTA, 2 mM dithiothreitol, 0.5 M KCl, and 10% glycerol to give a final concentration of protein between 6 and 7 mg/ml. Solubilization of Microsomal Membranes-Zwitterionic detergent CHAPS (1%; detergent/protein ratio, 2:l) was added to the microsomal membranes, and the mixture was stirred on ice for 30 min. The solubilized crude enzyme extract was recovered with a Pasteur pipette after centrifugation at 106,000 X g for 60 min.
The extract was desalted by a passage through a desalting column equilibrated with 50 mM potassium phosphate (pH 7.4), 10% glycerol, and 1% CHAPS.
Purification of Acyl-CoA Hydrolase-All chromatographic procedures were carried out at 4-7 "C except for HPLC, which was run at ambient temperature.
Affi-Gel Heparin-The desalted solubilized microsomal extract was mixed for 1 h with 10 ml of Affi-Gel heparin preequilibrated with 50 mM potassium phosphate (pH 7.4), 10% glycerol containing 0.5% CHAPS (Buffer A). Subsequently, the mixture was poured into a column, washed with Buffer A, and eluted with Buffer A containing 0.25 and 1 M NaCl, respectively.
Cibacron Blue A 3GA-Agarose-Fractions eluted at 14-18 min from DEAE-HPLC were dialyzed against Buffer A and mixed for 30 min with 5 ml of the dye ligand affinity medium preequilibrated with Buffer A. The mixture was transferred to a column, washed with Buffer A, and eluted with Buffer A containing 1 M NaCl.
Polyacrylamide Gel Electrophoresis-10% SDS-polyacrylamide gels (11) were run under non-reducing conditions and were silver-stained by the method of Rabilloud et al. (12).
Effect of Inhibitors and Cofactors-Purified acyl-CoA hydrolase was preincubated with selected inhihitors and cofactors for 15 min at 37 "C. Radiolabeled acyl-CoA was then added to the assay mixture and the acyl-CoA hydrolase activity was determined as indicated above. The activity is expressed as a percentage of the activity obtained with untreated enzyme (specific activity, 3-4 pmol/mg protein/minj. Other Enzyme Assays-Monoacylglycerol hydrolase was assayed by monitoring 2-oleoyl[2-3H]glycerol (100 p~; specific activity, 5 mCi/mmol) hydrolysis in the absence of acyl-CoA. The reaction mixture and assay conditions as well as the TLC solvent system were identical to those used for measuring acyl-CoA hydrolase activity. The monoacylglycerol was added in acetone (2.5% final concentration). After lipid extraction, residual monoacylglycerols were isolated by TLC and counted for radioactivity. Glycerophosphate, monoacylglycerol, diacylglycerol, and cholesterol acyltransferases were assayed essentially by the procedure used to assess acyl-CoA hydrolase activity except that sn-glycerol-3-phosphate (300 pM), 2-oleoylglycer01(60 p~) , 1,2-dioleoyl-rac-glycerol(250 p~) , and cholesterol (300 p M ) were added to the incubation mixture containing in addition 4 mM MgC12, 1 mM dithiothreitol, and 2 mg/ml bovine serum albumin.
Hydrolysis of non-radiolabeled acyl ester substrates was detected by a mass assay using gas-liquid chromatography. Incubations and lipid extractions were carried out essentially as outlined above except that the reaction volume was 0.5 ml. Tridecanoylglycerol ( 5 pg) and no other lipid carriers were added during the lipid extraction. The lipid extract was dried under nitrogen, converted to their corresponding trimethylsilyl esters/ethers by reaction with Sylon BFT (Supelco, Canada Ltd. Oakville, Ontario) in pyridine (l:l, v/v) and l/lOth of the sample was subjected to gas-liquid chromatography analysis on a non-polar capillary column with a 170-350 "C temperature program (14). The amount of fatty acid released by the enzyme during incubations and of non-hydrolyzed substrates was calculated after correction for differences in a flame ionization detector area response (15) and subtraction of control runs (substrates minus enzyme).
Analysis of the Acyl-protein Linkage-Labeled proteins were incubated for 1 h at 37 "C with 1 M hydroxylamine (pH 7.0). As a control, proteins were incubated with 1 M Tris-HC1 (pH 7.0). Susceptibility of the linkage to alkaline hydrolysis was tested by incubation of the labeled proteins with 0.3 M methanolic KOH for 1 h at 37 "C. The stability of the acyl-protein bond toward a reducing agent was evaluated by reaction with mercaptoethanol. Also, the acyl-proteins were extracted with chloroform/methanol 2:l (v/v), and the recovered proteins were analyzed by SDS-PAGE and fluorography.
Antibody Preparation-Purified acyl-CoA hydrolase (approximately 100 pg) in 0.5 ml of 10 mM Tris-HC1 (pH 7.5), 150 mM NaCl (Tris-buffered saline, TBSj was mixed with 0.5 ml of Freund's complete adjuvant, and the thick emulsion was injected subcutaneously into two rabbits (10-12 weeks old). Booster intradermal injections of 25 pg of antigen in Freund's incomplete adjuvant were given 4 and 8 weeks later. Rabbits were bled from the marginal ear vein 1 week prior to (preimmune) and 9 weeks after (immune) the initial immunization. Serum was prepared and stored at -70 "C.
Western Blotting-Proteins separated on 10% SDS-PAGE were elecroblotted onto an Immobilon (polyvinylidene difluoride) membrane in ice-cold 25 mM Tris, 192 mM glycine, pH 8.3, transfer buffer a t 370 mA constant current for 1 h. Following transfer, the membrane was blocked with 5% skim milk in TBS (Blotto) at room temperature for 1 h and then incubated for 1 h with a 1:4,000 dilution of rabbit serum in TBS containing 1% skim milk. The membrane was subsequently washed two times with Blotto for 15 min and incubated for 1 h with the second antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG (H + Lj, diluted 1:1,000 with TBS containing 1% skim milk. The membrane was then washed twice in 0.5% Tween 20 (TTBS) for 15 min and three times in TBS for 10 min. The bound antibody was detected by incubation solution containing a 100:1:1 mixture of 0.1 M sodium bicarbonate, 1 mM magnesium chloride, pH 9.5; 1.5% 5-bromo-4-chloroindoxyl phosphate in N,N-dimethylformamide, and 3% nitro blue tetrazolium in 70% N,N-dimethylformamide. After color development, the membrane was washed with water and air-dried.
Purification ofdntibodies-1 ml of serum was mixed for 3 h at 4 "C with 1 ml of protein A-agarose at pH 8.0 in a 10-ml Poly-Prep chromatography column (Bio-Rad). Following a 10-column volume wash with 100 and 10 mM Tris, pH 8.0, the bound antibodies were eluted with 100 mM glycine, pH 3.0, and neutralized by addition of 1 M Tris, pH 8.0. The purified antibodies were dialyzed for 16 h against TBS with one change after 8 h.
Preparation of Immunoaffinity Columns-Covalently linked antibody-protein A-agarose columns were prepared essentially as described by Schneider et al. (17). Antibodies were bound to protein A beads as described above, the beads were washed twice with 10-column volumes of 0.2 M sodium borate (pH 9.0) and were crosslinked to the protein A using 20 mM dimethylpimelimidate in 10 volumes of 0.2 M sodium borate (pH 9.0). The cross-linking reaction was stopped after 30 min by incubating the beads with 10-column volumes of 0.2 M ethanolamine (pH 8.0) followed by extensive washing with TBS.
Other Methods-Protein concentrations were determined after precipitation with deoxycholate and trichloroacetic acid (18) by bicinchonic acid assay (Pierce Chemical Co.). Unilamellar egg yolk phosphatidylcholine vesicles were prepared by sonication (Branson Sonic Power Co., 50% duty cycle, output control setting 5) of 10 mg phosphatidylcholine/ml suspended in 25 mM Tris, pH 7.8, 150 mM NaCl for 30 min on ice followed by ultracentrifugation at 42,000 rpm (TI 70 rotor) for 3 h. The supernatant was recovered with a Pasteur pipette, and the concentration of phosphatidylcholine was determined by gas-liquid chromatography after phospholipase C treatment using tridecanoylglycerol as an internal standard (15).
Purification of Acyl-CoA
Hydrohe-Microsomal membranes isolated from 8-10 rats were depleted of their lumenal content and peripheral proteins by treatment with a low concentration of detergent (0.1% CHAPS) and 0.5 M KC1, respectively. The membrane-bound enzyme was then solubilized by 1% CHAPS. CHAPS and other bile salt conjugates were superior to non-ionic detergents (Triton and Zwittergent series as well as n-alkyl-8-D-glucopyranosides and n-alkyl-Nmethylglucamides) in preserving the acyl-CoA hydrolase activity. However, addition of any detergent above its critical micellar concentration (a concentration required for extraction of proteins from their natural environment) led to a decrease of the enzyme activity. The detergent extract accounted for about 70% of the total recovered activity and a corresponding percentage of the microsomal protein. The desalted solubilized extract was mixed with Affi-Gel heparin for 1 h to allow maximal protein-ligand interactions. The acyl-CoA hydrolase activity bound to the affinity medium was displaced by 1 M NaCl (Fig. 1). The active fractions were dialyzed and chromatographed on a Spherogel TSK-DEAE-5PW HPLC column (7.5 X 75 mm). The column was eluted with a NaCl gradient (Fig. 1). The acyl-CoA hydrolase activity eluted between 0.2 and 0.3 M salt concentrations. The active fractions (14-18 min) were combined, dialyzed, and mixed with Cibacron blue 3GA-agarose. Most of the activity interacted strongly with the dye-ligand as it could not be released by CoA (100 p~) , acyl-CoA (100 p~) , or ATP (10 mM) but was eluted by 0.8 M NaCl (Fig. 1). A silver-stained SDSpolyacrylamide gel electrophoresis (PAGE) profile of the active fraction recovered from the Cibacron blue A-agarose chromatographic step showed a polypeptide of apparent molecular mass of 54 kDa (Fig. 2). The overall purification of the acyl-CoA hydrolase activity starting from the detergent solubilized microsomal extract was estimated a t 450-fold with 24% recovery of the original solubilized activity ( Table I).
Characterization of the Purified Acyl-CoA Hydrolase-The purified enzyme displayed closely similar activities toward a variety of acyl-CoA esters ( Table 11). The highest activity was obtained with arachidonoyl-CoA and the lowest with stearoyl-CoA. It did not hydrolyze long chain monoacylglycerols, short and long chain diacylglycerols, or glycerophospholipids. Furthermore, various isomers of long chain monoradylglycerols or short and long chain diradylglycerols had little or no effect on the acyl-CoA hydrolase activity up to 150 pM concentra-tions (Table 111). Higher concentrations of dioleoyl and dioctanoylglycerols (1 mM) resulted in approximately 50% decrease of the hydrolase activity. It is important to note that the activity was not affected by preincubation with sodium fluoride or phenylmethylsulfonyl fluoride, potent inhibitors of monoacylglycerol lipase and other carboxylester lipases and by divalent cations (Ca2+, M e ) which are necessary cofactors for optimal activities of most lipases. The enzyme was activated almost 2-fold by inclusion of dithiothreitol (5 mM) in the assay suggesting the presence of a critical cysteine residue at or near the active site. In agreement with this observation the enzyme binds to Affi-Gel501 (organomercurial agarose, Bio-Rad) specific for sulfhydryl containing proteins (result not shown). Furthermore, the activity can be inhibited by sulfhydryl-directed reagents such as iodoacetamide (Table 111).
The solubilized and purified enzyme did not exhibit phospholipid dependence as indicated by incubations with increasing concentrations of unilamellar phospholipid vesicles (Fig. 3). In fact, at concentrations in excess of 200 pg of phosphatidylcholine/ml, a substantial decrease of acyl-CoA hydrolysis (transfer) was observed.
Formation and Characterization of Acyl-proteins in Microsomal Fractions and Purified Acyl-CoA Hydrolase-We observed selective incorporation of fatty acid into microsomal proteins in the presence of ATP and CoA (Fig. 4). The protein acylation was time-dependent with significant levels of labeling observed after 5-min incubations (Fig. 5). Incubation of the purified acyl-CoA hydrolase with radiolabeled oleoyl-CoA also resulted in a time-dependent acyl-enzyme formation (Fig. 6). The inclusion of ATP and CoA was necessary but not sufficient for acyl-protein formation as it also depended on the presence of acyl-CoA ligase. No acyl-ezyme was obtained upon incubation of the purified hydrolase with radiolabeled oleic acid in the presence of ATP and/or CoA indicating that activated fatty acid was necessary to form the acyl-protein and that the enzyme did not possess acyl-CoA ligase activity (result not shown). A significant amount of the radioactive acyl moiety was lost from the acyl-enzyme upon subsequent incubation with methanolic KOH and neutral hydroxylamine (Fig. 7) or with 8-mercaptoethanol (Fig. 8). A 10-fold excess of free CoASH did not prevent protein acylation (Fig. 4). However, a futile cycle of protein acylation and regeneration of acyl-CoA in the absence of ATP by trans-thioesterification could not be eliminated. Extraction of the labeled protein with chloroform/methanol 2:l (v/v) did not remove the label thus confirming presence of a covalent acyl-enzyme linkage (result not shown). Similar results were obtained with the labeled microsomal proteins (not shown).
Immunochuracterization of Acyl-CoA Hydrolase-The purified enzyme was used to prepare polyclonal anti-acyl-CoA hydrolase antibodies in rabbits. Western blot analysis indicated that the antibody reacted with purified acyl-CoA hydrolase and did not show any apparent cross-reactivity with other rat intestinal proteins (Fig. 9). The antibody interacted with a protein of an apparently identical molecular mass of 54 kDa in rat liver, kidney, and heart total homogenates (Fig. 9) as well as in human cerebellum (Fig. 10). The colonic cancer cell line CaCo-2 displayed heterogenous cross-reactivity (Fig. 10) as did various other established cell lines (human melanoma 74-36 cells, colon carcinoma T-84 cells, rat hepatoma and rat glioma C-6 cells, not shown). Antibodies purified on protein A beads did not appear to inhibit the enzyme activity when added to the assay mixture containing purified enzyme or microsomes a t antibody/enzyme weight ratio of 1:l indicating that the polyclonal antibodies are not directed against the Substrate specificity of purified rat intestinal acyl-CoA hydrolase Enzyme activity was determined by gas-liquid chromatography analyses of released fatty acids as described under "Experimental Procedures." The standard assay medium contained, in 0.5 ml, 0.5 pg of purified enzyme, 100 p~ non-radiolabeled substrates indicated below, and 50 mM potassium phosphate, pH 7.4. The glycerolesters were added to the incubation mixture in acetone (final concentration of acetone 2.5%). The values represent averages of two independent experiments performed in duplicate.
Substrate
Fatty acid released CoA hydrolase activity Enzyme activity was determined as described under "Experimental Procedures." The standard assay medium contained, in 0.2 ml, <0.5 pg of purified enzyme, 50 p~ radiolabeled oleoyl-CoA, and 50 mM potassium phosphate, pH 7.4. The standard assay gave the specific activity of 3.5-3.8 pmollmg of protein/min. The values represent averages of two separate experiments. as was a corresponding decrease of the enzyme activity in the immunoprecipitation supernatant (not shown), thus indicating that the antibody population recognized and immunoprecipitated the native enzyme.
DISCUSSION
In this study a homogeneous preparation of acyl-CoA hydrolase has been obtained from solubilized rat intestinal microsomes by chromatography on Affi-Gel heparin, anion exchange, and Cibacron blue-agarose. Previous work has established the presence of the acyl-CoA hydrolases in tissues of several animal species (1-6) indicating an ubiquitous need for such activity in cells. Despite extensive studies, the role of the enzyme in intracellular processes and its regulation has not yet been elucidated. It appears that different tissues express different isozymes with various substrate specificities. For instance, the 57-60-kDa liver enzyme catalyzes hydrolysis of palmitoyl-CoA but exhibits only a marginal activity toward other long chain CoA esters (2,19). In addition, it hydrolyzes monoacylglycerols with similar efficiency (19). The 41-kDa bovine heart microsomal acyl-CoA hydrolase showed marked preference for arachidonoyl-CoA, but other medium to long chain saturated and polyunsaturated fatty acyl-CoAs were also suitable substrates (3). The enzyme was 80% inhibited by 1 p~ lysophosphatidylcholine or lysophosphatidylinositol but was not affected by up to 5 p~ concentrations of lysophosphatidylserine and lysophosphatidylethanolamine (3). Cytosolic and mitochondrial acyl-CoA hydrolase activities in various tissues with differences in specificities have also been reported (5,(20)(21)(22). The purified enzyme from intestinal microsomes appears to be a form of the enzyme different from those reported earlier. The apparent molecular mass of 54 kDa as estimated by denaturing gel electrophoresis is lower than that found for the rat liver enzyme (57-59 kDa). A polyclonal antibody raised against the purified hydrolase cross-reacts with a 54-kDa protein present in total cell homogenates of rat liver, heart, and kidney, as well as human cerebellum. More important, the enzyme does not hydrolyze glyceryl esters and their presence did not significantly activate or inhibit the activity. However, high concentrations of diacylglycerols and phospholipids in the assay mixture interfere with the hydrolysis reaction. This could be perhaps attributed to the interference of the phospholipid and diacylglycerol with the substrate availability. Inactivation of the activity due to asymmetrical incorporation of the enzyme into the phospholipid bilayer with 50150 outside/inside active site orientation may also be considered but it is unlikely as the lipidlprotein molar ratio required to obtain 50% inactivation is high. The acyl-CoA hydrolysis was not dependent on the presence of cations and was not inactivated by known lipase inhibitors. The enzyme was sensitive to thiol-directed reagents, suggesting that it contains important cysteine residues in or near its active site.
Several microsomal proteins including acyl-CoA hydrolase were found to form covalent acyl-proteins upon incubation with activated fatty acid. The rate of incorporation of the radioactive acyl groups into the proteins corresponded closely to the rate of acylglycerol synthesis (8). The nature of the acyl-enzyme bond appears to be consistent with the definition of a thioester or a "reactive" ester as the linkage is susceptible to neutral hydroxylamine mediated hydrolysis (23). The acylprotein formation may represent an intermediate step in the lipid biosynthetic pathway, where the enzyme acylation would precede acyl transfer to acyl acceptors. It has been recently suggested (24) that a common acyl-CoA-binding subunit (acyl-CoA hydrolase) may be a member of a hetero-oligomeric complex containing acyl acceptor subunits. Hence if substrates for the acyltransferases or the acyltransferase subunits are not present, only the hydrolysis (transfer to water) of acyl-CoA would occur. There exists some evidence for involvement of acylated cysteine residues in fatty acyl transfer/ hydrolysis. The reaction proceeding through a covalent cysteine-linked acyl-enzyme intermediate has been demonstrated for fatty acid synthetase (25), myristoyl-CoAprotein N-myristoyltransferase (26), and acyl-protein synthetase and acyl-CoA reductase of the fatty acid reductase complex from Photobacterium phosphoreum (27). A group of proteins which served as acceptors for fatty acids in cell-free extracts from mouse heart, kidney, and liver has also been recently identified (28). We are presently investigating the role of acyl-CoA hydrolase in intestinal triacylglycerol biosynthesis. We have purified by affinity (29) or hydrophobic interaction (30) chromatography a triacylglycerol synthetase complex from rat intestinal mucosa containing monoacylglycerol and diacylglycerol acyltransferases as well as acyl-CoA hydrolase and ligase. We have obtained evidence that acyl-CoA hydrolase inhibitors also inhibit triacylglycerol synthesis from monoacylglycerol and acyl-CoA substrates and prevent the formation of the covalent acyl-enzyme.' | v3-fos-license |
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